JP2003508011A - Compositions, kits and methods relating to a novel tumor suppressor gene that is the human FEZ1 gene - Google Patents

Abstract

The present invention relates to a human tumor suppressor gene, an isolated polynucleotide homologous to a portion of one strand of FEZ1 , and a tumor suppressor protein, Fez1, encoded thereby. Polynucleotides are useful, for example, as probes, primers, parts of expression vectors, and the like. The invention also relates to diagnostics, therapeutics, cell growth enhancement and screening methods involving such polynucleotides and proteins. The invention further includes kits useful for performing the methods of the invention.

Description

Detailed Description of the Invention

[0001] (Technical field)                                BACKGROUND OF THE INVENTION   The present invention relates generally to cancer and tumor suppressor genes.

Proliferation of normal cells is driven by growth-promoting protooncogenes, and growth-suppression (constr
aining) It is thought to be regulated by tumor suppressor genes (Weinberg, 1991, Sc)
ience 254: 1138). Genetic alterations that either inactivate the tumor suppressor gene or activate the protooncogene release cells from the suppression of growth forced by the unmodified gene, thereby allowing tumor growth. Accumulation of genetic aberrant forms in cells causes abnormal, rapid replication of cells in vivo, from normal growth or quiescent stages, and in some cases through discernable pre-neoplastic stages. Causes progression to a potentially cancerous condition in areas of the body not normally found (Knudson, 19
93, Proc. Natl. Acad. Sci. USA 90: 10914; Nowell, 1993, Adv. Ccancer Res. 62: 1).

The presence of tumor suppressor genes at specific chromosomal locations is often indicated by increased prevalence of heterozygous (LOH) deletions at chromosomal locations in tumor tissue relative to non-tumor tissue ( Weinberg, 1991, Science 254: 1138; Lasko et al., 1991, An
n, Rev. Genet. 25: 281; Knudson, 1993, Proc.Natl.Acad.Sci.USA 90: 10914; Nowell,
1993, Adv. Cancer Res. 62: 1). In allelotype studies, chromosome 8p, especially the band
Allele deletion (s) at 21-22 is associated with a variety of tumors including prostate, breast, head and neck squamous cell carcinoma, bladder cancer, hepatocellular carcinoma and hematological malignancies. (Kagan et al., 1995, Oncogene 11: 2121; Macoska et al., 1
995, Cancer Res. 55: 5390; Jenkins et al., 1998, Gene Chromosom.Cancer 21: 131
Yaremko et al., 1995, Gene Chromosom. Cancer 13: 186; Yaremko et al., 1996,
Gene Chromosom. Cancer 16: 189; Kerangueven et al., 1997, Cancer Res. 57: 5469.
Anbazhagan et al., 1998. Am.J. Pathol. 152: 815; E1-Naggar et al., 1998, Oncog
ene 16: 2983; Sunwoo et al., 1996, Gene Chromosom. Cancer 16: 164; Wu et al.,
1997, Gene Chromosom. Cancer 20: 347; Wagner et al., 1997, Am.J.Pathol. 151: 75.
3; Boige et al., 1997, Cancer Res. 57: 1986; Takeuchi et al., 1995: Cancer Res.
55: 5377).

Studies transferring chromosomal regions into tumor cells have provided evidence that one or more tumor suppressor genes reside at human chromosome position 8p (Gustafson et al., 1996, Cancer Res. 5).
6: 5238; Ichikawa et al., 1994, Cancer Res. 54: 2299; Kuramochi et al., 1997, Pr
ostate 31:14). These considerations suggest that chromosomal region 8p21-22 plays an important role in the development of various tumors.

In order to identify the tumor suppressor gene (s) located on chromosome 8p, studies by others have identified two candidate cancer suppressor genes, designated N33 and PRLTS (Bookstein et al. al., 1997, Br. J. Urol. 79 (Suppl. 1): 28; Bova et al.
., 1996, Genomics 35:46; MacGrogan et al., 1996, Genomics 35:55; Cher et al.
, 1994, Genes Chromosom.Cancer 11: 153; Bookstein et al., 1994, Genomics 24: 3
17; Fujiwara et al., 1995, Oncogene 10: 891; Komiya et al., 1997, Jpn. J. Cancer
r Res. 88: 389). The gene N33 is located near the MSR locus at position 8p22, but a point mutation in N33 was not associated with tumor. Four cancer-related point mutations have been reported in PRLTS, which are located at 8p21.3-22. However, the frequency of modification in this gene was very low. Thus, neither the N33 gene nor the PRLTS gene appears to be the tumor suppressor gene associated with many cancers.

Prior to the present disclosure, the tumor suppressor gene (s) located at chromosome position 8p has not been identified. The inability of others to identify this gene has delayed the development of diagnostics, therapeutics and other useful methods and compositions involving this tumor suppressor gene. The present invention enables such methods and compositions.

BRIEF SUMMARY OF THE INVENTION The present invention comprises a single, highly stringent, annealing (ie, substantially complementary) portion of 20 or more consecutive nucleotide residues of a chain of the human FEZ1 gene. Isolated polynucleotides. The exemplified human FEZ1 gene has the nucleotide sequence of SEQ ID NO: 1. The annealing moiety can be substantially homologous to residues in the human FEZ1 gene, or preferably can be completely homologous to such residues. Preferably, this portion is at least substantially at least 20 residues of the exon region of the human FEZ1 gene, i.e. nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550 of the chain of SEQ ID NO: 1. Are homologous.

In one aspect, the isolated polynucleotide of the invention comprises a portion having the nucleotide sequence of the strand of SEQ ID NO: 3, and optionally further comprising a promoter. The promoter may be, for example, a constitutive promoter, an inducible promoter or a tissue-specific promoter.

In another aspect of the isolated polynucleotide of the invention, the isolated polynucleotide is incorporated into a nucleic acid vector or is encoded by a nucleic acid that is incorporated into the nucleic acid vector. The isolated polynucleotide can have sequence homology with, for example, the strand of SEQ ID NO: 1 and can be detectably labeled. Examples of detectably labeled isolated polynucleotides are immobilized polynucleotides, protein-ligand pair protein linked polynucleotides, protein-ligand pair ligand linked polynucleotides, biotinylated polynucleotides, Fluorophore-linked polynucleotides, chromophore-linked polynucleotides, enzyme-linked polynucleotides and radio-labeled polynucleotides. When using the immobilized polynucleotide, the polynucleotide can be immobilized on the surface of the gene chip. Preferably the isolated polynucleotide of the invention is substantially purified.

An isolated polynucleotide of the invention need not comprise only naturally occurring bases and bonds. Isolated polynucleotides include, for example, phosphonates, phosphorothioates, phosphorodithioates, phosphoramidates, methoxyethyl phosphoramidates, formacetals, thioformacetals, diisopropylsilyl, acetamidates, carbamates, dimethylene-sulfides (-CH ₂ -S-CH ₂ )
, Dimethylene - sulfoxide _{(-CH 2 -SO-CH 2)} , dimethylene - sulfone _{(-CH 2 -SO 2 -CH 2)} , 2'-O- alkyl, and 2'-deoxy-2'-fluoro phosphorothioate, Hosuhoroto Reester, siloxane, carbonate, carboxymethyl ester,
Acetamidate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate bond, bridged sulfone bond and It may have at least two nucleotide residues linked by a non-naturally occurring bond other than a phosphodiester bond, such as a bond selected from the group consisting of combinations of such bonds. Furthermore, the ends of the isolated polynucleotide may be nucleolytically blocked.

The present invention also includes an isolated polynucleotide comprising a portion having a sequence that anneals with high stringency to at least 20 consecutive nucleotide residues of the strand of SEQ ID NO: 3.

[0012] In another aspect, the invention comprises a kit for amplifying a portion of the human FEZ1 gene. The kit comprises a first isolated polynucleotide and a second isolated polynucleotide. The first isolated polynucleotide comprises a portion that anneals with high stringency to at least 20 consecutive nucleotide residues of the coding strand of SEQ ID NO: 1, and the second isolated polynucleotide comprises the non-coding strand of SEQ ID NO: 1. Comprising a moiety that anneals with high stringency to at least 20 consecutive nucleotide residues of.

The invention further includes a kit for amplifying a portion of the cDNA produced from the transcript of the human FEZ1 gene. The kit comprises a first isolated polynucleotide and a second isolated polynucleotide. The portion of the first isolated polynucleotide is SEQ ID NO: 1.
Anneal with high stringency to at least 20 contiguous nucleotide residues of the coding strand of, and a portion of the second isolated polynucleotide has high stringency to at least 20 consecutive nucleotide residues of the non-coding strand of SEQ ID NO: 1. Anneal with.

The invention further includes a foreign DNA molecule having at least a portion of the strand of SEQ ID NO: 1 that is substantially homologous to nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550. Including animal cells consisting of. In one embodiment, the foreign DNA molecule further comprises a promoter operably linked to this portion,
The foreign DNA molecule is then expressed in the animal cell.

The invention also includes a genetically modified animal comprising cells into which a foreign DNA molecule has been artificially introduced. This foreign DNA molecule has a portion substantially homologous to at least the coding region of the chain of the human FEZ1 gene. The foreign DNA molecule may have a portion which is substantially homologous to at least nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550 of the strand of SEQ ID NO: 1, or It can comprise a portion having a sequence that is substantially homologous to the number 2 strand.

The invention also relates to an isolated human Fez1 protein, such as a protein having an amino acid sequence substantially or preferably completely homologous to SEQ ID NO: 4.
In one aspect, the protein is substantially purified.

The invention further includes isolated antibodies that specifically bind the human Fez1 protein and hybridoma cells that produce such antibodies.

The method further relates to a method of determining a cancerous state of sample tissue. The method comprises comparing FEZ1 expression in sample tissue to FEZ1 expression in control tissue of the same type. The reduced FEZ1 expressed in the sample tissue to FEZ1 expression in control tissue samples tissue indicative that is cancer. In one embodiment, the sample tissue is a phenotypically abnormal portion of human body tissue and the control tissue is a phenotypically normal portion of body tissue, such as epithelial tissue. Body tissue can also be selected from the group consisting of, for example, gastrointestinal tract tissue, esophageal tissue, stomach tissue, colon tissue, prostate tissue, breast tissue, hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue and ovarian tissue. In another aspect of the present invention, by comparing the relative amounts of the index of the sample in the tissue and control tissue, the FEZ1 expression of sample tissues compared to FEZ1 expression in control tissue. The indicator may for example be selected from the group consisting of FEZ1 mRNA, FEZ1 cDNA of mRNA was prepared using, DNA and Fez1 proteins prepared by amplification of any of these.

The invention also includes a method of determining the cancerous state of a sample tissue. The method FEZ1 was obtained from a sample tissue - comprising comparing the nucleotide sequence of the association polynucleotide - a nucleotide sequence of association polynucleotide, control FEZ 1. FEZ1- obtained from sample tissue-nucleotide sequence of the associated polynucleotide and control FEZ1-
The difference between the nucleotide sequences of the associated polynucleotides is an indicator that the sample tissue is cancerous.

The present invention includes another method for determining the cancerous state of human sample tissue. The method comprises comparing the length of the FEZ1 -transcript-association polynucleotide obtained from the sample tissue with the length of the control FEZ1 -transcript-association polynucleotide. FEZ1 -transcript from sample tissue-association polynucleotide length control FEZ1 -transcript
-If it is shorter than the length of the associated polynucleotide, this is an indication that the sample tissue is cancerous.

[0021]   The present invention includes yet another method of determining a cancerous condition in a sample tissue. This way
In the sample tissueFEZ1Comprising assessing expression. In the sample tissue FEZ1 Substantial absence of expression is an indication that the sample tissue is cancerous.FEZ1 Expression is for exampleFEZ1mRNA,FEZ1cDNA prepared using mRNA, any of these
Selected from the group consisting of Fez1 protein and DNA prepared by amplification of
It can be assessed by assessing the presence or substantial absence of the indicator.

The invention includes yet another method of determining a cancerous condition in a sample tissue. This method comprises detecting aberrant splicing of the FEZ1 transcript in sample tissue. Abnormal splicing of the FEZ1 transcript is an indicator that the sample tissue is cancerous. Aberrant splicing of FEZ1 transcript, for example, exon boundaries polynucleotide probe FEZ1 - transcripts - can be detected by assessing the ability to annealing at a meeting polynucleotide high stringency. Exon boundary polynucleotide probes can anneal with high stringency to the end portions of two consecutive FEZ1 exons when the end portions are adjacent, but not when the end portions are not adjacent.

In another aspect, the invention relates to a method of modulating aberrant proliferation of human cells having a modified FEZ1 gene. This method comprises providing a cell with a Fez1 protein of exogenous origin. Abnormal growth of cells is suppressed, delayed or prevented by this. The exogenous Fez1 protein may be a composition comprising an isolated human Fez1 protein, such as the human Fez1 protein having the amino acid sequence of SEQ ID NO: 4. An exogenous Fez1 protein is an expression vector comprising a polynucleotide having a coding region that encodes a functional Fez1 protein such as the human FEZ1 gene having the nucleotide sequence of the strand of SEQ ID NO: 3 (eg, the nucleotide sequence of SEQ ID NO: 60). Adenovirus vector) such as a vector comprising a vector nucleic acid having The polynucleotide may further comprise a constitutive, inducible or tissue-specific promoter operably linked to the coding region. When the promoter is an inducible promoter, the method further comprises administering to the cell an inducer of the inducible promoter. Of course, the polynucleotide can comprise the wild-type FEZ1 promoter region.

The present invention further includes a kit for amplifying a portion of the cDNA produced from the transcript of the human FEZ1 gene. The kit comprises a first isolated polynucleotide and a second isolated polynucleotide. The portion of the first isolated polynucleotide is SEQ ID NO: 1.
Anneal with high stringency to at least 20 contiguous nucleotide residues of the coding strand of, and a portion of the second isolated polynucleotide has high stringency to at least 20 consecutive nucleotide residues of the non-coding strand of SEQ ID NO: 1. Anneal with.

The invention further includes a foreign DNA molecule having at least a portion of the strand of SEQ ID NO: 1 that is substantially homologous to nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550. Including animal cells consisting of. In one embodiment, the foreign DNA molecule further comprises a promoter operably linked to this portion,
The foreign DNA molecule is then expressed in the animal cell.

The invention also includes a genetically modified animal comprising cells into which a foreign DNA molecule has been artificially introduced. This foreign DNA molecule has a portion substantially homologous to at least the coding region of the chain of the human FEZ1 gene. The foreign DNA molecule may have a portion which is substantially homologous to at least nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550 of the strand of SEQ ID NO: 1, or It can comprise a portion having a sequence that is substantially homologous to the number 2 strand.

The invention also relates to an isolated human Fez1 protein, such as a protein having an amino acid sequence substantially or preferably completely homologous to SEQ ID NO: 4.
In one aspect, the protein is substantially purified.

The invention further includes isolated antibodies that specifically bind to the human Fez1 protein and hybridoma cells producing such antibodies.

The method further relates to a method of determining a cancerous state of a sample tissue. The method comprises comparing FEZ1 expression in sample tissue to FEZ1 expression in control tissue of the same type. The reduced FEZ1 expressed in the sample tissue to FEZ1 expression in control tissue samples tissue indicative that is cancer. In one embodiment, the sample tissue is a phenotypically abnormal portion of human body tissue and the control tissue is a phenotypically normal portion of body tissue, such as epithelial tissue. Body tissue can also be selected from the group consisting of, for example, gastrointestinal tract tissue, esophageal tissue, stomach tissue, colon tissue, prostate tissue, breast tissue, hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue and ovarian tissue. In another aspect of the present invention, by comparing the relative amounts of the index of the sample in the tissue and control tissue, the FEZ1 expression of sample tissues compared to FEZ1 expression in control tissue. The indicator may for example be selected from the group consisting of FEZ1 mRNA, FEZ1 cDNA of mRNA was prepared using, DNA and Fez1 proteins prepared by amplification of any of these.

The invention also includes a method of determining the cancerous state of a sample tissue. The method FEZ1 was obtained from a sample tissue - comprising comparing the nucleotide sequence of the association polynucleotide - a nucleotide sequence of association polynucleotide, control FEZ 1. FEZ1- obtained from sample tissue-nucleotide sequence of the associated polynucleotide and control FEZ1-
The difference between the nucleotide sequences of the associated polynucleotides is an indicator that the sample tissue is cancerous.

The present invention includes another method for determining the cancerous state of human sample tissue. The method comprises comparing the length of the FEZ1 -transcript-association polynucleotide obtained from the sample tissue with the length of the control FEZ1 -transcript-association polynucleotide. FEZ1 -transcript from sample tissue-association polynucleotide length control FEZ1 -transcript
-If it is shorter than the length of the associated polynucleotide, this is an indication that the sample tissue is cancerous.

[0032]   The present invention includes yet another method of determining a cancerous condition in a sample tissue. This way
In the sample tissueFEZ1Comprising assessing expression. In the sample tissue FEZ1 Substantial absence of expression is an indication that the sample tissue is cancerous.FEZ1 Expression is for exampleFEZ1mRNA,FEZ1cDNA prepared using mRNA, any of these
Selected from the group consisting of Fez1 protein and DNA prepared by amplification of
It can be assessed by assessing the presence or substantial absence of the indicator.

The invention includes yet another method of determining a cancerous condition in a sample tissue. This method comprises detecting aberrant splicing of the FEZ1 transcript in sample tissue. Abnormal splicing of the FEZ1 transcript is an indicator that the sample tissue is cancerous. Aberrant splicing of FEZ1 transcript, for example, exon boundaries polynucleotide probe FEZ1 - transcripts - can be detected by assessing the ability to annealing at a meeting polynucleotide high stringency. Exon boundary polynucleotide probes can anneal with high stringency to the end portions of two consecutive FEZ1 exons when the end portions are adjacent, but not when the end portions are not adjacent.

In another aspect, the invention relates to a method of modulating aberrant proliferation of human cells having a modified FEZ1 gene. This method comprises providing a cell with a Fez1 protein of exogenous origin. Abnormal growth of cells is suppressed, delayed or prevented by this. The exogenous Fez1 protein may be a composition comprising an isolated human Fez1 protein, such as the human Fez1 protein having the amino acid sequence of SEQ ID NO: 4. An exogenous Fez1 protein is an expression vector comprising a polynucleotide having a coding region that encodes a functional Fez1 protein such as the human FEZ1 gene having the nucleotide sequence of the strand of SEQ ID NO: 3 (eg, the nucleotide sequence of SEQ ID NO: 60). Adenovirus vector) such as a vector comprising a vector nucleic acid having The polynucleotide may further comprise a constitutive, inducible or tissue-specific promoter operably linked to the coding region. When the promoter is an inducible promoter, the method further comprises administering to the cell an inducer of the inducible promoter. Of course, the polynucleotide can comprise the wild-type FEZ1 promoter region.

In yet another aspect, the invention relates to a method of preventing tumor formation in human cells. The method comprises providing to the cell an expression vector comprising a polynucleotide having a coding region encoding a functional Fez1 protein. When the expression vector is provided to the cell, tumor formation in the cell is suppressed.

The present invention also includes a method of inducing cell proliferation in reverse. The method comprises providing an inhibitor of FEZ1 expression inside the cell. Cell growth is thereby induced in the presence of the inhibitor inside the cell but not in the absence of the inhibitor inside the cell. The inhibitor can be, for example, an isolated polynucleotide comprising a portion that anneals with high stringency to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. The isolated polynucleotide can be delivered intracellularly by administering to the cell a gene vector comprising a promoter operably linked to the isolated polynucleotide. The cell can be located in the body of an animal such as a human.

In another aspect, the invention features a method of determining whether a test compound is an inducer of cell proliferation. The method comprises incubating cells comprising a functional FEZ1 gene in the presence of test compound and assessing FEZ1 expression in the cells. If FEZ1 expression in cells is reduced compared to the FEZ1 expression of the same type of cells that incubation in the absence of the test compound, the test compound is an inducer of cellular proliferation.

The invention also includes a method of determining if a test compound is effective to slow aberrant growth of cells having a modified FEZ1 gene. The method comprises incubating the cells in the presence of the test compound and assessing FEZ1 expression in the cells. If FEZ1 expression in cells is increased compared with FEZ1 expression of the same type of cells that incubation in the absence of the test compound, the test compound is effective to slow the abnormal growth of cells And

The invention further relates to a method of determining whether a Fez1 protein binds to a polynucleotide having a test nucleotide sequence. This method comprises: a) contacting a Fez1 protein with a test polynucleotide having a test nucleotide sequence, and b) subsequently assessing whether a detectably labeled Fez1-polynucleotide complex is formed. Consists of At least one of the Fez1 protein and polynucleotide is detectably labeled. The formation of the complex is used as an indicator that the Fez1 protein binds to the polynucleotide having the test nucleotide sequence.

The invention further relates to a method of identifying an inducer of cell proliferation. The method comprises: a) contacting a Fez1 protein and a polynucleotide to which the Fez1 protein binds in the presence and absence of a test compound, and b) assessing the formation of a Fez1-polynucleotide complex. The formation of the complex in the presence of the test compound as opposed to the formation of the Fez1-polynucleotide complex in the absence of the test compound is an indicator that the test compound is an inducer of cell proliferation.

The present invention includes a kit for selecting anti-cancer therapeutic compounds for administration to humans suffering from cancer. The kit comprises a plurality of candidate anti-cancer therapeutic compounds and reagents for assessing FEZ1 expression in cells.

The present invention also includes methods of inducing cell proliferation. The method comprises suppressing FEZ1 expression in the cell. This induces the cells to proliferate. In one aspect, the cells are removed from a human. The cells can be returned to humans after suppressing FEZ1 expression in the cells. Alternatively, the cells can be cells that are present in the human body. For example, expression of FEZ1 in cells is suppressed by providing inside the cell an isolated polynucleotide comprising a portion that anneals with high stringency to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. be able to.

The present invention further includes enhanced human cell culture techniques. This technique comprises incubating human cells and suppressing FE Z1 expression in the cells according to known human cell culture techniques.

The method further includes detecting FEZ1 expression in the sample tissue. The method is: a) labeling an isolated antibody that specifically binds to human Fez1 protein and contacting a preparation of the isolated antibody with sample tissue, and b) rinsing the tissue sample thereafter, thereby non-specifically. Rinsing the specifically bound antibody from the tissue sample, and c) assessing the presence of labeled antibody in the tissue sample. The presence of labeled antibody in the tissue sample is an indicator that FEZ1 was expressed in the tissue sample.

The present invention includes methods for determining whether a test compound is useful for ameliorating disorders associated with aberrant tubulin polymerization. The method comprises: (i) tubulin polymerization in a first assay mixture comprising tubulin, Fez1 and a test compound, and (ii) a second assay mixture comprising tubulin and Fez1 but no test compound. Comparing tubulin polymerization in. First and second
The difference between tubulin polymerization in the assay mixture (eg, rate or extent of tubulin polymerization) is an indication that the test compound is useful in ameliorating the disorder. Preferably the first and second assay mixtures are substantially the same, but the presence or absence of the test compound is different. Disorders include, for example, disorders associated with aberrant initiation of mitosis, disorders associated with aberrant modulation of rates and stages of mitosis, disorders associated with aberrant modulation of cell proliferation initiation and rate, of cell growth. Disorders associated with aberrant initiation and velocity modulation, disorders associated with aberrant modulation of cell shape, disorders associated with aberrant modulation of cell hardness, disorders associated with aberrant modulation of cell motility, cellular DNA replication Disorders associated with aberrant modulation of velocity, disorders associated with aberrant modulation of cellular DNA replication stages, disorders associated with aberrant modulation of organelle intracellular distribution, and aberrant modulation of cell metastatic capacity Transformation of cells from impaired and non-cancerous to cancerous phenotype It may be a tubulin overpolymerization disorder or tubulin oligomeric disorders, such as disorders related to abnormal modulation. For example, the disorder can be tumorigenesis, tumor survival, tumor growth and tumor metastasis. Examples of test compounds include fragments of Fez1, fragments of Fez1, fragments of tubulin, fragments of tubulin, fragments of EF1-γ and fragments of EF1-γ. .

The present invention also includes methods of determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1. The method comprises: (i) phosphorylation of Fez1 in a first assay mixture comprising Fez1, at least one kinase, a phosphate source and a test compound; and (ii) Fez1, a kinase and a phosphate source. Comparing with phosphorylation of Fez1 in the second assay mixture without test compound. The difference in Fez1 phosphorylation between the first and second assay mixtures is an indicator that the test compound is useful in ameliorating the disorder. As in the method described in the preceding paragraph, the disorder can be selected from the group consisting of tumorigenesis, tumor survival, tumor growth and tumor metastasis.

The present invention includes methods of determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1. This method comprises (i) phosphorylation of Fez1 in a first assay mixture comprising phosphorylated Fez1, at least one phosphatase and a test compound, and (ii) phosphorylated Fez1 and phosphatase, wherein the test compound is Comparing with phosphorylation of Fez1 in the second assay mixture without. Differences in phosphorylation of Fez1 between the first and second assay mixtures (eg dephosphorylated Fez1 de-
The difference in the rate or degree of phosphorylation) is an indicator that the test compound is useful in ameliorating the disorder.

The invention further includes methods of determining whether a test compound is useful for ameliorating a disorder associated with aberrant binding of Fez1 and a protein to which Fez1 normally binds. This method comprises: (i) binding between Fez1 and a protein in a first assay mixture comprising Fez1, a protein and a test compound; and (ii) a second assay comprising Fez1 and a protein but no test compound. Comparing the binding between Fez1 and the protein in the mixture. First
And the difference in Fez1 and protein binding between the second assay mixture (eg, rate or extent of binding) is an indication that the test compound is useful in ameliorating the disorder. Examples of proteins of this method include tubulin and EF1-γ. The obstacle is
For example, it can be any of the obstacles listed above.

The invention further includes a method of determining whether a test compound is an inhibitor of cell proliferation. This method comprises incubating cells comprising a functional FEZ1 gene in the presence of a test compound and assessing FEZ1 expression in the cells. If the FEZ1 expression in the cells is increased relative to the FEZ1 expression in the same type of cells incubated in the absence of the test compound, the test compound is indicative of an inhibitor of cell expansion.

[0050] The present invention includes a method of inhibiting tumor formation in humans, inducer of the method FEZ1 gene expression in humans, enhancers FEZ1 gene expression, the Fez1 phosphorylation inhibitors, the phosphorylation Fez1 dephosphorylation Enhancers, Fez1 and EF-γ
Administering a compound selected from the group consisting of an agent that inhibits the binding of Fez1 and an agent that inhibits the binding of Fez1 to tubulin.

[0051]

Detailed Description of the Invention

The present invention is based on the discovery, isolation, and sequencing of FEZ1, a tumor suppressor gene located on human chromosome position 8p22. It has been observed that reduced or no expression of FEZ1 can be detected in various cancer cells obtained from cancer cell lines and tissue samples taken from human patients. The forms of cancer in which abnormal (ie, reduced or absent) expression of FEZ1 is detected include epithelial cancer, digestive system cancer, esophageal cancer, gastric cancer, colon cancer, prostate cancer, breast cancer, as described herein. Includes but is not limited to hematopoietic organ cancer, lung cancer, melanoma, and uterine cancer. It is believed that the expression of FEZ1 will also be associated with other cancers when tested for modified FEZ1 expression.

Expression of FEZ1 suppresses tumor growth and proliferation both in vitro and in vivo.
The ability of the Fez1 protein to interact with tubulin, microtubulin, and the protein FEZ1-γ indicates that intracellular expression of FEZ1 is associated with physiological processes associated with microtubulin, such as mitosis, cell proliferation, It shows that it regulates cell motility. Furthermore, post-translational phosphorylation and dephosphorylation of the Fez1 protein can regulate the effects that the Fez1 protein has on these physiological processes. Definitions As used herein, each of the following terms has the meaning set forth in this chapter.

As used herein, the articles “a” and “an” refer to one or more (ie, at least one) of the grammatical object of the article. For example, "an ele
“Ment” represents one element or one or more elements.

The terms “cancereous” (eg cells, tissues, conditions, etc.) and “tumor (t
umor) ”(eg, cells, tissues, conditions, etc.) are used interchangeably herein.

“Polynucleotide” means a single strand or parallel or antiparallel strands of nucleic acid. Thus, the polynucleotide can be either single-stranded or double-stranded nucleic acid.

An “isolated” polynucleotide refers to a nucleic acid segment or fragment that is separated from sequences that naturally flank it, eg, sequences that normally flank the fragment, eg, flanking the fragment in its naturally occurring genome. The DNA fragment is not adjacent to the sequence. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, such as RNA, DNA, or proteins that naturally accompany it in the cell. Thus, this term is incorporated into, for example, a vector, into a plasmid or virus that replicates autonomously, or independently of other sequences into prokaryotic or eukaryotic DNA, or into another molecule ( For example
Recombinant DNA present as cDNA or PCR or genomic or cDNA fragments produced by restriction enzyme digestion). It also includes recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

An “isolated” protein or antibody is one that is separated from one or more components that naturally accompany it in its naturally occurring state. By way of example, an isolated protein can be prepared by separating the protein from at least one other naturally associated protein. As another example, the isolated protein may include at least one other naturally associated protein.
The protein can be prepared synthetically in the absence of the seed protein.

“Substantially purified” polypeptide nucleotides, proteins, or antibodies are those that are evaluated from at least most of the components that naturally accompany them in their naturally occurring state, preferably by weight or molar basis. At least 75%, 80% of the ingredients of
It is also separated from up to 90% or 95%.

As used herein, "homologous" means between two polymer molecules, such as between two nucleic acid molecules, such as between two DNA molecules or two RNA molecules or between two. It refers to subunit sequence similarities between polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomer subunit, eg if a position in each of the two DNA molecules is occupied by adenine, these Homologous in position. The homology between two sequences is a direct function of the number of matching or homologous positions, eg half of the positions in two compound sequences (eg 5 positions in a polymer of 10 subunits length). Are homologous, the two sequences are 50% homologous, and 90% of the positions, ie, 9 out of 10 are matched or homologous, the two sequences are 90% homologous. Have. As an example, the DNA sequences 3'-ATTGCC-5 'and 3'-TATGGC-5' are 50
Both share% homology.

“Substantially homologous” means at least 70%, 75%, 80%, 85%, 90%, 95%, 97.
It is meant to have%, 98%, or at least 99% homology.

[0061]   "Completely homologous" means 100% homology.

“Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. An adenine residue in the first nucleic acid region forms a specific hydrogen bond (“base pairing”) with a residue in the second nucleic acid region that is antiparallel to the first region when the residue is thymine or uracil. It is known to have the ability to do. It is also known that cytosine residues in the first nucleic acid strand are capable of base pairing with residues in the second nucleic acid strand that are antiparallel to the first strand when the residue is guanine. . A first region of a nucleic acid if at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region when the two regions are arranged in antiparallel fashion. Are complementary to a second region of the same or another nucleic acid. Preferably, the first region comprises the first part and the second region comprises the second part, whereby the first and second parts are arranged in antiparallel form, At least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with the nucleotide residues of the second portion. More preferably, all nucleotide residues in the first part are base pairable with nucleotide residues in the second part,
In this case, the two parts are described as "fully complementary". "Substantially complementary" means having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or even at least 99% complementarity.

If the nucleotide residues of at least one region of each of the two polynucleotides are involved in base pairing when the two regions are arranged in antiparallel form in a suitable solution, One polynucleotide "anneals" to the second polynucleotide. Such solutions are well known in the art and include, for example, standard citrate saline (SSC) buffer.

At least about 75%, and preferably at least about 90% or at least about 95
Under conditions where only oligonucleotides that are% complementary anneal to each other,
A first polynucleotide anneals with "high stringency" to a second polynucleotide when two polynucleotides anneal. The stringency of the conditions used to anneal two polynucleotides depends on temperature, ionic strength of the annealing medium, incubation time, length of the polynucleotide, length of the polynucleotide, among other factors, if known among other factors. It is a function of the G-C content and the degree of heterology expected between the two polynucleotides. Methods for adjusting the stringency of annealing conditions are known (eg Sambrook et al., 1989, Molecul.
ar Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York
reference). As an example, high stringency hybridization conditions include (1) low ionic strength and high temperature such as 0.015 molar NaCl, 1.5 mmol sodium citrate, and 0.1% (w / v) sodium dodecyl sulfate ( 5 of SDS)
Used for washing at 0 ° C .; (2) denaturing agent during hybridization,
For example formamide, eg 50% (v / v) formamide, 0.1% (w / v)
) Bovine serum albumin, 0.1% (w / v) ficoll, 0.1% (w / v) polyvinylpyrrolidone, and 50 mM sodium phosphate buffer, pH 6.5.
Is used at 42 ° C. with 750 mmol NaCl, 75 mmol sodium citrate; or (3) 50% (v / v) formamide, 5 × SSC (0.75).
Molar NaCl, 75 mM sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg per milliliter), 0.1% (w / v
) SDS, and 10% (w / v) dextran sulfate at 42 ° C.,
Hybridization conditions in 0.2 x SSC and 0.1% (w / v) SDS at. Under stringent hybridization conditions, only highly complementary nucleic acids hybridize.

A “functional” or “engineered” protein is a protein that is in a form that exhibits at least one biological activity characterized in its naturally occurring state.

A “functional” or “engineered” gene is one that is present within a functional gene-expressing protein that compromises the environment (eg, inside a human cell or in vitro gene expression of the type described in the art). Within a mixture), a gene that is expressed to produce a gene product that is encoded or designated by the gene.

A first polynucleotide is where the first polynucleotide is either homologous or complementary to a transcribed polynucleotide produced by either transcription or reverse transcription by at least a portion of the second polynucleotide, "Designated" by the second polynucleotide. The first polynucleotide can be homologous or complementary to the transcribed polynucleotide either before or after the transcribed polynucleotide is acted upon by a eukaryotic mRNA splicing component.

A “portion” or “region” of a polynucleotide is at least two consecutive nucleotide residues of the polynucleotide, and preferably at least 10, 11, 12,
...., 20, 21, 22, ...., 30, 31, 32, ...., 40, 41, 42, ..., or 50 or more consecutive nucleotide residues Means

A first portion of a polynucleotide is “adjacent” to a second portion of the same polynucleotide when the nucleotide sequences of the first and second portions are directly attached to each other and have no intervening nucleotides. As an example, pentanucleotide 5'-AA
Both of AAA-3 'are 5'-AAAAATTT-3' or 5'-TTTAA.
Trinucleotide 5'-TTT-3 when linked as AAA-3 '
This is not the case when it is adjacent to 'and is connected as 5'-AAAAACTTT-3'.

The two parts are adjacent to each other or the two parts are about 1000, 999, 998,
...., 900, 899, 898, ...., 750, 749, 748, ...., 500, 499, 498,
...., 250, 249, 248, .... "Flank" to the part.

When describing two polynucleotides as “operably linked,”
A single strand comprising two polynucleotides, wherein at least one of the two polynucleotides is arranged within the nucleic acid portion in such a way that the physiological effect being characterized can act on the other. Or, it means a double-stranded nucleic acid portion. By way of example, a promoter operably linked to the coding region of a gene can drive transcription of the coding region.

As used herein, the term "promoter" means a nucleic acid sequence necessary for the expression of a gene product operably linked to a promoter sequence. In some cases, this sequence may be a nuclear promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. For example, the promoter sequence may be one that expresses the gene product in a tissue-specific manner.

A “constitutive” promoter is one which, when operably linked to a polynucleotide encoding or designating a gene product, produces the gene product in human cells living under most or all physiological conditions of the cell. Is a nucleotide sequence that produces

An “inducible” promoter is a promoter that, when operably linked to a polynucleotide encoding or designating a gene product, is only live if an inducer corresponding to the promoter is substantially present in the cell. Is a nucleotide sequence that produces a gene product in human cells.

A “tissue specific” promoter is only operably linked to a polynucleotide encoding or designating a gene product and only if the cell is of a tissue format that substantially corresponds to the promoter. A nucleotide sequence which produces a gene product in living human cells.

A “substantially no expression” of a gene means that the level of expression of the gene is undetectable or at least significantly lower than that of the gene in its naturally occurring state (
For example, 100 times or 1000 times or more).

An “expression vector” comprises an isolated nucleic acid and is intended to deliver the isolated nucleic acid to the interior of the cell such that the gene product encoded or designated by the isolated nucleic acid is produced intracellularly. It is a combination of substances that can be used for. Many expression vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. The expression vector generally comprises a promoter operably linked to a portion of the isolated nucleic acid encoding or designating the gene product, or the isolated nucleic acid is placed within a cellular nucleic acid in which the portion is operably linked to the cellular promoter. Either can be inserted.

“Foreign” polynucleotides within an organism are those that do not exist in the same form as the polynucleotide in the organism's naturally occurring forms. By way of example, an exogenous oligonucleotide may comprise a nucleic acid sequence that the organism's genome does not include, or a form that does not exist in the organism's naturally occurring forms (eg, plasmids or artificial chromosomes). ), A part of the genome of the organism in

A “analog” of a gene is substantially homologous to the gene and has substantially the same biological activity as that expressed by the gene product coded for or designated by the gene. It codes or specifies the gene product.

A “FEZ1-related polynucleotide” is at least about 20, 21, 22, .. either a human FEZ1 gene or a spliced mRNA designated by the human FEZ1 gene.
.., 30, 31, 32, ..., 40, 41, 42, ..., Or 50 or more consecutive homologous or substantially complementary nucleic acid residues A polynucleotide comprising a portion.

A “FEZ1-transcript-association polynucleotide” refers to at least about 20 of either the splice or the unspliced mRNA designated by the human FEZ1 gene.
, 21, 22, ...., 30, 31, 32, ..., 40, 41, 42, ..., or substantially homologous to 50 or more consecutive nucleic acid residues Or, a polynucleotide comprising a portion that is substantially complementary.

A “contig” of genomic region is usually contained within a yeast, bacterial, or phage vector and contains all or substantially all (ie> 95%) of the sequence of the genomic region.
, And preferably> 99%) together.

An “exon boundary polynucleotide probe” is a polynucleotide that is complementary or homologous to at least 5 nucleotide residues of an exon of the FEZ1 gene flanked by introns of the gene.

“Protein-ligand pair” refers to a protein and other molecules to which the protein specifically binds to other molecules. Examples of protein-ligand pairs include antibodies and their corresponding epitopes and avidin proteins such as streptavidin, and biotin.

A protein or polynucleotide is “detectably labeled” when it comprises or is linked to a composition of matter that is detectable after contacting the protein or polynucleotide with another protein or polynucleotide. ". There are numerous methods known in the art for detectably labeling proteins or polynucleotides, such as the surface to which such compounds are linked, the radionuclide incorporated within such proteins, such compounds. Included are chromophores and fluorophores linked to.

A “gene chip” is an article that comprises a surface having an ordered array of polynucleotides permanently or reversibly attached. For example, an ordered array can consist of four parts, where one of the four polynucleotides is anchored to the surface of each part, and the four polynucleotides with the exception of one nucleotide residue. Have identical nucleotide sequences (eg 5'-A
ACCAAAAAAA-3 ', 5'-AACCCAAAAAT-3', 5'-A
ACCAAAAAAC-3 'and 5'-AACCAAAAAG-3').

An “inducer of cell proliferation” is one that, when contacted with a cell, causes the cell to grow, divide or replicate at a higher rate than would otherwise be the case in the absence of the composition.
A composition of matter.

[0088]   When the rate of cell growth slows, cell growth is "delayed".

The “cancerous state” of a tissue or cell is whether cells or one or more cells within the tissue currently exhibit one or more characteristics of tumor cells or tissue (eg, uncontrolled cell growth. Or metastasis) or more without genomic damage and accumulating sufficient genomic mutations to exhibit one or more characteristics of a tumor cell or tissue upon incubation or maintenance of the cell. ..

A “phenotypically aberrant” portion of tissue is one that comprises one or more properties of a tissue-shaped cancer cell, such as cells having an abnormal morphology or an abnormal growth or proliferation rate. is there.

A “phenotypically normal” portion of tissue is one that is not considered phenotypically abnormal.

A “candidate anti-cancer compound” is a compound that exhibits potential anti-cancer activity in a relevant assay or a compound that has substantial structural similarity to such compound. Methods for identifying compounds with potential anti-cancer activity and designing structurally similar compounds are well known in the art.

The term “pharmaceutically acceptable carrier” refers to a chemical composition in which one or more ingredients can be combined and, in combination, can be used to administer one or more active ingredients to a patient. Means a thing.

The term “physiologically acceptable” ester or salt refers to an ester or salt form of the active ingredient that is tolerable with the other ingredients of the pharmaceutical composition and is not deleterious to the patient to whom the composition is administered. means.

“Explanatory material” is used to inform the utility of an isolated polynucleotide, isolated protein, or pharmaceutical composition of the invention to practice one or more methods of the invention. Means any publication, record, chart or other medium of expression that can be expressed. The instructional material may be, for example, a method of using these pharmaceutical compositions to carry out the therapeutic methods of the invention, a therapeutic method of the invention, or a screening assay of the invention, or a suitable dose of, for example, a pharmaceutical composition of the invention. May be described.

A “tubulin overpolymerization disorder” is a disorder associated with the extent and rate of tubulin polymerization in cells or animals afflicted with this disorder that is greater than in cells or animals afflicted with this disorder.

A “tubulin hypopolymerization disorder” is a disorder associated with the extent and rate of tubulin polymerization in cells or animals afflicted with the disorder that are smaller than cells or animals afflicted with the disorder.

[0098]

[Detailed description]

As a tumor suppressor gene, FEZ1 is closely involved in controlling the cancerous or non-cancerous phenotype of normally expressing cells. The characteristics of normal, FEZ1-expressing tumor cells are abnormal cell proliferation, abnormal cell growth, and abnormal cell differentiation.

In normal (ie non-cancerous) cells, FEZ1 expression limits cell proliferation.
Without wishing to be bound by any particular theory of manipulation, within the putative structure of the Fez1 protein, the leucine zipper region described herein is Fez1 and one of the physiological polynucleotides (eg genomic DNA) or It is thought to be involved in binding between further regions, which may inhibit or inhibit expression (ie, transcription or translation) of the polynucleotide. The binding of Fez1 to one or more regions on the human genome can repress transcription of one or more closely located genes on the genome, and for FEZ1 regulation of cell proliferation. It is considered to be a mechanism capable of acting. However, the possibility that the Fez1 protein binds to mRNA and regulates its transcription cannot be ruled out. Regardless of how FEZ1 expression or non-expression is involved in the regulation of cell growth, the compositions and methods described herein serve the purposes described herein.

Nucleotide sequence of a portion of the human genome encoding wild-type FEZ1 (SEQ ID NO: 1)
Is shown in FIG. 5A. The nucleotide sequence (SEQ ID NO: 2) of the cDNA produced using the full-length mRNA transcribed from wild-type FEZ1 is shown in FIG. 5B. The nucleotide sequence of the open reading frame (ORF) of wild type FEZ1 (SEQ ID NO: 3) is shown in Figure 5I. The deduced amino acid sequence of the wild-type Fez1 protein (SEQ ID NO: 4) is shown in FIG. 5J. Nucleotide sequence of cDNA produced using truncated FEZ1 mRNA species (SEQ ID NOs: 9-14)
And the corresponding truncated amino acid sequence of Fez1 protein (SEQ ID NOs: 15-20)
Are shown in Figures 5C-5H and 5K-5P, respectively. Isolated Polynucleotides of the Invention The present invention provides isolated polynucleotides that anneal with high stringency to at least one strand of the human FEZ1 gene, eg, at least 20 consecutive nucleotide residues of the human gene having SEQ ID NO: 1. Including. Preferably, the isolated polynucleotide of the present invention is at least 20, 21, 22, ..., 30 of at least one strand of the human FEZ1 gene.
, 31, 32, ...., 50, 51, 52, ..., 75, 76, 77, ..., or 100 consecutive nucleotide residues with high stringency annealing, or It is substantially complementary to these residues. In one aspect, the isolated polynucleotide of the invention comprises about
200, 199, 198, ...., 150, 149, 148, ...., 100, 99, 98, ...., 50,
It has a length that is not longer than 49, 48, ...., 40, 39, 38, ..., or 35 nucleotide residues.

The isolated polynucleotide of the present invention preferably comprises at least 20, 21, 22, ...., 30, 31, 32 ,. 41, 42, ....
, Or having a sequence that is substantially homologous to 50 consecutive nucleotide residues. More preferably, the isolated polynucleotide is at least one of the human FEZ1 genes.
At least 20, 21, 22, .... 30, 31, 32, ..., 40, 41, 42, ..., or 50 contiguous nucleotide residues of this strand, and more preferably Is at least 20,21,22, .... 30,31,32, ...., 40,41,42 of at least one strand of SEQ ID NO: 1
...., or having a sequence that is completely homologous to 50 consecutive nucleotide residues.

An isolated polynucleotide of the invention can be selected as homologous to either the coding or non-coding strand of FEZ1. Alternatively, the isolated polynucleotide is FEZ1
Can comprise both a first part that is homologous to one strand and a second part that is homologous to the other strand of which the first part is annealed to the second part to form a hairpin-shaped structure. An isolated polynucleotide capable of Depending on the application for which the isolated polynucleotide of the invention is used, skilled artisans will, with reference to this disclosure, appreciate that the isolated polynucleotide is homologous to the coding strand of FEZ1, the non-coding strand. It will be possible to decide whether or not it should include both parts or both.

Depending on the application for which the polynucleotides of the invention are used and the length of the isolated polynucleotide, the degree of homology between the isolated polynucleotide and at least one strand of human FEZ1 will be It is understood that it may be more or less important in the various embodiments described herein.

At least one of the isolated polynucleotide and FEZ1 when the isolated polynucleotide of the invention is hybridized or annealed to a nucleic acid having a sequence in which at least one portion is complementary to the isolated polynucleotide. The degree of homology required with the present strand depends on the length of the polynucleotide. As is well known, longer polynucleotides reduce the degree of complementarity required to anneal the polynucleotide to other polynucleotides with high stringency. Numerous methods, algorithms, computer programs, etc. are known which allow the skilled practitioner to predict the stringency of binding between two polynucleotides (eg Suhai,
Ed., 1992, Computational Methods in Genome Research, Plenum Press, New
York; Swindell, Ed., 1997, Sequence Data Analysis Guidebook, Humana Pres
s, New Jersey; Bishop, Ed., 1998, Guide to Human Genome Computing, Acade
See mic Press, New York). In any of these methods, skilled artisans will refer to this disclosure to design or select isolated polynucleotides of various lengths that anneal with high affinity to at least one strand of the human FEZ1 gene. Can be used to All such isolated polynucleotides are within the scope of the invention.

The determination of the match rate between two nucleotide or amino acid sequences can be performed using a mathematical algorithm. For example, a mathematical algorithm that can be used to compare two sequences is described by Karlin and Altschul (1990 ,, Proc. Natl. Acad. Sci. USA.
87: 2264-2268), modified by Karlin and Altschul (1993 ,, Proc. Natl. Acad. Sci.
USA 90: 5873-5877). This algorithm is based on Altwschul et
al., (1990, J. Mol. Biol. 215: 403-410) NBLAST and XBLAST.
Incorporated into the program, eg National Center for Biotechnology Inform
ation (NCBI) World Wide Web site, it has a universal resource locator "http://www.ncbi.nlm.nih.gov/BLAST/". BLAS
A T nucleotide search can be performed with the NBLAST program (called "blastn" on the NCBI website) using the following parameters: gap penalty = 5 to obtain nucleic acid sequences homologous to the nucleic acids described herein. , Gap extension
penalty = 2, mismatch penalty = 3, match reward = 1, expectation value
10.0 and word size = 11. BLAST Protein Search is XBRAS
T program (called "blastn" on the NCBI website) or NCBI
It can be run with the "blastp" program using the following parameters: To obtain an amino acid sequence homologous to the protein molecule described herein, expectation value
10.0, BLOSUM62 scoring matrix. To obtain a gapped algorithm for comparison purposes, gapped BLAST is described by Altschul et al. (1997, Nu.
cleic Acid Res. 25: 3389-3402). Or PSI
-Blast or PHI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.) And relationships between molecules that share a common pattern. BLAST, gapped BLAST, PSI-Blast, and PHI-Bl
When using the ast program, the respective program (eg XBLAST
And NBLAST) default parameters can be used. http: //www.ncb
See i.nlm.nih.gov.

The percent match between two sequences can be determined using techniques similar to those described above, with or without gaps. In calculating the concordance rate, accurate matches are typically counted.

When the isolated polynucleotide of the present invention is used to express all or part of the human Fez1 protein in vitro or in vivo, (i) the isolated polynucleotide and the human FEZ1 gene (eg, The homology with SEQ ID NO: 1) is such that the amino acid sequence encoded by the isolated polynucleotide corresponds to the corresponding region of FEZ1, and (ii) the sequence of the isolated polynucleotide and the corresponding region of FEZ1. Differences between the two do not result in differences in the encoded amino acid sequences (ie, any sequence difference in the coding region is simply replaced by a codon encoding the amino acid with another codon encoding the same amino acid). Or (iii) all differences in the encoded amino acid sequence between the isolated polynucleotide and the corresponding region of FEZ1,
It is important to bring about only one or more conservative amino acid substitutions, which are elaborated elsewhere in this specification. The human codon table below can be used to select or identify alternative codons that encode the same amino acid.

[0108]

[Table 1]

When it is necessary or desirable to introduce nucleotide residue changes into a polynucleotide, such as an isolated polynucleotide of the invention, or into a Fez1 protein or portion thereof, various well-known techniques, such as specific site Mutagenesis can be used. Site-directed mutagenesis, for example, allows the production of mutants using specific oligonucleotides encoding the sequence of the desired mutation as well as a sufficient number of contiguous nucleotides to modify the nucleotide sequence ( It results in a primer sequence that is of sufficient size and sequence complementarity to form a stable duplex on both sides (eg, codons). Typically, primers of about 17-25 nucleotides in length with about 5-10 residues modified on either side of the junction of sequences are preferred. This technology
Typically, phage vectors that exist in both single-stranded and double-stranded form are used. A typical vector that can be used for site-directed mutagenesis is, for example, the M13 phage vector. Such vectors are commercially available and their use is well known in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis protocols, eliminating the need to transfer the gene of interest from the plasmid to the phage vector. Site-directed mutagenesis involves first obtaining a single-stranded vector, or dissociating the two strands of a double-stranded vector containing within its sequence a DNA sequence comprising the desired site of mutation. Do it. The oligonucleotide primers described above are annealed to the single-stranded vector and DNA polymerized to produce the strand with the mutation. A heteroduplex is formed between the strand with the mutation and either the original unmutated strand of the double-stranded vector or the addition or synthetic strand that is substantially complementary to the strand with the mutation. . This heteroduplex is then used to transform appropriate cells, such as E. coli or cultured human cells, and a clone is selected which comprises the recombinant vector with the mutated sequence arrangement. The preparation of sequence variants of the isolated polynucleotides of the invention using site-directed mutagenesis is a method that may produce such variants, as there are other well known methods of producing such variants. Is submitted as an example and is not intended to be limiting. By way of example, a recombinant vector comprising or encoding the desired isolated polynucleotide can be treated with a mutagenizing agent such as hydroxylamine to obtain sequence variants.

An isolated polynucleotide of the invention can be single-stranded or double-stranded, and is in single-stranded form when describing the annealing of the isolated polynucleotide of the invention with other nucleic acids. Is the form referred to herein.

The isolated polynucleotides of the present invention may consist of deoxyribonucleosides or ribonucleosides and may also have phosphodiester bonds or modified bonds such as phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, Acetamidate, carbamate, thioether, crosslinked phosphoramidate, crosslinked methylenephosphonate, crosslinked phosphoramidate,
Bridged phosphoramidates, bridged methylene phosphonates, phosphorothioates, methyl phosphonates, phosphorodithioates, bridged phosphorothioates, or sulfone bonds, and combinations of such bonds, can be virtually any nucleic acid. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically present bases (adenine, guanine, thymine, cytosine, and uracil). The isolated polynucleotide of the present invention is preferably in a purified form.

The present invention is not intended to be limited by the nature of the nucleic acid used. An isolated polynucleotide of the invention may be an isolated naturally occurring nucleic acid, or it may be a synthetic nucleic acid. Isolated naturally occurring nucleic acid can be obtained from viral, bacterial, animal, human, or plant sources. The polynucleotide can be DNA or RNA. In addition, nucleic acids can be isolated, synthesized, or assembled as part of a virus or other macromolecule. See, eg, Fasbender et al., 1996, J. Biol. Chem. 272: 6479-89 (polylysine condensation of DNA in the form of adenovirus).

Nucleic acids that can be used in the present invention include, but are not limited to, oligonucleotides and polynucleotides such as antisense DNA and / or RNA.
, Ribozymes, DNA for gene therapy, viral fragments containing viral DNA and / or RNA, DNA and / or RNA chimeras, mRNA, plasmids,
Cosmid, genomic DNA, cDNA, gene fragment, single-stranded DNA, double-stranded DNA,
Includes various structural forms of DNA, including supercoiled and / or triple-stranded DNA, Z-DNA, and the like. Nucleic acids can be prepared by any conventional means that is normally used to prepare nucleic acids in large quantities. For example, DNA and RNA can be chemically synthesized by methods well known in the art using commercially available reagents and synthesizers (eg,
Gait, 1985, Oligonucleotide Synthesis; A Practical Approach (IRL Press,
Oxford, England))). RNA is transferred to plasmids such as S via in vitro transcription.
It can be prepared in high yield using P65 (Promega Corporation, Madison, WI).

[0114]   For example, if high nuclease stability is desired, denatured nucleotides
Nucleic acids having interosidic linkages may be preferred. Modified nucleoside ligation
Nucleic acids, including compounds, can also be synthesized using reagents and methods well known in the art. example
For example, phosphonate, phosphorothioate, phosphorodithioate, phosphoramid
Date, methoxymethyl phosphoramidate, formacetal, thioformua
Cetal, diisopropylsilyl, acetamidate, carbamate, dimethyle
-Sulfide (-CH₂ -S-CH₂ ), Dimethylene-sulfoxide (-CH ₂  -SO-CH₂ ), Dimethylene-sulfone (-CH₂ -SO₂ -CH₂ ) 2
'-O-alkyl, and 2'-deoxo-2'-fluorophosphorothioate
Methods for synthesizing nucleic acids containing internucleotide linkages are well known in the art.
(Ullmann et al., 1990, Chem. Rev. 90: 543-584; Schneider et al., 1990,
 Tetrahedron Lett. 31: 335). The stability of the isolated polynucleotide of the invention is
Block one or both ends (if linear) of nucleotides by nucleolytic degradation
Can be treated with at least one reagent to enhance. Such reagents are
Known in the art (eg Oligonucleotides as Therapeutic Agents, 1997
, John Wiley & Sons, New York).

The isolated polynucleotide can be purified using any suitable means, such as those well known in the art. For example, isolated polynucleotides can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography, or gel electrophoresis. Of course, the skilled artisan will recognize that the method of purification will depend in part on the size and format of the nucleic acid to be purified and the properties of any molecule, structure or organism with which it can associate. It is further contemplated that the isolated polynucleotides of the present invention may also comprise five naturally occurring bases, adenine, guanine, thymine, cytosine, and other nucleotide residues of uracil.

In one aspect, the isolated polynucleotide of the invention is detectably labeled. Any known method of labeling nucleic acids can be used to label the polynucleotide. By way of example, well known methods of detectably labeling polynucleotides include the incorporation of radionuclides within the polynucleotide, the attachment of the polynucleotide to surfaces such as latex beads or nylon membranes, the attachment of proteins to the polynucleotide such as enzymes. , A type of binding of a protein-ligand pair (eg, avidin-biotin pair or antibody-antigen pair) to a polynucleotide, binding of a chromophore to a polynucleotide, and binding of a fluorophore to a polynucleotide. In one embodiment that can be used for the quantification of nucleic acids capable of annealing the isolated polynucleotides of the invention,
An isolated polynucleotide reversibly binds to both a fluorophore and a molecule capable of quenching the fluorescence of the fluorophore, thereby causing either the fluorophore or the quencher molecule to dissociate from the isolated polynucleotide. , Fluorophore enhanced fluorescence can be detected, which has been described (Livak et al., 1995, "Guidelines for Designing TaqMan ^™ Fluorogenic Probes for 5'Nuclease Assays", Perkin Elmerm Norwalk, CT,
US Pat. Nos. 5,210,015, 5691,146, Heid et al., 1996, Genome
Res. 6: 986-994).

The isolated polynucleotide of the present invention has many uses. For example, such an isolated polynucleotide encodes a different polynucleotide (eg, a genome, genomic fragment, mRNA, cDNA, DNA, or human FEZ1 that is detectably labeled and has a sequence that comprises an annealing moiety. Library clone)
Can be used as a probe to detect the presence of Such probes can be used, for example, to detect or quantify FEZ1 expression in human cells or tissues. It is understood that numerous methods using polynucleotide probes for the detection and quantification of nucleic acids to which the probe anneals are known in the art (eg Sa
mbrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York; Ausbel et al., 1992, Current Protocols in
Molecular Biology, John Wiley & Sons, New York; Gerhardt et al., Eds., 1
994, Methods for General and Molecular Bacteriology, American Society fo
r Microbiology, Washington, DC), and thus these methods are not described in detail here. When the probe is used for the detection or quantification of nucleic acids encoding all or part of FEZ1, it is preferably detectably labeled.

The isolated polynucleotides of the invention can also be used to detect the presence of non-human analogs of the human FEZ1 gene in polynucleotides obtained or derived from non-human sources (eg, animals such as A library of genomic fragments obtained from a mammal or a cDNA library derived from its mRNA). It is well known that gene sequences are conserved in animals, and the degree of sequence conservation is generally related to the degree of evolutionary relatedness in the animal. Therefore, at least 20, 21, 22, .... 30, 30, 31, of human FEZ1
Isolations that anneal with high stringency to 32, ...., 40, 41, 42, ...., or 50 consecutive nucleotide residues or are substantially complementary to these residues. The polynucleotides can be used to identify genomic fragments, cDNAs, mRNAs, or other polynucleotides that comprise a portion of an animal FEZ1 gene that is similar to the portion of the human FEZ1 gene that an isolated polynucleotide of the invention anneals. Think Given that human FEZ1 regulates at least one important physiological function (eg, cell proliferation), the nucleotide sequence of FEZ1 may be more highly conserved in organisms than less important genes. Be expected. Thus, the isolated polynucleotides of the invention are not only for isolation and identification of primate and other mammalian FEZ1 analogs, but also for other vertebrates, other eukaryotes, and possibly any FEZ1.
It is believed that it can also be used to isolate and identify analogs. Preferably, when a non-human analog of FEZ1 is isolated or identified, multiple isolated polynucleotides of the invention are used, each polynucleotide being complementary to a different portion of human FEZ1. is there. Also preferred is that at least one isolated polynucleotide of the invention is complementary to a portion of human FEZ1 that is expected to be particularly conserved, eg, the portion encoding the leucine zipper region of the Fez1 protein.

Also considered is a product comprising a plurality of isolated polynucleotide probes of the invention immobilized in an ordered array on the surface. Such products are routinely called "gene chips". Each of the multiple probes anneals with high stringency to a portion of the human FEZ1 gene. By including probes that differ by a single nucleotide residue within the corresponding portion of the FEZ1 gene, nucleic acids whose positions within the FEZ1 gene comprise various nucleotide residues can be distinguished. Therefore, missense and deletion mutations within the FEZ1 sequence can be detected using methods well known in the art. Furthermore, by incorporating into the array probe that binds with high affinity to the sequence portion of the wild-type FEZ1 gene, each sequence portion now contains one nucleotide residue that was not included in the previous sequence portion. , The nucleotide sequence of all or any part of the FEZ1 gene can be determined. Preferably, the wild type human FEZ1 gene sequence used is SEQ ID NO: 1. Similarly ordered arrays can be designed to detect mRNA sequence alterations, preferably using SEQ ID NO: 2 or SEQ ID NO: 3 as the wild type human FEZ1 mRNA sequence. Products of this type are manufactured by Affymetrix, Inc. (Santa Clar
a, CA), which is similar to the GeneTip ^™ device, which has multiple primers that bind with high stringency to, for example, a portion of the human p53 gene or a portion of the HIV-1 protease or reverse transcriptase gene. Comprises. Methods of preparation and use of such products are described separately and within the FE described in this disclosure.
Only modifications by skilled experts are required to include the Z1 gene sequence (Wallraff et al., February 1997, Chemtech 22-23; Lockhart e
t al., 1996, Nature Biotechnol. 14: 1675-1680: Pease et al., 1994, Proc.
Natl. Acad. Sci. USA 91: 5022-5026; Fodor et al., 1993, Nature 364: 555-55.
6).

One or more isolated polynucleotides of the invention may be used as primers for the replication or amplification of all or part of a nucleic acid comprising all or part of the human FEZ1 gene or non-human FEZ1 analog. Can also be used. For example, the nucleic acid may be a strand of the human genome, a human chromosome, a fragment of the human genome, or all or part of a non-human genome, or it may be an mRNA produced by transcription of the human FEZ1 gene or a non-human analog thereof or It may be either strand of cDNA produced using such mRNA. With reference to the present invention, the skilled artisan will be able to replicate or reproduce virtually any nucleic acid comprising a portion of homology or complementarity to all or part of the human FEZ1 gene, for example having the nucleotide sequence SEQ ID NO: 1. Can be amplified. Methods such as DNA transcription, RNA reverse transcription, DNA replication, polymerase chain reaction (PCR) are well known and are not explained beyond the citation of standard textbooks below (Sambrook et al., 1989, Molecular Cloning: A Laboratoy
Manual, Cold Spring Harbor Laboratory, New York; Ausbel et al., 1992, Cu
rrent Protocols in Molecular Biology, John Wiley & Sons, New York; Gerha
rdt et al., eds., 1994, Methods for General and Molecular Bacteriology,
American Society for Microbiology, Washinton, DC). Methods for amplifying genomic regions flanking previously sequenced genomic regions are likewise known, and so long as amplification of genomic regions flanking the human FEZ1 gene or its non-human analogues is concerned, Included within the range.

When the pair of isolated polynucleotides of the present invention is used for the amplification of all or part of the human FEZ1 gene, its transcript, or cDNA produced using this transcript, it is well known in the PCR method. As such, one polynucleotide anneals with high stringency to one strand near one end of the region to be amplified, and another polynucleotide with the other strand of the other region of the region to be amplified with high stringency. The polynucleotide should be chosen to anneal near the ends. Of course, as is also well known, when the nucleic acid to be amplified is mRNA or other RNA, a cDNA complementary to the mRNA.
Must be generated prior to performing the PCR reaction.

Virtually any region of the human FEZ1 gene, or non-human analog thereof, can be amplified using one or more isolated polynucleotides of the invention. In one embodiment, at high stringency, at least near the opposite end and on the opposite strand of the human FEZ1 gene.
Polynucleotides that anneal with 20, 21, 22, ...., 30, 31, 32, ..., 40, 41, 42, ..., or 50 nucleotide residues are all human FEZ1 Used to amplify a gene, or non-human analog thereof, from one or more parts of the human or non-human genome.

In another embodiment, one or more pairs of isolated polynucleotide primers are selected, each of the pairs being 5'- or 3'- of the exon on the coding strand of the nucleic acid encoding the exon. A first primer that anneals with high stringency to the intron portion adjacent to the end and a second primer that anneals with high stringency to the intron portion adjacent to the respective 3'- or 5'-ends of the same exon on the non-coding strand of the nucleic acid. Comprising a primer. Optionally, each of the two primers of each pair flank the designated end of the exon. Thus, according to this method, amplification of a nucleic acid encoding the human FEZ1 gene, or at least one exon of a non-human analog thereof, using one or more primer pairs optionally does not include any intron sequences. It results in the amplification of one or more exon sequences of a gene or analog. Amplification of both the exon sequence and its adjacent intron sequence is believed to be more informative than the amplification of the exon sequence alone, which appears in the intron but nevertheless encodes the encoded protein (e.g. mR
This is because sequence alterations that are thought to affect the amino acid sequence of mutations that affect NA splicing) can appear.

In another embodiment of the amplification method of the invention, the pair of isolated polynucleotide primers of the invention comprises a wild-type human genomic FEZ1 region (eg SEQ ID NO: 1), the corresponding wild-type mRNA.
, Or amplification of the cDNA produced from wild-type human FEZ1 mRNA with these primers is selected to produce a mixture of amplification products having a determined length. Selection of these amplification products by size (eg by gel electrophoresis or chromatography) shows a characteristic pattern for wild type sequences. Amplification of the same nucleic acid obtained from an individual having a mutation that affects the length or presence of any of the amplification products,
It produces a pattern different from wild type, and the presence of mutations within an individual can thereby be identified.

In yet another aspect of the amplification method of the present invention, a pair of isolated polynucleotide primers of the present invention is known to have been modified in tumor cells (ie, known to have a deletion or missense mutation). Selected) to amplify the region of the nucleic acid encoding the human Fez1 protein, or a non-human analog thereof. Several of such regions are described herein in Example 1, and primers that can be used to amplify these regions are included within the invention. Identification of the presence of such a modification is
It is an indicator that the cell or tissue from which the nucleic acid was obtained is cancerous. Examples of primers that can be used within this embodiment are, for example, primer pairs G12 and G13, G14.2 and G15, and G16 and IntABR, exon 2 to amplify the coding region of exon 1.
In order to amplify the coding region of IntABF and G17, G20 and G21, and G32 and IntBCR, the primer pair IntB to amplify the coding region of exon 3
CF and Nut6, G1 and G2, G75 and G82, G5 and G6, and G7 and G8. These primers have the nucleotide sequences listed in the table below.

[0126]

[Table 2]

For quantitative amplification of nucleic acids homologous to all or part of the human FEZ1 gene,
Also contemplated is the use of an isolated polynucleotide primer that comprises both a fluorophore and a molecule capable of quenching the fluorescence of the fluorophore. The use of such labeled primers is described elsewhere (Livak et al., 1995, "Guidelines for Designing TaqM.
an ^TM Fluorogenic Probes for 5'Nuclease Assays ", Perkin Elmer, Norwalk,
CT; U.S. Pat.No. 5,210,015; U.S. Pat.No. 5691,146; Heid et al., 1
996, Genome Res. 6: 986-994).

The isolated polynucleotide of the invention can also be used as an antisense oligonucleotide (ASO) for suppressing the expression of the human FEZ1 gene or its non-human analogues. As is well known in the art, ASOs can be complementary to either the coding or non-coding strand of the gene. ASO is used by delivering ASO inside the cell, and preferably inside the nucleus of the cell, whereby ASO
SO is capable of interacting with one or more nucleic acids that encode a protein. When the isolated polynucleotide of the present invention is used as ASO, this means that human FEZ1 is used even when ASO is used in vitro or in non-human animals.
A gene, eg at least 20, 21, 22 ... Of a single chain having the sequence of SEQ ID NO: 1.
., 30, 31, 32, ...., 40, 41, 42, ..., Or 50 consecutive nucleotide residues with high stringency. When the recipient of ASO is a human cell, either in vitro or in vivo, the isolated polynucleotide ASO of the invention is preferably substantially homologous, and more preferably the human FEZ1 gene. At least 20, 21, 22, ...., 30, 31, 32, ..., 40, 41, 42, ... of (SEQ ID NO: 1)
, Or substantially homologous to 50 consecutive nucleotide residues. Furthermore, the isolated polynucleotide ASO is preferably substantially or completely homologous to the translation start site, the transcription start site, the exon-intron boundaries for splicing immature mRNA, or the coding sequence of the human FEZ1 gene. . Other preferred ASO
Is homologous or complementary to or close to the FEZ1 ORF (SEQ ID NO: 3) or most (eg 100-500 nucleotides) thereof. ASO can be administered in either single-stranded or double-stranded form, although the single-stranded form is preferred. ASO can be administered to either animals or cells in the form of a pharmaceutical composition comprising ASO, as described herein.

The isolated polynucleotide of the present invention can be used as a template, either in vitro or in vivo, for the expression of human Fez1 protein. If in vitro expression of the human Fez1 protein is desired, an intron region of FEZ1 such as a strand of at least one cDNA produced using a spliced human mRNA encoding the Fez1 protein (eg, SEQ ID NO: 2 or SEQ ID NO: 2). It is preferred to use an isolated polynucleotide which does not comprise an isolated polynucleotide which comprises a part which is complementary to the cDNA having the nucleotide sequence of 3). Methods and compositions that can be used for in vitro expression of proteins from nucleic acids are well known in the art and are described elsewhere (eg Sambrook et al., 1989, Molecular Cloning: A Laboratoy Manual, Cold Spr.
ing Harbor Laboratory, New York; Ausbel et al., 1992, Current Protocols
in Molecular Biology, John Wiley & Sons, New York).

When the isolated polynucleotide of the present invention is used as a template in vivo for the expression of human Fez1 protein, the isolated polynucleotide has at least one nucleotide residue of at least one strand of SEQ ID NO: 1. Group 112-456, nucleotide residue 1707
-2510, and a sequence substantially homologous to nucleotide residues 4917-5550.
It is not necessary to remove the intron region of FEZ1 from the isolated polynucleotide if the cells in which the Fez1 protein is expressed are mammalian cells, and especially human cells. However, preferably, the intron region of FEZ1 is removed from the isolated polynucleotide prior to providing it to the cell.

When the isolated polynucleotide of the present invention is used as a template in vivo for the expression of human Fez1 protein, the isolated polynucleotide is provided to the cell, preferably in the form of an expression vector, wherein The region encoding the Fez1 protein is operably linked to the promoter region. The promoter region may be the human FEZ1 promoter region, or it may be virtually any other region. In various aspects, the promoter region of the expression vector is a constitutive promoter, an inducible promoter, or a tissue-specific promoter. Many constitutive promoters are known in the art and are included within the scope of this invention. Examples of constitutive promoters are, for example, retrovirus LTR promoter, cytomegalovirus immediate early promoter, SV40 early promoter, simple help swish thymidine kinase promoter, adenovirus based promoters, elongation factor 1 alpha promoter, SV40-HTLV-1 LTR. Fusion promoters, and CMV-beta actin enhancer fusion promoters.

Operable ligation of an isolated polynucleotide of the invention with an inducible promoter includes
Allows controlled expression of the Fez1 protein following delivery of the expression vector to the cells. Such regulated expression is regulated by providing a promoter of the promoter to the cell, or removing or removing such an inducer from the cell. An example of an inducible promoter that can be operably linked to the isolated polynucleotide of the invention is the tetracycline promoter, which is well known in the art as an inducible promoter.

Operable ligation of the isolated polynucleotides of the invention with a tissue-specific promoter permits localization of Fez1 protein expression to relevant tissues, thereby resulting in non-tissue specific expression of Fez1 protein. Minimize all relevant side effects. The tissue-specific promoter is selected from the group consisting of, for example, an epithelial-specific promoter, a tumor-specific promoter, a milk-specific promoter, a prostate-specific promoter, and an esophageal-specific promoter. By way of example, a prostate specific antigen promoter can be operably linked to the isolated polynucleotide of the invention to achieve Fez1 protein prostate specific expression.

The isolated polynucleotide of the present invention can be provided to cells in vitro or in vivo using various gene supply vectors. The nature of the vector is not important. Virtually any vector known in the art for delivering nucleic acids to the interior of a cell can be used for this purpose. Exemplary vectors include, but are not limited to, naked DNA vectors, plasmids, condensed nucleic acids, proprietary nucleic acid-coated microparticles or macroparticles, and viral vectors.

The invention also includes animal cells comprising a foreign DNA molecule having at least one portion that is substantially homologous to at least the coding region of the human FEZ1 gene. Outpatient
The DNA molecule comprises nucleotide residues 112-456 of at least one strand of SEQ ID NO: 1.
, Nucleotide residues 1707 to 2510 and nucleotide residues 4912 to 5550, individually or together, may comprise one, two, three or more regions of substantial homology. Preferably, the foreign DNA molecule is at least one SEQ ID NO: 2.
Comprises a region of substantial homology with the chain of. More preferably, the foreign DNA molecule is completely homologous to the coding region of the human Fez1 gene. Also preferably foreign DNA
The molecule comprises a promoter operably linked to the FEZ1 coding region, where
The Fez1 protein is expressed in cells containing foreign DNA molecules.

The cell may be a human cell, a non-human animal cell, or a non-animal cell, such as a plant cell,
It can be a yeast cell, a fungal cell, or a bacterium. The cells can also be cells in culture, cells in the body of an animal, or cells that are removed from the body of an animal for the purpose of providing foreign DNA molecules and later return the cells to the body of the same or a different animal.

The present invention also relates to an animal comprising a cell comprising a foreign DNA molecule having at least one portion that is substantially homologous to at least the coding region of the human FEZ1 gene. Preferably, the animal is a human comprising a tissue lacking a copy of the human wild-type FEZ1 gene, eg, some tumor tissue. Such an animal (eg, mouse) can be prepared by disrupting the FEZ1 gene in the animal using a known gene targeting method. As an example, exon 1 of FEZ1 can be replaced with a neomycin resistance cassette. Animal embryonic stem cells are transfected with a targeting DNA vector and cells are selected for neomycin resistance. In these cells, homologous recombination between the target-constructed DNA and one of the animal genomic copies of the FEZ1 gene occurs. In rare cases, recombination of both FEZ1 copies can occur, but most but not all selected cells are heterozygous for the recombinant FEZ1-neomycin resistance gene, And it grows as a heterozygous growth animal. These heterozygous animals are characterized by animals that have only a single functional FEZ1 gene per cell, such as atypical cells or cell differentiation, atypical cell proliferation, increased incidence of cancer or other cell proliferative disorders. , And unregulated gene expression. Furthermore, breeding heterozygous FEZ1 animals gives rise to animals that are heterozygous for the recombinant FEZ1-neomycin resistance gene (ie FEZ1 “knockout” animals). These FEZ1 “knockout” animals display the trait characteristics that result from the loss of the functional FEZ1 gene within the animal's cells. Such properties include, for example, abnormal cell or tissue differentiation, abnormal cell proliferation, increased cancer development and other cell proliferative disorders, and uncontrolled gene expression.

Isolated Fez1 Protein of the Invention The invention also relates to an isolated Fez1 protein. The deduced amino acid sequence of human Fez1 protein (SEQ ID NO: 4) is shown in Figure 5D. Preferably, the isolated human Fez1 protein is substantially purified. Isolated human Fez1 protein can be a suspension of native or denatured protein in a liquid such as water, buffer, etc., lyophilized powder, protein and one or more adjuvants or It can be in the form of an immunogenic composition comprising immunogenicity enhancers as known in the art, or a pharmaceutical composition as described elsewhere herein.

The isolated Fez1 protein of the present invention can be produced by various techniques. For example, the protein can be expressed in an in vitro expression mixture using the isolated polynucleotide of the invention. The isolated polynucleotide of the invention can also be operably linked to a constitutive promoter or other promoter, and the Fez1 protein is overexpressed in human or non-human cells and then purified from it. You can also Alternatively, the Fez1 protein can be purified using standard chromatographic techniques, eg, from a naturally occurring source of the human Fez1 protein (eg, normal human brain or testis tissue).

The present invention also includes fragments of the isolated Fez1 protein of the present invention. Such fragments can be obtained, for example, by expressing an isolated polynucleotide of the invention wherein the polynucleotide encodes only a portion of the human Fez1 protein,
Alternatively, proteolytic deproteinization of human Fez1 protein
gradation).

Although it is preferred that the isolated human Fez1 protein has an amino acid sequence that is completely homologous to SEQ ID NO: 4, the isolated Fez1 protein has one or more amino acid sequences relative to SEQ ID NO: 4. Many conservative amino acid substitutions (con
servicing amino acid substitution).

For example, certain amino acids of the human Fez1 protein can be replaced with other amino acids without appreciably affecting the biological activity of the protein. Preferably, the amino acid sequence of the isolated Fez1 protein of the present invention is substantially homologous to SEQ ID NO: 4. The hydropathic index of naturally occurring Fez1 amino acid residues was used to determine the possible alternative amino acid residues (potential subst
It can be compared with the hydropathic index of itute amino acid residues. The significance of the amino acid hydropathic index similarity between naturally occurring amino acid residues and possible alternative amino acid residues is generally understood in the art as it relates to retention of biological function of proteins. ing. Relative hydropathic characteristics of amino acids
It is recognized that ter) contributes to the secondary structure of the resulting protein, which defines its interaction with other molecules such as enzymes, substrates, receptors, DNAs, antibodies, antigens, etc. There is.

Each naturally occurring amino acid residue is as described (Kyte et al.
al. , 1982, J. Am. Mol. Biol. 157: 105), and have been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These hydropathic index values are isoleucine (
+4.5), valine (+4.2), leucine (+3.8), phenylalanine (
+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8). ), Tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), Aspartate (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5). Amino acid residues can be replaced with other amino acid residues having a similar hydropathic index without significantly affecting the biological activity of the protein. Preferably, the alternative amino acid residue has a hydropathic index that differs from that of the naturally occurring amino acid residue by less than 2.0, preferably less than 1.0, more preferably less than 0.5. For example, if the naturally occurring amino acid residue has a hydropathic index of 1.8, the alternative amino acid residue is in the range 3.8 to -0.2, preferably 2.8 to 0.8. More preferably 2
． It should have a hydropathic index ranging from 3 to 1.3.

Regions of the Fez1 protein that are predicted to interact with other molecules (eg DN
Alternative methods can be used to predict amino acid residues that can be substituted for the naturally occurring Fez1 amino acid residues in the leucine zipper region of Fez1 that are believed to interact with A). This method has been described in the art (Hoop et al., 1981, Proc. Natl. Acad.
Sci. USA 78: 3824) and the following hydrophilicity values (hy
Drophilicity values: arginine (+3.0), lysine (+3.0), aspartate (+3.0), glutamate (+3.0), serine (+0.3), asparagine (+0.2), glutamine (+0) .2), glycine (0), proline (0.0), threonine (-0.4), alanine (-0.5).
), Histidine (-0.5), cysteine (-1.0), methionine (-1.3).
), Valine (-1.5), leucine (-1.8), isoleucine (-1.8),
Tyrosine (-2.3), Phenylalanine (-2.5), Tryptophan (-3
． 4), with allocating Amino acid residues can be replaced with other amino acid residues having similar hydrophilicity values without significantly affecting the biological activity of the protein. Preferably, the alternative amino acid residue has a hydrophilicity value that differs from the hydrophilicity value of the naturally occurring amino acid residue by less than 2.0, preferably less than 1.0, more preferably less than 0.5. For example, if the naturally occurring amino acid residue has a hydrophilicity value of 1.8, the alternative amino acid residue is 3.8 to -0.2).
Should have a hydrophilicity value in the range of, preferably in the range of 2.8 to 0.8, more preferably in the range of 2.3 to 1.3.

As mentioned above, amino acid substitutions can be based on amino acid side chain substituents, eg, their relative similarity in hydrophobicity, hydrophilicity, charge, size, and the like. For example, conservative amino acid substitutions can include substitutions within the following groups: Glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine.

Modifications, which usually do not alter the primary sequence, include in vivo or in vitro chemical derivatization of the polypeptide, eg, acetylation or carboxylation. Modifications of glycosylation, eg, during synthesis and processing of the polypeptide or in further processing steps, eg, an enzyme that affects glycosylation of the polypeptide, eg, mammalian glycosylation or deglycosylation.
Modifications of glycosylation made by modifying the glycosylation pattern of the polypeptide by exposure to a sylating enzyme are also included. Also included are sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine or phosphothreonine. Polypeptides modified using conventional molecular biology techniques to improve the proteolytic resistance of the polypeptide or to optimize its solubility properties or to further render the polypeptide more suitable as a therapeutic agent Is also included. Analogs of such polypeptides include analogs containing residues other than naturally occurring amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. The isolated Fez1 protein and fragments thereof of the present invention are not limited to products of any of the specific exemplary methods described herein.

It will, of course, be appreciated that the isolated Fez1 protein and fragments thereof can incorporate the modified amino acid residue without affecting activity. For example, the termini may block blocking groups, ie, N and C termini, by "undesirable degradation.
on) and can be derivatized to include the appropriate chemical substituents to protect and / or stabilize it. "Undesired degradation" means a compound at the end of a compound that is likely to affect the function of the compound (eg, as an anti-proliferative agent) by sequential degradation of the compound at the end of the compound. Is a term meant to encompass any type of enzymatic, chemical or biological degradation of.

Blocking groups include protecting groups conventional in the field of peptide chemistry that do not adversely affect the in vivo activity of Fez1 protein or fragments thereof. For example, a suitable N-terminal blocking group can be introduced by N-terminal alkylation or acylation. Examples of suitable N-terminal blocking groups include C ₁ -C ₅ branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups and substituted forms thereof such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups and can be coupled to the N-terminus of the peptide or can be used in place of the N-terminal residue. Suitable C-terminal blocking groups with or without incorporation of a C-terminal carboxyl group include esters, ketones and amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl, and propyl, and amide-forming amino groups such as primary amines (-NH ₂₎ and mono and dialkylamino groups, such as methylamino, Ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogs, such as agmatine, are also useful C-terminal blocking groups and can be coupled to the C-terminal residue of the peptide or used instead. Furthermore, the free amino and carboxyl groups at the ends are completely removed from the Fez1 protein or fragments thereof, so that its desamino form (desamino forms) without affecting biological activity.
) And descarboxylated forms
It will be appreciated that it can occur.

Other modifications can also be incorporated without adversely affecting biological (eg, antiproliferative) activity, and these can be replaced by amino acid in the D isomeric form of amino acids in the natural L isomeric form. It includes, but is not limited to, one or more substitutions. Thus, the Fez1 protein or fragment thereof may contain one or more D-amino acid residues, or may all comprise amino acids in D-form. Retro-inverso forms of the protein peptides according to the invention, eg inverted peptides in which all amino acids are replaced by D-amino acid forms.
ptides) are also included.

Acid addition salts of the Fez1 protein of the present invention or fragments thereof are also expected to be functional equivalents. Thus, the protein or peptide according to the present invention may be an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid or the like or an organic acid such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, Malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
Treatment with methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, or salicylic acid can give water-soluble salts of peptides suitable for use as antiproliferative agents.

The isolated Fez1 protein or fragment thereof of the present invention can be used to raise polyclonal or monoclonal antibodies using known methods. As is well known, administration of the Fez1 protein of the present invention to an animal results in a soluble immune response to the protein or fragment in the animal.
mune response) can be induced. Preferably the protein or fragment is mixed with an adjuvant or other immune system enhancer. Such adjuvants include, but are not limited to, mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols and polyanions, other peptides and oil emulsions. Antibodies that specifically bind the Fez1 protein or fragment can be identified and isolated using well known methods (eg, Harlow et al., 1988. Antibo.
diees: A Laboratory Manual, Cold Spring
g Harbor, New York). Similarly, immortal hybridomas can be generated using known methods to provide a supply of such antibodies.

Diagnostic Methods of the Invention As described herein, reduced or no expression of the human FEZ1 gene has been demonstrated in numerous cancer cell lines and tumor samples. These data indicate that evaluation of the level of FEZ1 gene expression in human cells or tissues can be an indicator of the cancerous state of the cells or tissues. Diagnostic techniques based on this relationship allow for tumorigenesis before other physiological changes associated with cancer can be detected in the same cell or tissue.
has the advantage that it can be detected in cells and tissues at an early stage. In addition, these diagnostic techniques can be used to corroborate or prove a preliminary diagnosis of tumorigenesis made by visual or cytological examination of potentially cancerous tissues.

The diagnostic methods described in this section are useful for diagnosing cancer in human body tissue, especially when the body tissue is epithelial tissue. By way of example, body tissue may be gastrointestinal tissue, esophageal tissue, stomach tissue, colon tissue, prostate tissue, breast tissue.
sue), hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue (cervical)
Tissue) and ovarian tissue.

The present invention includes a method of determining the cancerous state of a sample tissue. The method comprises comparing FEZ1 expression in sample tissue to FEZ1 expression in control tissue of the same type. Reduced FEZ1 expression in sample tissue compared to FEZ1 expression in control tissue is an indication that the sample tissue is cancerous. The sample tissue is a phenotypically abnormal tissue.
tissue) (e.g., a biopsy sample obtained from a potential cancerous lesion in human tissue such as breast or prostate) or it can be phenotypically normal tissue. The control tissue is the same type of non-cancerous tissue and can be obtained from the same person from whom the sample tissue was obtained or from one or more persons different from the person from whom the sample tissue was obtained. FEZ1 expression in the sample tissue is the same as that in the same or different humans if a panel of data exists or if the data creates a representative value for expression of FEZ1 in non-cancerous tissue of the same type as the sample tissue.
Rather than make a separate determination of ez1 expression, one can compare to this representative value.

Expression of FEZ1 in sample tissues is compared to FEZ1 expression in control tissues (or data sets) by comparing the relative amounts of at least one indicator in sample tissues and control tissues (or data sets). It The indicator used may be any indicator capable of correlating with the transcription of the FEZ1 gene in tissues or the translation of this transcript in such tissues. For example, the indicator can be selected from the group consisting of FEZ1 mRNA, cDNA produced using FEZ1 mRNA, DNA produced by amplification of any of these, and Fez1 protein.

The present invention includes other methods of determining the cancerous state of a sample tissue. The method comprises comparing the nucleotide sequence of the FEZ1-associated polynucleotide obtained from the sample tissue to the nucleotide sequence of a control FEZ1-associated polynucleotide. The difference between the nucleotide sequence of the FEZ1-associated polynucleotide obtained from the sample tissue and the nucleotide sequence of the control FEZ1-associated polynucleotide is an indication that the sample tissue is cancerous. The FEZ1-associated polynucleotide is, for example, at least part of a chromosome, unspliced mR
NA, partially spliced mRNA, fully spliced m
RNA, cDNA produced using unspliced mRNA,
Selected from the group consisting of cDNA produced using partially spliced mRNA, cDNA produced using fully spliced mRNA, and DNA produced by amplification of any of these Can be one. As an example, the FEZ1-associated polynucleotide can be DNA produced by amplification of cDNA produced using fully spliced mRNA obtained from humans, in which case the control FEZ1-associated polynucleotide has the sequence It should be the DNA with number 3. As a further example, the FEZ1-associated polynucleotide is a D produced by amplification of at least a portion of human chromosome 8.
It can be NA, in which case the control FEZ1-associated polynucleotide should be DNA having the sequence of SEQ ID NO: 1.

According to this method, both sample and control tissue can be obtained from the same human, in which case the sample tissue is expected to have an abnormal phenotypic portion of human body tissue or tumorigenesis. It should be part of a tissue and the control FEZ1-associated polynucleotide should be obtained from a part of the same bodily tissue with a normal phenotype or a tissue in which tumor formation is not expected. The sample and control tissue can also be obtained from the same tissue, but from different humans, in which case the control tissue should be obtained from a human whose relevant tissue is not cancerous. Alternatively, as mentioned above, the control tissue can be a panel of data collected from tissues of the type obtained from humans whose tissues were not cancerous. In this case, only the nucleotide sequence of the sample FEZ1-associated polynucleotide needs to be determined empirically, and this sequence can be compared to the consensus or other sequences shown by the population of data. For example, the FEZ1 gene sequence described herein (SEQ ID NO: 1), the FEZ1 cDNA sequence described (SEQ ID NO: 2) or the FEZ1 O described herein.
The RE sequence (SEQ ID NO: 3) can be used as a control FEZ1-associated polynucleotide sequence.

The present invention encompasses yet another method for determining the cancerous state of a sample tissue. As described herein, certain mutations in the human FEZ1 gene are:
Transcript production results from this gene having a length different from that of the wild-type FEZ1 gene transcript. This method correlates this transcript length difference with the cancerous state in the sample tissue. This method uses FEZ1-transcript-association polynucleotides (FEZ1-transcript-associi) obtained from sample tissues.
Comparing the length of the aged polynucleotide with that of the control FEZ1-transcript-associated polynucleotide. FEZ1-transcript obtained from sample tissue-control FEZ1-transcript with length of associated polynucleotide
If shorter than the length of the associated polynucleotide, this is an indication that the sample tissue is cancerous. FEZ1-transcript-associated polynucleotides are, for example, from the group consisting of fully spliced mRNA, cDNA produced using fully spliced mRNA, and DNA produced by amplification of any of these. Can be chosen. In one aspect of this method, the FEZ1-transcript-association polynucleotide is DNA produced by amplification of cDNA produced using fully spliced mRNA obtained from humans,
And the control FEZ1-transcript-association polynucleotide is DNA having the sequence of SEQ ID NO: 2.

In another aspect of this method, the FEZ1-transcript-associated polynucleotide is a fully spliced mRNA obtained from a human patient, and a control FEZ.
1-Transcript-associated polynucleotide is at least part of a nucleic acid complementary to SEQ ID NO: 2, wherein binding of patient mRNA and control polynucleotide is detected by standard RNA blot or Northern blot analysis. You can

As in the methods described above, the sample and control FEZ1-transcript-associated polynucleotides can be obtained from the same or different humans, and control FEZ1.
The transcript-associated polynucleotide may instead be a consensus or other relevant sequence as described herein or FEZ1 obtained from a human whose tissue was not cancerous. -Transcript-Association Polynucleotide sequences can be formulated using.

The present invention includes yet another method for determining the cancerous state of a sample tissue.
The method comprises assessing FEZ1 expression in sample tissue. The substantial absence of FEZ1 expression in the sample tissue is an indication that the sample tissue is cancerous. FEZ1 expression is FEZ1 mRNA, FEZ1mRN
CDNA produced using A, D produced by amplification of either of these
It can be evaluated by evaluating the presence or the substantial absence of at least one indicator selected from the group consisting of NA and Fez1 protein.

The present invention also includes other methods for determining the cancerous state of a sample tissue. This method comprises detecting aberrant splicing of the FEZ1 transcript in sample tissue. Abnormal splicing of the FEZ1 transcript is an indicator that the sample tissue is cancerous. Aberrant splicing of FEZ1 transcripts can occur, for example, in FE with high stringency when at least one exon boundary polynucleotide probe is
It can be detected by assessing the ability to anneal to the Z1-transcript-associated polynucleotide. Such an exon boundary polynucleotide probe is
It can anneal with high stringency to the ends of two sequential FEZ1 exons when the ends are adjacent, but not when the ends are not adjacent. By way of example, such an exon-bounded polynucleotide probe has two moieties, one that binds with high stringency to the 3'-end of the first exon of DNA, mRNA or cDNA encoding the strand of FEZ1, and DNA encoding the strand of FEZ1,
It may comprise other moieties that bind with high stringency to the 5'-end of the second exon of the mRNA or cDNA. If the two parts of the probe are contiguous, then the probe is the mRNA, or cDNA generated using that mRNA, only if the two exons are contiguous in the mRNA or cDNA.
It will bind to the coding strand of A with high stringency. Thus, if the mRNA is aberrantly spliced such that the first and second exons of FEZ1 are not contiguous in the spliced mRNA (and therefore not in the corresponding cDNA), the probe is It will not bind with high stringency to the corresponding cDNA. The design of such primers would be well within the level of ordinary skill in the art in light of the present disclosure.

Immunohistological Diagnostic Methods The present invention provides immunohistological (immunohistological) for detecting the expression of Fez1 protein in cells or tissue samples obtained from human patients.
l) Method is also included. This method involves the use of an isolated Fez1 protein (or fragment thereof) of the invention in accordance with standard antibody generation methods to generate antibody preparations (an
It involves the use of tibody preparations (eg monoclonal or polyclonal antibody preparations). This preparation contains one or more types of antibodies that specifically bind the human Fez1 protein. The antibody preparation is contacted with a cell or tissue sample and the Fez1-binding antibody is labeled before or after contact with the sample. Non-specifically bound antibody is washed from the sample and the presence of labeled antibody in or on the sample is assessed. The presence of labeled antibody is an indication that the sample comprises human Fez1 protein. Thus, this immunohistological method can be used to detect Fez1 expression, or a decrease in such expression, for example, which is associated with an increased likelihood of tumorigenesis.

Therapeutic Methods of the Invention Aberrant expression of the human FEZ1 gene is not merely a symptom of epithelium and other cancers in human tissues. It is also a contributing cause of tumorigenesis in these tissues and in some cases the only cause. Inactivation of all genomic copies of the FEZ1 gene in one or more cells of human tissue, especially epithelial tissue, can result in aberrant proliferation of these cells. Normal control of cell proliferation is restored by reactivating the genomic copy of the FEZ1 gene in abnormally proliferating cells or by providing the abnormally proliferating cells with at least one exogenous source of the Fez1 protein. be able to. An exogenous source of Fez1 protein is
For example, it can be a nucleic acid encoding a Fez1 protein or a composition comprising a Fez1 protein. An exogenous source of Fez1 protein can either cause further abnormal growth of tumor cells before tumorigenesis (ie for the purpose of suppressing, delaying or preventing tumorigenesis) or at any time after the onset of tumorigenesis. Suppress or
Cells for the purpose of slowing or preventing or reversing abnormal growth).

The present invention thus includes a method of modulating the proliferation of human cells having an altered FEZ1 gene. The method comprises providing the cell with an exogenous source of Fez1 protein. When a protein is given to a cell, the abnormal growth of the cell is suppressed, delayed or prevented.

A cell given an exogenous source of the Fez1 protein may have one, two or more copies of the altered FEZ1 gene, and no normal functioning copy of this gene. There is. In most cases, this method is intended to be used in situations in which it is observed that the tissue of a human patient comprises cells that do not express the wild-type FEZ1 gene or express at abnormally low levels. Expression of FEZ1 in cells was observed in non-cancerous cells of the same type FE
About the level of expression of Z1 is about 50, 49, 48, ... 40, 39, 38, ...
································· Or less than 1% is considered to be abnormally low when observed in cells. The cells are, for example, cells that can be recognized as tumor cells, cells that are abnormally proliferating but cannot be recognized as tumor cells yet, metastatic cancer cells (metastatic cancer cells).
ls), and is predisposed to abnormal proliferation (predisposed to abno)
It can be a cell for which rmal proliferation is not yet recognizable as a tumor cell, or a cell that has an altered FEZ1 gene but does not grow abnormally when an exogenous source of the Fez1 protein is given to the cell. The cells are preferably epithelial cells, such as breast epithelial cells, prostate epithelial cells, esophageal epithelial cells, lung epithelial cells or epidermal epithelium cells.
ial cells).

The modified FEZ1 gene may be, for example, a FEZ1 gene that is not transcribed in cells, a FEZ that is transcribed to generate an incorrectly spliced transcript.
1 gene, reduces or abolishes the normal function of the Fez1 protein (a
b) a FEZ1 gene comprising at least one mutation, a FEZ1 gene transcribed but not translated, or a FEZ1 gene partially or deleted from the genome of the cell.

An exogenous source of Fez1 protein can be, for example, a composition comprising the isolated human Fez1 protein of the invention described herein. Alternatively, the Fez1 protein can be a functional fragment or analog of the Fez1 protein (ie, a fragment of Fez1, a peptidemimetic having a similar structure to all or part of the Fez1 protein,
The fragment or analogue then exhibits one or more of the physiological activities of the Fez1 protein, such as the inhibition of tubulin polymerization. Fez
The one protein is preferably a human Fez1 protein, or a human Fez1 protein having one or more conservative amino acid residue substitutions. Preferably, the amino acid sequence of the Fez1 protein is completely homologous to the amino acid sequence of the Fez1 protein normally encoded by the cellular FEZ1 gene. In one aspect, the amino acid sequence of the Fez1 protein is SEQ ID NO: 4. The isolated Fez1 protein provided to the cell can be expressed in vitro, isolated from an organism transformed with the FEZ1 gene or is naturally occurring, as described herein. It can be isolated from the source. For example, Fez1
The protein can be isolated in vitro or in vitro from the patient's cultured cells to give to other cells of the same patient. As a further example, the Fez1 protein is at least a coding portion of the human FEZ1 gene (eg, SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO: 3), preferably the human FEZ1 gene obtained from a patient to be administered the Fez1 protein. Can be isolated from cultured human or bacterial cells transformed with an expression vector comprising a polynucleotide encoding at least the coding portion of

As mentioned herein, the Fez1 protein can be administered to humans in a number of pharmaceutical compositions. Preferably, the composition is such that the interior of cells (eg liposomes, membrane vesicles, microspheres having an aqueous core).
es), protein-coated protruding particles, etc.) are compositions known in the art for providing proteins.

The exogenous source of Fez1 protein can also be, for example, an expression vector comprising a polynucleotide having at least one coding region encoding a functional Fez1 protein. The Fez1 protein is provided to the cell when the polynucleotide is expressed in the cell. Preferably, the polynucleotide encodes a human Fez1 protein, or a human Fez1 protein having one or more conservative amino acid residue substitutions. Preferably, Fez1
The amino acid sequence of the protein is F, which is normally encoded by the cellular FEZ1 gene.
It is completely homologous to the amino acid sequence of ez1 protein. In one aspect, Fe
The amino acid sequence of the z1 protein is SEQ ID NO: 4. In another aspect, the polynucleotide comprises a portion having the nucleotide sequence of SEQ ID NO: 2. Also preferably, the polynucleotide is substantially homologous, preferably completely homologous, to the wild type genomic sequence of the patient's FEZ1 gene for which the patient's cells are provided with the polynucleotide. For example, the polynucleotide can comprise a portion that is substantially or completely homologous to SEQ ID NO: 1. The polynucleotide can, of course, be the isolated polynucleotide of the invention described elsewhere herein, so long as the isolated polynucleotide encodes a functional Fez1 protein.

Nucleic acid-containing vectors, including expression vectors, are preferentially or exclusive to such types of cells, such as cells of a certain type, or predominantly located within a tissue or only within a tissue. Is known in the art as a method of targeting such a vector to specifically provide the nucleic acid of the vector.
Exemplary expression vectors include non-viral vectors (eg, plasmids, naked DNA).
, DNA complexed with polycations such as polylysine, etc.)
And viral vectors such as retroviruses, adenoviruses and adeno-associated virus vectors. The use of all such vectors is intended and the choice of the appropriate vector will depend on the disclosure provided herein, the size, composition and characteristics of the nucleic acid, the condition and condition of the patient to whom the nucleic acid is to be given and the characteristics of the vector. Within the level of ordinary skill in the art.

As described elsewhere herein, the polynucleotide can be an expression vector in which the portion of the polynucleotide encoding the Fez1 protein is operably linked to a promoter. . Promoters include constitutive promoters, inducible promoters, tissue-specific promoters, or virtually any other promoter. However, mammals,
A human promoter is particularly preferable. In one aspect, the promoter of the expression vector is the normal human FEZ1 promoter region. In other embodiments, the promoter is an inducible promoter, and the method of treatment further comprises administering an inducer of the promoter to cells that are provided with the polynucleotide. In another aspect, the promoter is a tissue-specific promoter, which normally promotes expression of a gene operably linked thereto in epithelial tissue. In another embodiment, the expressible portion of the FEZ1 gene is contained within an expression vector and is operably linked to genetic elements that can be used to stop FEZ1 expression. Many genetic elements of this type are known, including, for example, genetic elements associated with the Cre-LoxP system (Pluck, Intl. J. Exp. Pathol.
． 77: 269-278; Li et al. , 1997, Human Gen
e Ther. 8: 1695-1700: Lewandski et al.
, 1997. Nature Genet. 17: 223-225; Russ e.
t al. , 1996. J. Virol. 70: 4927-4932; Saka.
i et al. , 1995, Biochem. Biophys. Res. Co
mm. 217: 393-401; de Wit et al. , 1998, Nu
cl. Acids Res. 26: 676-678).

The present invention also includes methods of preventing tumor formation in human cells. This method
Providing the cell with an expression vector comprising a polynucleotide having at least one coding region encoding a functional Fez1 protein.
Thereby the Fez1 protein is expressed in the cell and thereby tumorigenesis is prevented in the cell. The cell is, for example, a cell in which the altered FEZ1 gene is detected, a tissue cell in which the altered FEZ1 gene is detected, or FEZ1.
It can be a normal cell in a predisposed individual (human with a family history of FEZ1 gene modification), or a normal cell of a normal individual. Preferably the cells are epithelial cells. The polynucleotide may be any of the polynucleotides described herein for modulating the growth of human cells having an altered FEZ1 gene.

The present invention also relates to a method of inducing cell proliferation in reverse. This method uses FEZ1
It comprises providing an inhibitor of gene expression inside the cell. Cell proliferation is induced when the inhibitor is inside the cell, but not when the inhibitor is not inside the cell. This method is useful for promoting desired cell growth in vitro or in vivo. An example of a situation where it is advantageous to induce cell proliferation is when tissue is transplanted from one location in one animal to another in the same or a different animal (eg, skin allograft).
graft) or bone marrow transplantation), when a mixture of desirable and unwanted cells is treated to remove or kill unwanted cells (eg, radiotherapy or chemotherapy of partially cancerous tissue), or wounds. Healin of received tissue
g) includes, but is not limited to, when desired (eg, fusion of skin perforations or incisions).

The inhibitor used in this method can be an ASO, such as one of the isolated polynucleotides of the invention, or it can be expressed using one of the screening methods of the invention in FEZ1 gene expression. Can be a compound identified as an inhibitor of If the inhibitor can diffuse across the cell membrane, then it is not necessary to use the vector to deliver the inhibitor inside the cell; otherwise, the inhibitor should be delivered inside the cell. Use of vector for. Any vector known in the art, such as any of the vectors described herein, can be used for this purpose.

The use of ASO reversely suppresses FEZ1 gene expression (reversible
preferred for inhibiting). Useful ASO compositions are described elsewhere herein. According to this method, ASO is, for example, a naked nucleic acid, a polycation or other nucleic acid complexed with a condensing agent,
It can be administered to cells in the form of a nucleic acid vector such as a plasmid or viral vector. ASO can be provided directly inside the cell, or an expression vector encoding ASO can be provided inside the cell. When such an expression vector is used, it is preferable that the expression of ASO can be regulated.
By way of example, the polynucleotide encoding ASO can be operably linked to an inducible promoter such that ASO is produced only when an inducer of the promoter is given to the cell. Alternatively, the expression vector may not be able to replicate. Such a replication defect vector (replicat)
Examples of ion-deficient vectors include, but are not limited to, plasmids lacking an origin of replication and replication defective viral vectors (eg, replication defective adenovirus vectors). The mechanism by which ASO expression is regulated is not critical, instead it is important that ASO expression be stopped or severely restricted when desired.

When the isolated polynucleotide of the invention or the isolated FEZ1 protein of the invention is administered to an animal such as a human for diagnostic, therapeutic or other purposes, the polynucleotide or protein has a pharmaceutical composition. It is preferably in the form of a product.

The present invention includes methods of inhibiting human tumorigenesis. Inducer of FEZ1 gene expression, enhancer of FEZ1 gene expression, inhibitor of Fez1 phosphorylation, enhancer of phosphorylated Fez1 dephosphorylation, agent inhibiting the binding of EF1-γ and Fez1 and Fez1 and tubulin Comprising administering to a human a compound selected from the group consisting of agents that inhibit binding.

Pharmaceutical Compositions of the Invention The present invention is directed to a Fez-1 protein or another one described herein as an active ingredient.
And the preparation and use of pharmaceutical and pharmaceutical compositions comprising any of the compounds of the class. The isolated polynucleotides of the invention described herein can be provided in the form of nucleic acid vectors, including but not limited to expression vectors.

A pharmaceutical composition of the invention may consist of only one or more active ingredients in a form suitable for administration to a subject, or the pharmaceutical composition may comprise one or more active ingredients and one or more active ingredients. It may comprise a pharmaceutically acceptable carrier, one or more additional ingredients or any combination thereof. Administration of one of these pharmaceutical compositions to a subject is useful in practicing any of the methods of the invention, as described elsewhere herein. The active ingredient is combined with a physiologically acceptable cation or anion, as is well known in the art,
It may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt.

The preparations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed in the future. In general, these methods of preparation involve mixing the active ingredient with one carrier or one or more other accessory ingredients and then, if necessary or desired, bringing the product into the desired single or Multiple-comprising the steps of molding or packaging into dosage units.

Although the description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for ethical administration to humans, these compositions are generally suitable for administration to animals of all species. It will be understood by those skilled in the art. Modifications of pharmaceutical compositions suitable for administration to humans, in order to render the composition suitable for administration to a variety of animals, are well understood, and animal pharmacologists of ordinary skill will understand, if any. Again, these modifications can be planned and implemented by mere routine experimentation. Subjects envisioned for administration of the pharmaceutical compositions of this invention include, but are not limited to, humans and other primates, cattle, pigs, horses, sheep,
Of cats and mammals including market related mammals such as dogs, birds including market related birds such as chickens, ducks, geese and turkeys, fish including farmed and aquarium fish, cultured shellfish Including such crustaceans.

The pharmaceutical compositions useful in the methods of the invention are oral, rectal, vaginal, parenteral, topical,
It can be prepared, packaged, or sold as a preparation suitable for intrapulmonary, intranasal, cheek, eye, or other routes of administration. Other envisioned preparations include projected nanoparticles containing the active ingredient, liposomal preparations, resealed red blood cells and immunologically based preparations.

The pharmaceutical compositions of the present invention may further comprise, in addition to the active ingredient, one or more additional pharmaceutically active substances.

Controlled-release or sustained-release preparations of the pharmaceutical compositions of this invention may be prepared using conventional techniques.

Preparations of the pharmaceutical composition of the invention suitable for oral administration include, but are not limited to, tablets, hard or soft capsules, cachets, each containing a predetermined amount of active ingredient. , Troches or lozenges may be prepared, packaged or sold in the form of discrete solid dosage units. Other preparations suitable for oral administration include, but are not limited to, powdered or granular preparations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions.

An “oleaginous” liquid, as used herein, is one that comprises carbon-containing liquid molecules and exhibits less polar character than water.

A tablet comprising the active ingredient can be prepared, for example, by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets suitably contain the active ingredients in a free-flowing form, such as a powder or granule preparation, optionally mixed with one or more binders, lubricants, excipients, surfactants and dispersants. It can be prepared by compressing in any device. Molded tablets may be made by molding in a suitable machine, a mixture of the active ingredient, a pharmaceutically acceptable carrier and a liquid sufficient to at least moisten the mixture. Pharmaceutically acceptable excipients used in the preparation of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to,
Includes gelatin, acacia, pregelatinized corn starch, polyvinylpyrrolidone and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid,
Includes silica and talc.

The tablets may be uncoated or they may be coated using known methods to achieve delayed degradation in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. . For example, materials such as glyceryl monostearate or glyceryl distearate can be used to coat tablets. Further, for example, the methods described in US Pat. Nos. 4,256,108, 4,160,452 and 4,265,874 for forming tablets into osmotically controlled release tablets. Can be used to coat. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative or some combination thereof in order to provide a pharmaceutically fine and palatable preparation.

Hard capsules comprising the active ingredient may be prepared using a physiologically degradable composition such as gelatin. These hard capsules comprise the active ingredient and may further comprise additional ingredients including inert solid diluents such as calcium carbonate, calcium phosphate or kaolin.

Soft gelatin capsules comprising the active ingredient may be prepared using a physiologically degradable composition such as gelatin. These soft capsules comprise the active ingredient which can be mixed with water or an oil solvent such as peanut oil, liquid paraffin or olive oil.

Liquid preparations of the pharmaceutical composition of the invention suitable for oral administration may be in liquid form or in the form of dry products intended for reconstitution with water or another suitable carrier before use. Either can be prepared, packaged, and sold.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily carrier. Aqueous carriers include, for example, water and isotonic saline. Oily carriers include, for example, almond oil, oily esters, ethyl alcohol, peanuts, vegetable oils such as olive, sesame or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin. Liquid suspensions further include one or more including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulsifying agents, preservatives, buffers, salts, flavors, coloring agents and sweetening agents. It may comprise additional ingredients. The oily suspension may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, tragacanth gum, acacia gum and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. . Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides such as lecithin, fatty acids, long chain fatty alcohols, partial esters derived from fatty acids and hexitols, or fatty acids and hexitols. Partial products derived from anhydrides (for example, polyoxyethylene stearate, heptadecaethyleneoxycetanol, sorbitol polyoxyethylene monooleate and sorbitan polyoxyethylene monooleate), and condensation products of alkylene oxides. Including. Known emulsifiers include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl or n-propyl-para-hydroxybenzoic acid esters, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in a manner substantially similar to liquid suspensions, with the major difference that the active ingredient is not suspended in the solvent. , To be dissolved. A liquid solution of a pharmaceutical composition of the invention may comprise each component described for liquid suspensions, which suspension does not necessarily assist in the dissolution of the active ingredient in the solvent. It is understood that it will be. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, peanuts, vegetable oils such as olive, sesame or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin.

The powdered and granulated preparations of the pharmaceutical preparations according to the invention can be prepared using known methods. These preparations may be administered directly to a subject and used to prepare aqueous or oily suspensions or solutions, for example by forming tablets, filling capsules, or adding aqueous or oily carriers thereto. it can. Each of these preparations may further comprise one or more dispersing or wetting agents, suspending agents and preservatives. Additional excipients such as fillers and sweeteners, flavoring agents or colorants can also be included in these preparations.

The pharmaceutical compositions of this invention may also be prepared, packaged, or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oily phase can be a vegetable oil such as olive or peanut oil, a mineral oil such as liquid paraffin or a combination thereof. These compositions further comprise naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, a combination of fatty acids and hexitol anhydrides such as sorbitan monooleate. It may comprise one or more emulsifiers such as esters or partial esters derived from ## STR00003 ## and condensation products of these partial esters with ethylene oxide, such as sorbitan monooleate polyoxyethylene. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

The pharmaceutical compositions of this invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. These compositions can be in the form of, for example, suppositories, retention enemas, and rectal or colonic lavage solutions.

Suppository preparations are non-irritating pharmaceutical preparations which are active ingredients, solid at normal room temperature (ie about 20 ° C.) and liquid at the rectal temperature of the subject (ie about 37 ° C. in healthy humans). It can be prepared by combining with a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycol and various glycerides. Suppository preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or rectal or colonic lavage solutions can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations can be administered or packaged using a delivery device adapted for the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

The pharmaceutical compositions of this invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. These compositions may be in the form of, for example, suppositories, impregnated or coated vaginal inserts such as tampons, press preparations or vaginal irrigation solutions.

Methods for impregnating or coating substances with chemical compositions are known in the art,
Methods of attaching or binding a chemical composition to a surface, including but not limited to, incorporating the chemical composition into the structure of the material during the synthesis of the material (ie, as in a physiologically degradable material) And a method of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Pressurized preparations or vaginal irrigation solutions can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, pressure preparations can be administered using, and packaged within, a delivery device adapted to the subject's vaginal anatomy. Pressurized preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungals and preservatives.

“Parenteral administration” of a pharmaceutical composition, as used herein, refers to the physical breaching of tissue of a subject and the administration of the pharmaceutical composition through tissue crevices. Includes all routes of characterizing administration. Thus parenteral administration includes, but is not limited to, pharmaceutical compositions by injection of the composition, application of the composition by surgical incision, application of the composition through a non-surgical laceration through tissue, and the like. Including administration of the product. Above all, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular or intrasternal injection and intravenous, intraarterial or renal dialysis infusion.

Preparations of pharmaceutical compositions suitable for parenteral administration comprise the active ingredient in association with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. These preparations can be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable preparations may be prepared, packaged, or sold in unit dose form such as an ampoule or in multi-dose containers containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes and implantable sustained or biodegradable preparations. These preparations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents. In one embodiment of preparations for parenteral administration, the active ingredient is reconstituted with a suitable carrier (eg sterile, pyrogen-free water) prior to parenteral administration of the reconstituted composition. Are provided in a dry (ie powder or granule) form.

The pharmaceutical compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oleaginous suspensions or solutions. This suspension or solution may be prepared according to the known art and comprises, in addition to the active ingredient, additional ingredients such as dispersants, wetting agents or suspending agents as described herein. be able to. These sterile injectable preparations can be prepared, for example, with water or a nontoxic parenterally acceptable diluent or solvent such as 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution and fixed oils such as synthetic mono- or di-glycerides. Other useful parenterally administrable preparations include microcrystalline forms, liposomal preparations, or those which comprise the active ingredient as a component of a bioerodable polymer system. Sustained-release or implantable compositions may comprise pharmaceutically acceptable polymers such as emulsions, ion exchange resins, sparingly soluble polymers or sparingly soluble salts, or hydrophobic substances. .

Formulations suitable for topical administration include, but are not limited to, oil-in-water or water-in-oil emulsions such as ointments, lotions, creams, ointments or pastes and liquids such as solutions or suspensions. Includes semi-liquid preparations. While the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent, topically administrable preparations contain, for example, from about 1 to about 10% (w / w) active ingredient. May consist of Preparations for topical administration may further comprise one or more of the additional ingredients described herein.

The pharmaceutical composition of the invention is prepared as a formulation suitable for pulmonary administration via the oral cavity,
It can be packaged or sold. These preparations may comprise the active ingredient and comprise dry particles having a particle size in the range of about 0.5 to about 7 nanometers, and preferably about 1 to about 6 nanometers. . These compositions are conveniently
Use of a device comprising a reservoir of dry powder to which a propellant stream can be directed to disperse the powder, or the active ingredient dissolved or suspended in a low boiling propellant in a closed container. In the form of a dry powder for administration using a self-propelling solvent / powder-dispersion container, such as a device comprising Preferably these powders comprise at least 98% by weight of the particles having a diameter greater than 0.5 nanometers and at least 95% of the particles comprise particles having a diameter less than 7 nanometers.
More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% of the particles have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dosage form.

Low boiling propellants generally include liquid propellants having a boiling point at atmospheric pressure of less than 65 ° F (about 18.3 ° C). The propellant is generally 50-99.9% (w / w) of the composition.
The active ingredient may constitute 0.1-20% (w / w) of the composition. The propellant may also be an additional component such as a liquid nonionic or solid anionic surfactant or a solid diluent, preferably having the same degree of fineness as the particles comprising the active ingredient. May comprise.

A pharmaceutical composition of the invention prepared for delivery to the lung may also provide the active ingredient in the form of drops of a solution or suspension. These preparations may be prepared, packaged or sold, optionally sterile, as an aqueous or dilute alcoholic solution or suspension comprising the active ingredient, and are conveniently provided by any spray or atomizer device. Can be used and administered. These preparations further include one or more additional agents including, but not limited to, perfumes such as sodium saccharin, volatile oils, buffers, surfactants or preservatives such as methyl hydroxybenzoate. It may comprise an active ingredient. The droplets provided by this route of administration preferably have an average particle size in the range of about 0.1 to about 200 nanometers.

The formulations described herein as useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of this invention.

Another formulation suitable for intranasal administration comprises the active ingredient and comprises about 0.2-5
It is a coarse powder containing average particles of 00 micrometer. These preparations are administered by nasal inhalation, that is, by immediate inhalation through the nasal cavity from a container of powder held near the nostril.

Formulations suitable for intranasal administration may comprise, for example, small amounts, such as about 0.1% (w / w), and large amounts up to 100% (w / w) of the active ingredient. There is
And, in addition, it may comprise one or more additional ingredients described herein.

The pharmaceutical compositions of this invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. These preparations may, for example, be in the form of tablets or lozenges prepared using conventional methods, eg 0.1-20% (w / w
) Active ingredient, the balance comprising orally soluble or degradable compositions and optionally one or more of the additional ingredients described herein. Alternatively, a formulation suitable for buccal administration may comprise a powder or aerosolized or nebulized solution or suspension comprising the active ingredient. These powdered, aerosolized or nebulized preparations, when dispersed, preferably have a concentration of about 0.
． It has an average fineness or droplet size in the range of 1 to 200 nanometers and may further comprise one or more additional components described herein.

The pharmaceutical compositions of this invention may be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. These preparations can be in the form of eye drops, for example, containing a 0.1% to 1.0% (w / w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. These eye drops may further comprise buffering agents, salts or one or more of the additional ingredients described herein. Other useful ophthalmic preparations include those comprising the active ingredient in microcrystalline form or in liposomal preparations.

“Additional ingredients” as used herein include, but are not limited to,
Such as: excipients, surfactants, dispersants, inert diluents, granulating and degrading agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, preservatives, gelatin ,
Physiologically degradable compositions, aqueous carriers and solvents, oily carriers and solvents, suspending agents,
Dispersing or wetting agents, emulsifiers, demulsifiers, buffers, salts, thickeners, fillers, emulsifiers,
It includes one or more of antioxidants, antibiotics, antifungal agents, stabilizers, and pharmaceutically acceptable polymers or hydrophobic substances. Other “additional ingredients” that may be included in the pharmaceutical compositions of the present invention are known in the art, eg, Genaro, ed., 1985, incorporated herein by reference. , Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, PA.

The pharmaceutical compositions of this invention may be prepared, packaged, or sold in bulk as a single unit dose or in multiple unit doses. A "unit dose" as used herein is a discrete amount of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of active ingredient will generally be equal to the dose of the active ingredient which will be administered to a subject or a convenient fraction of the dose, eg 1/2 or 1/3 of that dose.

The relative amounts of active ingredient, pharmaceutically acceptable carrier and any additional ingredients in the pharmaceutical compositions of the present invention will depend on the identity, size and condition of the subject being treated. And further, it will depend on the route of administration of the composition. For example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.
The unit dose of the pharmaceutical composition of the present invention will depend on the kind of active ingredients contained therein. Generally, the pharmaceutical composition comprising the expression vector must be administered to the cell or cells being treated in an amount sufficient to provide at least one expression vector. The exact dose of vector will be determined by those of skill in the art in light of the efficiency with which the vector enters and transforms the target cells, the number of those cells to be treated, the physical accessibility of the cells to the vector, and the specification. It is understood that it will depend on other factors that will be understood. The pharmaceutical composition comprising the expression vector is preferably administered in an amount sufficient to provide individual cells with a 2-fold, 5-fold, 10-fold or 50-fold excess or more than the minimum recommended amount of vector. To be done. The pharmaceutical composition comprising ASO must be administered in an amount sufficient to provide at least an amount of ASO molecules equal to at least the expected or determined number of genomic copies of the ASO target or transcript thereof. I won't. The pharmaceutical composition comprising ASO is preferably 2 times, 10 times, 100 times or 100 times the minimum recommended amount of ASO for target cells.
An amount sufficient to provide a 0-fold excess or more is administered.

It is understood that a physician or veterinarian having ordinary skill will readily determine and prescribe the effective amount of the active ingredient (s) for carrying out the methods of the invention in a subject. At that time, the physician or veterinarian can, for example, prescribe a relatively low dose initially and then increase the dose until the appropriate response is obtained. But,
Furthermore, the specific dose level for any particular subject will include the activity of the particular compound used, the subject's age, weight, general health, sex and diet, time of administration,
It is understood that it will depend on a variety of factors including the route of administration, excretion rate, any drug combination and the severity of the condition being treated.

Another aspect of the invention relates to a kit comprising the pharmaceutical composition of the invention and the instructional material. The instructional material of the kit of the invention can, for example, be affixed to a container containing the pharmaceutical composition of the invention or shipped with the container containing the pharmaceutical composition. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the pharmaceutical composition be used together by the recipient.

The present invention also includes a kit comprising the pharmaceutical composition of the present invention and a delivery device for delivering the composition to a subject. For example, the delivery device can be a squeezable spray bottle, a metering spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, an automatic spray solvent / powder-dispensing device, a syringe, a needle, a tampon, or a dose measuring container. is there. The kit may further comprise instructional material as described herein.

Screening Methods of the Invention Identification of the human FEZ1 gene as a tumor suppressor gene as described herein provides a means for identifying compounds that induce cell proliferation. In addition, it can be demonstrated that some modified FEZ1 genes associated with human cancers are capable of normal or near normal expression in the presence of certain compounds, thus reducing tumor cell and tissue levels. Methods are provided for identifying compounds that are capable of suppressing aberrant cell growth in cells carrying such modified FEZ1 genes.

The invention thus includes a method of determining whether a test compound is an inducer of cell proliferation. This method comprises incubating cells comprising a functional FEZ1 gene in the presence of a test compound and assessing expression of FEZ1 in the cells. If the expression of FEZ1 in the cell is reduced compared to the expression of FEZ1 in the same type of cells incubated in the absence of the test compound,
The test compound is an inducer of cell proliferation. Particularly contemplated test compounds include the isolated polynucleotides of the invention described herein. Therefore, this method is a useful method for identifying ASOs that suppress the expression of FEZ 1 and thus induce cell proliferation.

[0223] The cells used in this method has substantially the possibility of any cells expressing FEZ1 genes such as those carried out both transcription and translation of those or FEZ1 transferring the FEZ1 gene. Preferably the cell is a human cell, more preferably it is an epithelial cell. When it is desired to identify a compound that induces the proliferation of certain cells, the cells used in this screening method are preferably those cells.

Expression of FEZ1 in cells can be assessed by any known method for assessing gene expression. For example, the amount of FEZ1 transcript accumulation or steady state in cells of the screening method or the production rate of these transcripts can be assessed using known methods. Alternatively, the amount of Fez1 protein accumulation or steady state or the rate of Fez1 protein production can be assessed using similarly known methods, including immunological methods involving the antibodies of the invention. .

The test compound can be administered to the cells in virtually any manner. Preferably, the cells are incubated in medium containing the test compound. If the test compound does not readily pass from the medium to the inside of the cell (eg, the test compound is a protein or a large nucleic acid in a form that normally does not cross cell membranes), a vector may be used to deliver the test compound to the inside of the cell. Can be used. However, because the screening method is intended to identify compounds that can be administered to cells in the most convenient and physiologically acceptable form, the test compound must be delivered to the interior of the cell in a vector. It is preferable not to request. Of course, it may be advantageous to evaluate the efficacy of vector-derived test compounds if it is not possible to identify valid test compounds that do not require the vector to gain cell entry.

Expression of FEZ1 need not be assessed in the same cell type each time a test compound is assessed. Instead, a series of data (a body of da) related to the level of FEZ1 expression in such cells under the conditions used to evaluate the test compound.
ta) can be developed.

[0227]   The invention also includes modified test compoundsFEZ1Slows the growth of cells that carry the gene
It relates to a method of determining whether or not to be effective. The method is performed in the presence of a test compound.
ModifiedFEZ1Incubate cells with the geneFEZ 1 Comprising evaluating the expression of In the cellFEZ1Expression of test compound
Of the same type (ie, also with the same modification, incubated in the absence of FEZ1 In the cell)FEZ1Is increased compared to the expression of
, The test compound is effective in slowing cell growth. This is also a test compound
In the tissue comprising the type of cells used in this screening assay
It is shown to be a useful cancer therapeutic compound for treating cancer. This screenini
The cells used in the screening method are modifiedFEZ1To have a gene
The screening method is substantially the same as the screening method described in the previous section except for the above.

The presence of the leucine-zipper-like portion of the deduced amino acid sequence of the Fez1 protein, as described herein, suggests that the Fez1 protein is a nucleic acid binding protein. This information is obtained by contacting the Fez1 protein with a test nucleic acid sequence and evaluating whether the protein and nucleic acid form a complex.
It is shown that at least one nucleic acid sequence to which the ez1 protein binds can be identified. Any known method of detecting these complexes including, but not limited to, nucleic acid footprinting, modified gel electrophoresis migration, modified chromatography migration, immunological methods involving the antibodies of the invention. Can be used. Once these sequences have been identified, the nucleic acids comprising those sequences can be used as an inducer of cell proliferation by delivering such nucleic acids to cells comprising the functional Fez1 protein. Nucleic acid is Fez1 in cells
It binds proteins and prevents Fez1 from binding its normal physiological binding partner, thereby inducing cell proliferation. In such a method, the nucleic acid is preferably in a significantly excess amount (eg 10-fold, 1-fold) of the intracellular concentration of Fez1 protein.
00 times or 1000 times or more) is used.

[0229]   The screening method of the present invention modifies a test compound.FEZ1Gene expression index
Anti-Cancer for Administration to Humans with Cancer by Identifying It
It can be used to identify therapeutic compounds. HumanFEZ1Gene is like
It can be modified in a number of ways in different cancers and different individuals, so
Performing the screening method of the present invention using cells obtained from the patient to be placed
Is convenient. Used in these assays to facilitate these treatments
Conveniently, the plurality of candidate anti-cancer therapeutic compounds and FEZ1 Can be packaged in the form of a kit containing the expression evaluation reagents of
It In one embodiment, the reagent comprises at least one strand of SEQ ID NO: 1.
An isolated polynucleotide that both anneals with high stringency to 20 consecutive nucleotide residues
Human like cleotideFEZ1An isolated poly that anneals with high stringency to a gene
It is a nucleotide. In another embodiment, the reagents are as described herein.
The antibody of the present invention.

The present invention includes screening methods to determine whether test compounds are useful in ameliorating disorders associated with aberrant tubulin polymerization. The method comprises: (i) tubulin polymerization in a first assay mixture comprising tubulin, Fez1 and a test compound; and (ii) a second tubulin and Fez1 but not test compound.
Of tubulin polymerization in the assay mixture of. A difference (eg, a difference between the tubulin polymerization rates in the first and second assay mixtures, or a difference in the degree of tubulin polymerization in the first and second assay mixtures) causes the test compound to ameliorate the disorder. It is an indicator that it is useful for. Preferably the first and second assay mixtures are substantially identical except for the presence or absence of test compound.

Disorders for which test compounds can be tested include both tubulin overpolymerization disorders and tubulin hypopolymerization disorders. For example, the disorder may be a disorder associated with aberrant initiation of mitosis, a disorder associated with aberrant modulation of mitotic rate and stage, a disorder associated with aberrant modulation of cell proliferation initiation and rate, cell growth. Disorders associated with aberrant modulation of onset and velocity of cells, disorders associated with aberrant modulation of cell shape, disorders associated with aberrant modulation of cell hardness, disorders associated with aberrant modulation of cell motility, cellular DNA Disorders associated with aberrant modulation of replication rate, disorders associated with aberrant modulation of cellular DNA replication stages, disorders associated with aberrant intracellular distribution of organelles, and aberrant modulation of cell metastatic capacity Disorders and transformation of cells from non-cancerous to cancerous phenotype There is a possibility to abnormal modulation is selected from the group consisting of related disorders. Specific examples of these disorders include tumorigenesis, tumor survival, tumor growth and tumor metastasis.

The test compound used in the screening method can be virtually any compound. Compounds that are expected to be particularly useful for ameliorating these disorders include fragments of Fez1, fragments of Fez1 fragments, fragments of tubulin, peptidomimetics of tubulin fragments, EF1-γ Fragments and peptidomimetics of fragments of EF1-γ are included.

The present invention includes another screening method for determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1.
The method comprises: (i) phosphorylation of Fez1 in a first assay mixture comprising Fez1, at least one kinase, a phosphate source and a test compound; and (ii) Fez1, a kinase and a phosphate source. And phosphorylation of Fez1 in a second assay mixture, but without the test compound. Differences in the phosphorylation of Fez1 in the first and second assay mixtures (eg, differences in the rate and extent of phosphorylation in the first and second assay mixtures) are useful for the test compound to ameliorate the disorder. Is an index of being. This screening method can be used to assess the utility of compounds for ameliorating similar disorders as described above.

The present invention includes yet another screening method for determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1. The method comprises (i) phosphorylation of Fez1 in a first assay mixture comprising phosphorylated Fez1, at least one phosphatase and a test compound, and (ii) phosphorylated Fez1 and phosphatase, Phosphorylation of Fez1 in the second assay mixture without test compound. The difference between the phosphorylation of Fez1 in the first and second assay mixtures (eg, the difference in the rate or extent of dephosphorylation of phosphorylated Fez1) is useful for the test compound to ameliorate the disorder. Is an index of. This screening method can be used to assess the utility of compounds for ameliorating similar disorders as described above.

The invention also provides that the test compound is a Fez with a protein to which Fez1 normally binds.
1. A method of determining whether it is useful for ameliorating a disorder associated with aberrant binding of 1, comprising: (i) a first assay mixture comprising Fez1, a protein and a test compound. Comparing the binding between Fez1 and the protein in (ii) the binding between Fez1 and the protein in a second assay mixture comprising Fez1 and the protein, but without the test compound. Comprising. The difference between the binding (eg, its rate or extent) of Fez1 to the protein in the first and second assay mixtures is an indication that the test compound is useful for ameliorating the disorder. The protein can be selected, for example, from the group consisting of tubulin and EF1-γ. This screening method is useful for assessing the usefulness of a test compound for ameliorating a disorder such as tumorigenesis, tumor survival, tumor growth and tumor metastasis.

The present invention includes a method of determining whether a test compound is an inhibitor of cell proliferation. The method comprises incubating a cell comprising a functional FEZ1 gene in the presence of a test compound and assessing expression of FEZ1 in the cell. If the expression of FEZ1 in the cells is increased compared to the expression of FEZ1 in the same type of cells incubated in the absence of the test compound, this is an indication that the test compound is an inhibitor of cell proliferation. .

Cell Proliferation Method of the Present Invention As described herein, the human FEZ1 gene was discovered to be a tumor suppressor gene. Thus, inactivation of this gene or suppression of expression of this gene results in an increased rate of cancer-associated cell proliferation. However, in some situations increased cell proliferation is desirable. For example, some in vitro cell culture methods are limited by the rate of cell proliferation and the effect of cell density on this rate. Further, in certain medical procedures, such as bone marrow transplants and skin allografts, it is desirable that cells proliferate at a faster than normal rate for a period of time and then only at normal rates. These methods will be improved if cell proliferation is promoted, especially in a reversible manner.

Providing cells with an inhibitor of FEZ1 expression increases the rate of proliferation of the cells and
This method can be used to improve on various known methods where the rate of cell proliferation was a limiting factor. In vitro of cell proliferation by, for example, including an inhibitor of FEZ1 expression in the cell culture medium, or by treating cells growing on or in these media (ie human epithelial cells) with these inhibitors. The rate of can be increased and can allow for faster and denser cell growth than would otherwise be possible.

Similarly, human cells, especially epithelial cells, can be treated in vivo with an inhibitor of FEZ1 expression to increase the proliferation rate of these cells. This method can be used, for example, to improve bone marrow remodeling in individuals exposed to radiation levels or cytotoxic chemicals sufficient to promote transplant integration into the transplant site or cause bone marrow loss. it can. Topical administration of the inhibitor to one or more tissues or one or more regions where increased cell proliferation is desired minimizes unwanted cell proliferation in other tissues and other body parts. Withdrawal of the inhibitor eventually leads to normal cell proliferation of the treated cells due to degradation of the inhibitor.

Similarly, cells obtained from humans were ex vivo with an inhibitor of FEZ1 expression to enhance their proliferation rate prior to transplantation of these cells into the same human from whom they were obtained or into a different human. Can be treated. The same or different inhibitors of FEZ1 expression can be administered locally or systemically to human cell recipients, or alternatively, ex vivo, in order to maintain an increased proliferation rate of the treated cells. Cells can be allowed to maintain their increased growth rate for as long as the inhibitors delivered to them withstand. In both cases, the ex vivo treated cells are degraded by one or more inhibitors,
Regains normal growth rate after not being replaced.

In the method of promoting cell growth described herein, an inhibitor of FEZ1 expression is optionally a molecule capable of replicating in human cells, eg, a viral vector encoding such an inhibitor. Could be
Inhibitors are preferably not able to be replicated in human cells if it is considered desirable to be able to induce reversibly stimulated cell proliferation. Further, in some embodiments, the inhibitor is added to cells in the form of a vector comprising a polynucleotide encoding the inhibitor such that production of the inhibitor is initiated and terminated by administration and withdrawal of the promoter inducer, respectively. It is preferred that the polynucleotide be provided and operably linked to an inducible promoter.

Kits of the Invention The present invention provides any two or more of the isolated polynucleotides of the invention, isolated Fez1 proteins of the invention, pharmaceutical compositions and diagnostic, therapeutic or screening methods of the invention. Included are various kits comprising descriptive material describing the use of these polynucleotides and proteins for practicing. Although representative kits are described below, the contents of other useful kits will be apparent to those of skill in the art in light of this specification. Each of these kits is included in the present invention.

One example of the kit of the present invention is a kit for amplifying at least a part of human FEZ1 gene. The kit comprises a first isolated polynucleotide and a second isolated polynucleotide, wherein the first isolated polynucleotide is at least 20 consecutive nucleotides of one strand of the human FE Z1 gene. Anneal with high stringency to the residue and the second isolated polynucleotide anneals with high stringency to at least 20 consecutive nucleotide residues of the other strand of the gene. For example, the first
An isolated polynucleotide of SEQ ID NO: 1 may anneal with high stringency to at least 20 contiguous nucleotide residues of the coding strand, and a second isolated polynucleotide of SEQ ID NO: 1 It is possible that it anneals with high stringency to at least 20 consecutive nucleotide residues in the chain. The kit further includes a DNA polymerase (eg, Thermus aquati
cus) DNA polymerase) or other components of the reaction mixture for amplifying a portion of the nucleic acid such as deoxyribonucleotides. Alternatively or in addition, the kit can include descriptive material describing polynucleotides useful for amplifying a portion of the gene or explaining how to perform these amplifications.

A second example of a kit of the invention is c prepared from transcripts of the human FEZ1 gene.
A kit for amplifying at least a part of DNA. The kit comprises a first isolated polynucleotide and a second isolated polynucleotide. The first isolated polynucleotide anneals with high stringency to at least 20 consecutive nucleotide residues of the cDNA, and the second isolated polynucleotide anneals with high stringency to at least 20 consecutive nucleotide residues of the cDNA. To do. In one aspect of this kit, the first isolated polynucleotide anneals with high stringency to at least 20 contiguous nucleotide residues of the coding strand of SEQ ID NO: 1 and the second isolated polynucleotide has SEQ ID NO: 1. Anneal with high stringency to at least 20 contiguous nucleotide residues of the non-coding strand of.

The invention will now be described with reference to the following examples. These examples are provided for purposes of illustration only, and the invention is not limited to these examples,
Rather, it includes all variations that are apparent as a result of the teachings provided herein. Example 1 The FEZ1 Gene at Chromosome Location 8p22 Encodes a Leucine Zipper Protein and Its Expression Is Altered in Multiple Human Tumors Loss of Heterozygosity (LOH) at 8p22 is a mammary, prostate and esophageal carcinoma Is a universal feature of epithelial tumors including In experiments presented in this example, been demonstrated in a number of cancer cell lines and tumor samples have altered expression and mutation of FEZ1 gene 8p22, Thus, FEZ1 gene, at least, 8P21-22 near It has been demonstrated to be one of the previously hypothesized tumor suppressor genes.

As first described herein, FEZ1 is a D designated Atf-5.
It encodes a leucine zipper protein that has substantial amino acid sequence similarity to NA-binding proteins. Expression of FEZ1 could not be detected in more than 60% of various types of epithelial tumors and tumor cell lines. Furthermore, analysis of the transcripts of tumor cells expressing FEZ1, the presence of mutations in FEZ1 as evidenced by the presence of abnormal sequences in FEZ1 transcripts, and the presence of the shortened FEZ 1 transcript The presence of frameshift mutations as demonstrated by Based on the results described in this example, it is concluded that alteration or inactivation of FEZ1 is involved in the development of multiple human tumors including epithelial tumors.

[0247]   The materials and methods used in the experiments presented in this example will now be described.

The esophageal cancer cell line was treated with RPMI 16 supplemented with 10% (v / v) fetal bovine serum.
Cultured in 40 medium. Prostate cancer cell line, breast cancer cell line, hematological cell line and H
eLa cells are from the American Type Culture Collection (Americ
an Type Culture Collection) and cultured as described (Negrini, 1996). Tumor and non-tumor tissue samples, 72 patients afflicted with primary esophageal cancer, 39 afflicted with breast cancer
Obtained from 1 patient, 24 patients afflicted with prostate cancer, and 8 patients afflicted with ovarian cancer.

Chromosomal DNA was isolated from 53 primary esophageal squamous cell tumors and matched normal tissue samples from the same patient. These DNA samples
Allele loss at 22 microsatellite loci on chromosome 8p was analyzed.

PCR amplification of microsatellite loci using FAM- or TET-labeled primers (Research Genetics, Huntsville, Ala.) With minor modifications, as described (Niederacher et al., 1997). , Genes Chromosom
． Cancer 18: 181). Briefly, PCR was performed using AmpliTaq ™ Gold (Perkin Elmer Cetus, Norwalk, Conn.) Using the following reaction conditions. After heating the reaction mixture to 95 ° C. for 12 minutes, a total of 30 PCR cycles were performed. The first 10 cycles are 1 at 94 ° C
5 seconds, 55-58 ° C for 15 seconds (to anneal DNA strands) and 7
Consisted of maintaining the reaction mixture at 2 ° C. for 30 seconds. The next 20 cycles are 89
The reaction mixture consisted of maintaining the reaction mixture at 55 ° C for 15 seconds, 55-58 ° C for 15 seconds (to anneal the DNA strands) and 72 ° C for 30 seconds. After these 30 cycles, the reaction mixture was maintained at 72 ° C for 30 minutes. After heat denaturation, the amplified reaction mixture was applied to Applied Biosystems.
tems) was loaded on a 6% (w / v) polyacrylamide denaturing gel on a model 373 DNA sequencer. Data collection and fragment analysis were performed using ABI Prism [Prism] ™ Genescan and AB.
I Prism ™ Genotyper analysis software (Perkin Elmer Perkin Elmer
Cetus), Norwalk, Connecticut; Applied Biosystems
It was carried out using Ink (Applied Biosystems, Inc., Foster City, CA).

LOH is 50% of the peak signal of the allele in DNA obtained from tumor samples with respect to the peak signal of the same allele in the corresponding normal tissue.
It was detected as the above deterioration. If the tumor sample demonstrated a 40-60% reduction in peak signal of the allele with respect to the corresponding normal tissue, the analysis was repeated two more times and the average reduction was used as the final data.

A contig of yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) in the region of the genome near the D8S261 marker was constructed. The relative positions of the YAC and BAC contigs for certain microsatellite loci are shown in Figure 1C.

Primers T7 and SP6 (Research Genetics (Research
h Genetics), Huntsville, Ala.) was used to sequence a BAC DNA sample of human chromosome 8p. Southern blot hybridization and PCR analysis showed that the BAC clones overlapped and contigs were constructed.

To screen a human YAC library obtained from Research Genetics (Huntsville, Ala.), STS (sequence tagged sequences) was screened.
PCR amplification was performed using the primers). A mixture of YAC clones was embedded in an agarose gel and described (Ausubel).
Et al., 1989, Current Protocols in Molecular Biology , Wiley-Interscience.
Science), New York; Bookstein et al., 1994, Gen.
mics 24: 317) and pulsed field gel electrophoresis (PFGE).
). After PFGE, YAC DNA was transferred to a nylon membrane in the presence of 0.4 molar sodium hydroxide and human genomic DNA was used to hybridize the membrane. DNA from individual YAC clones was digested in gel using MboI for 4 hours at 37 ° C. The DNA of the digested YAC clone was prepared according to the instructions of the kit by using Bio 101 ink (BIO
101, Inc. ) (La Jolla, Calif.) GeneClean I
Extracted from the gel using the II [Gene Clean III] ™ kit.

To clone the gene present in the DNA of the YAC clone, two deoxyoligonucleotides, 5'-GATCTCGACG AATTCGTGA.
G ACCT-3 ′ (SEQ ID NO: 44) and 5′-TGGTCTCACG AA
TTCGTCGA-3 ′ (SEQ ID NO: 45) was annealed to form a partially double stranded adapter linker. This adapter linker was digested with YA
It was ligated to the DNA of C clone. Fifteen cycles of PCR amplification were performed using the 5'-biotinylated primer corresponding to the adapter linker. The PCR product was sequenced and confirmed to be non-yeast genomic DNA.

Poly (A) of the prostate using NotI primer adapter / oligo dT primer (Superscript ™ plasmid system: Gibco-BRL, Grand Island, NY) according to a GC-rich protocol. ⁺ CDNA was synthesized by reverse transcription of RNA. Sal I adapters (Gibco-BRL, Grand Island, NY) were ligated to the cDNAs and the cDNAs were subjected to 20 rounds of PCR amplification using adapter primers.

Blocking, hybridization and washing methods are described in the procedure (
Bookstein et al., 1997, Br. J. Urol. 79 (Suppl. 1
): 28; Bova et al., 1996, Genomics 35:46; MacGr.
ogan et al. 1996, Genomics 35:55; Cher et al. 1994.
, Genes Chromosom. Cancer 11: 153; Books
tein et al., 1994, Genomics 24: 317; Akiyama et al.
1997, Cancer Res. 57: 3548). 1-2 micrograms of the amplified cDNA were added to an equal amount (by weight) of Cot-1 DNA.
Repeated by hybridizing with (Gibco-BRL, Grand Island, NY) to achieve a final DNA concentration of 80 micrograms per milliliter in 120 mM NaPO ₄ buffer, pH 7. The sequence was blocked. The reaction mixture is covered with mineral oil, heat denatured and 6
Incubated at 0 ° C. for 20 hours (Cot = 20). A biotin-labeled genomic DNA sample was heat denatured and the blocked cDNA (1 microgram, Co
Centricon ™ 100 Centrifuge Ultrafiltration Device (Amicon, Beverly, Mass.) with T-1 DNA excluded) and concentrated by centrifugation at 1000 × g for 25 minutes. And washed twice with 2 milliliters of 1 mM NaPO ₄ buffer, pH 7. The following concentrations at pH 7: 120 mM NaPO ₄ , 1 mM EDTA, and about 160 micrograms DN per milliliter.
Samples were adjusted to achieve A (excluding Cot-1 DNA). The reaction mixture was covered with mineral oil and incubated at 60 ° C. for 60 hours (Cot
= 120).

To prevent non-specific attachment of PCR amplifiable cDNA to beads, 10
Microliter avidin-coated magnetic bead suspension (Dynabeads [Dy
nabeads] ™ M-280; Dynal, Lake Success, NY), 100 micrograms of sonicated salmon sperm DN
Mix with A at room temperature for 30 minutes. The beads were pre-washed with TE buffer containing 1 molar NaCl and then incubated with 200 μl of the same hybridization reaction mixture in the same buffer for 30 minutes at room temperature. The beads were collected and the supernatant removed by using a magnetic concentrator (Dynal, Lake Success, NY). 0.1% (
w / v) The beads were washed twice for 15 minutes at room temperature using 0.1 × SSC buffer supplemented with SDS and then three times at 65 ° C. using the same buffer. Binding was performed by mixing the beads with 100 microliters of 50 mM NaOH for 15 minutes, and then neutralizing the mixture using 100 microliters of 1 molar Tris-HCl buffer at pH 7.5. C
DNA was eluted from the beads. The cDNA was purified using a PCR purification column (Qiagen ™, Chatsworth, CA) according to the manufacturer's instructions. The cDNA was reamplified by PCR using the same method and the same conditions. The resulting amplified cDNA product was purified and blocked, and a second round of cDNA selection was performed as described herein.

Amplified DN using the restriction endonucleases SalI and NotI
A was digested with pSPORT1 vector (Gibco-BRL).
, Cloned directly Grand Island NY), and used E. coli (E .coli) cells for transformation. CpG island cloning and shotgun sequencing was performed using the vector containing this cDNA. Using these methods, 87 potentially expressed clones were mapped in the YAC contig as shown in Figure 1C.

In CpG island cloning experiments, BAC D was constructed using the restriction endonucleases BssHI I and SacII , which specifically cleave the CpG island.
NA was digested. After digestion with Sau3A I, the cleavage product was described (E
lvin et al., 1992, Techniques for the analyzes is of complex genomes: Transcribed S sequences in YACs , edited by The Academic Press (A).
academic Press), London, p. 155) as per pBK-CM
V vector (Stratagene, La Jolla, CA) was ligated.

Shotgun sequencing has been described (Inoue et al., 1997, Proc.
Natl. Acad. Sci. USA 94: 14584). B
600 clones per AC were picked and sequenced to identify candidate cDNA sequences. cDNA selection was performed on three YAC templates as shown in Figure 1C. 400 clones per YAC were picked from a selected library of cDNAs and all clones were sequenced using vector primers. Sequences were analyzed using BLAST computer software and the NCBI / BLAST database to exclude genes associated with ribosomes or mitochondria. Fifty percent of the clones were genes associated with ribosomes or mitochondria, and the rest were sorted and analyzed.

Two candidate CpG islands were identified from the region near marker D8S233 by CpG island cloning. BACs were partially sequenced by the shotgun method to express expressed sequence tags in the NCBI / BLAST nucleotide / EST database.
The presence of sequences matching the (EST) was determined and two ESTs from the BAC genomic region were identified thereby.

Using these approaches, 123 clones totaling 400-800 base pairs in length were selected and characterized, and 87 of those clones were mapped.

Using 2 micrograms of total RNA from human brain, esophagus, or tumor cells, or using Superscript II [Superscript II] (
CDNA was synthesized from 150 nanograms of poly (A) ⁺ RNA obtained from one of these cell types using the Trademark) plasmid system (Gibco-BRL, Grand Island, NY). cDNA and adapter (catalog number K1802-1; Clontec Ink)
h, Inc. ), Palo Alto, Calif.) To generate a RACE template, and the template was used in PCR amplification of cDNA. Gene Bridge 4 [G
ene Bridge 4] (trademark) (Research Genetics (Rese
The chromosomal location of the F37 gene was confirmed by identification of the presence of the F37 gene sequence at 8p22 in a radiative hybrid panel designated as Arch Genetics), Huntsville, AL).

FEZ1 cDNA with full-length and 3'-truncated expression vector pcDN
RT ligated to A3HisA (Invitrogen, Carlsbad, Calif.) And using human brain cDNA as template
-Cloned by PCR. The complete nucleotide sequence of the insert cDNA is D
Confirmed by NA sequencing. The truncated cDNA (nucleotides 1-1128 in the FEZ1 ORF) is an OR located 3'to the leucine zipper region.
I was missing the F part.

Rabbit reticulocyte system (Quick TNT [trademark], Pharmacia LKB Biotechnology Ink (Pharmacia LKB)
Biotechnology Inc. ), Piscataway, NJ) and performed in vitro transcription and translation and
Monitored by GE. Full length and 3'truncated FEZ1 cDNA
To the glutathione-S-transferase (GST) fusion expression vector (pG
EX; Pharmacia LKB Biotechnology Ink (Pharmaci)
a LKB Biotechnology Inc. ), Piscataway, NJ, and proteins encoded by their cDNAs have been described (Ausubel et al., 1992, Current Protocols in Molecular Biology , John Wiley & Sons, NY). Expressed as above, extracted, separated by PAGE and visualized by Coomassie staining. The image of the result of SDS-PAGE is shown in FIG. 2C. The SDS-PAGE lane 1 protein was translated from a pcDNA vector containing a truncated FEZ1 cDNA lacking the portion of the cDNA located 3 ′ to the leucine zipper region. The protein in lane 2 is pcD containing the full-length FEZ1 cDNA.
Translated from NA vector. The protein in lane 3 is pc with no insert
Translated from a DNA vector. The protein in lane 3 is a truncated FEZ1 cDNA lacking the portion of the cDNA located 3'to the leucine zipper region.
Was translated from the pGEX vector containing

The nucleotide sequence of the open reading frame (ORF; exons 1-3) of the FEZ1 gene was analyzed in samples obtained from 194 cancer tissues, regardless of whether FEZ1 was expressed in the tissue. Tissues sampled were 72 primary esophageal cancer, 18 esophageal cancer cell lines, 24 primary prostate cancer, 3 prostate cancer cell lines, 39
Primary breast cancer, 25 breast cancer cell lines, 8 primary ovarian cancers, 4 leukemia cell lines and 1 cervical cancer cell line. Nucleotide sequence information was obtained by PCR and sequencing. FEZ1 encoding exons 1-3 using 11 pairs of PCR primers as described herein in the primer nucleotide sequence listing.
Was amplified.

4% DMSO (w / w) was added to the reaction mixture and PCR amplification was performed for 35 cycles (5 additional cycles are identical to 20 cycles described herein). Genomic PCR was performed using the same conditions described herein for the LOH study. DNA sequencing was performed directly using the purified PCR amplification product. ABI Prism [trademark] 377 DNA sequencing system (Applied Biosystems, Inc.)
tems, Inc. ), Foster City, Calif.), Sequencing reactions and analyzes were performed by using ABI Prism BigDye ™ terminator reaction chemistry. Sequence data was confirmed by sequencing the duplicated PCR amplification product and sequencing the antisense strand using the reverse primer.

[0269]   The results of the experiments presented in this example are now described.

Primary esophageal cancer tissue samples obtained from 23 of 53 patients (43%) showed alleles at one or more loci on 8p, as shown in FIGS. 1A and 1B. The disappearance of For example, a tissue sample obtained from patient E26 has LOH with markers designated D8S264 , LPL and D8S136.
, Also represented allelic retention with a marker designated FGFR1. The tissue sample obtained from patient E46 displayed LOH with the markers designated D8S264 and D8S136, and the markers designated LPL and FGFR1 loci are homozygous and alleles from one chromosome It meant that when the disappearance of the could occur it could not be detected.

Of the 23 tumor samples in which allelic loss was observed, 16 (70%) represented a universally lost 1.5 megabase region located near the D8S261 locus, and Fourteen of those 23 patients (61%) displayed a potential common LOH region located near D8S254 . These data suggest that the two tumor suppressor genes are located in a chromosomal region designated 8p22-23. Experiments described in this example, focused on more frequently affected by that 8p22 region around D8S261. This region is 4-6 megabases centromere to the MSR region and overlaps with target regions of other tumors, including prostate and breast cancer (Kagan et al., 1995, Oncogene 11: 212.
1; Macoska et al., 1995, Cancer Res. 55: 5390; J
enkins et al., 1998, Genes Chromosom. Cancer
21: 131; Yaremko et al., 1995, Genes Chromosom.
． Cancer 13: 186; Yaremko et al., 1996, Genes C.
homosom. Cancer 16: 189; Kerangueven et al.,
1997, Cancer Res. 57: 5469; Anbazhagan et al.,
1998, Am. J. Pathol. 152: 815; El-Nagar et al.,
1998, Oncogene 16: 2983; Sunwo et al., 1996, G.
enes Chromosom. Cancer 16: 164; Wu et al., 199.
7, Genes Chromosom. Cancer 20: 347).

To clone the genes present in this region, cDNA selection, CpG island cloning and shotgun sequencing were performed. Using these treatments, 87 potentially expressed clones were mapped in the YAC contig as illustrated in Figure 1C. Using RT-PCR amplification,
Clones displaying reduced expression in tumor cells were selected and shown to have 43 of 87 clones expressed in normal adult tissues including prostate. 9
Clones showed reduced or no expression in cancer cells. Rapid amplification of cDNA ends (RACE) was performed and 6 out of 9 clones
The sequence was successfully extended. Northern blot analysis showed that expression of 5 clones was not significant in cancer cells. In contrast, RACE analysis using the F37 clone obtained from the hybrid selection revealed that a 6.5 kilobase transcript was expressed in non-cancerous tissue, but F37 expression was found in the LNCaP prostate cancer cell line. It could not be detected by.

Approximately 6 × 10 ⁶ clones from a human testis cDNA library obtained from Clontech (Palo Alto, Calif.) Were cloned into F37.
Was screened using a probe specific for and the nucleotide sequence at the 5'end of the cDNA was obtained by RACE treatment. The F37 probe used is Figure 5Q.
Had the sequences listed in. Gene Bridge 4
(Research Genetics (Research Genetics), Huntsville, Alabama) by the presence of F3 7 gene sequence in 8p22 in radiation hybrid panel called a confirmed the chromosomal location of the F37 gene. These results show that the F37 gene is a gene marker of 3.36c designated WI-5962.
It was shown to be located within R (centirad). The F37 cDNA comprises a 1791 base pair open reading frame (ORF) encoding a 597 amino acid residue protein with a molecular weight of approximately 67 kilodaltons. Homology searches of protein sequence databases have revealed that the amino acid sequence of F37 comprises a single leucine zipper motif and that this region is Atf-5 (Hai et al., 1989, Gen.
es Development. 3: 2083) and has a 32% identity (68% similarity) to the DNA binding domain of the cAMP responsive activation transcription factor. Homology search was performed using KIA consisting of 673 amino acids of F37 protein.
A protein designated A0552 (Nagase et al., 1998, DNA Re
s. 5: 31-39) and also showed 38% identity.

Motif analysis software (Searching Protein and
Nucleic Acid Sequence Motif in Geno
me Net) is predicted cAMP-dependent phosphorylation sites located Ser 29 of F 37, and the predicted tyrosine kinase phosphorylation sites located Tyr 67 of F 37. The ORF contained 3 coding exons. The F37 gene was designated as FEZ1 (F37 / which encodes a leucine zipper motif).
Esophageal cancer gene (F 37 / E sophageal cancer gene en
coding leucine- z ipper motif). The deduced amino acid sequence of the protein Fez1 encoded by FEZ1 is listed in Figure 2A. Kozak's recognition rules (Kozak, 1989, J. Cell. Biol. 108:
229-241) was used to adapt the nucleotide residues around the first methionine codon in the FEZ1 cDNA. The stop codon in the same reading frame of 5'cD
Identified in NA and located at -111 to -109 from the first methionine codon.

Northern blot analysis showed that FEZ1 gene expression was nearly ubiquitous in normal tissues. Expression of FEZ1 was most prominent in the testis as shown in Figure 2D. FEZ1 gene expression was determined as shown in FIG. 3A and summarized in Table 1, 41
In a human tumor tissue sample containing 25 cancer-derived cell lines and 25 primary tumors,
Analyzed by Northern blotting and RT-PCR amplification. FEZ1 expression was undetectable in 31 cancer cell lines (76%) and 16 primary tumor samples (64%). FEZ1 expression was not detected in any of the 15 breast cancer cell lines studied or in the 10 primary breast cancer samples studied. However, FEZ1 was expressed in normal tissues.

[0276]

[Table 3]

^{2 At} E16, E26 and E41, normal tissue from a single patient organ was analyzed and showed no changes in the sequence of the coding region. In addition, the coding sequences from normal prostate, as well as four other samples from normal esophagus, were analyzed and 1 of 12 sequenced clones from testis cDNA were analyzed.
No changes were found except that one showed a deletion of nucleotides.

To exclude the possibility that normal stromal cells (but not normal epithelial cells) may express FEZ1 , normal breast epithelial cells and fibroblasts,
And normal prostate epithelial cells (these three types of cells are clontics (Cl
FEZ1 expression was assessed in ethics), obtained from San Diego, Calif.). RT-PCR amplification showed that FEZ1 was expressed in these 3 normal cells. No FEZ1 expression could be detected in breast and prostate (LNCaP) cancer cells.

To eliminate the possibility that the apparent differences in FEZ1 expression observed between cell types may be attributed to alternative splicing of FEZ1 transcripts,
Northern blot analysis was performed using 3 different probes. The three probes, complementarity to a region of the ORF of FEZ1 transcript, 3 immediately downstream from the ORF of FEZ1 transcripts 'complementary to non-coding regions, or 3 FEZ1 transcript'
It was constructed to be complementary to the non-coding end region. No differences were observed between Northern blots made using these three probes, suggesting that FEZ1 expression was absent in the cell lines and tumors examined.

The nucleotide sequence of the FEZ1 gene ORF was analyzed in a total of 194 cancer tissue samples, regardless of whether FEZ1 was expressed in the tissue. These tissue samples included 72 primary esophageal cancer tissue samples, 18 esophageal cancer cell lines, 24
Primary prostate cancer tissue sample, 3 prostate cancer cell lines, 39 primary breast cancer tissue samples, 25 breast cancer cell lines, 8 primary ovarian cancer tissue samples, 4 leukemia cell lines and 1 cervical cancer cell line did. As shown in Figure 3B, three point mutations were identified, two in two primary esophageal cancer tissue samples and one in one prostate cancer cell line. These point mutations are summarized in Table 2.

[0281]

[Table 4]

In one primary esophageal tumor tissue sample, designated E44, the point mutation resulted in a serine (normal) to proline (mutant) amino acid substitution at amino acid residue 29. Amino acid residue 29 is a predicted cAMP-dependent kinase phosphorylation site, as described herein. In another primary esophageal cancer tissue sample, designated E50, the second point mutation resulted in a different amino acid substitution, lysine at amino acid residue 119 (normal) to glutamic acid (mutant). The LOH study described herein showed that the two patients who obtained samples E44 and E50 displayed allelic loss at the D8S261 marker, respectively. Thus, tumor cells obtained from a patient of these two cases holds the mutated F EZ1 allele, and had disappeared normal FEZ1 allele.

The third point mutation detected was a change from a codon encoding a glutamine residue to a stop codon in the normal FEZ1 transcript of codon 501 in a prostate cancer cell line designated PC3. . This mutation resulted in a FEZ1 transcript encoding a putative 166 amino acid residue protein lacking the normal carboxyl-terminal region of the wild-type FEZ1 protein. Northern blotting, RT-PCR
And nucleotide sequencing showed that these three mutated DNA sequences were expressed in the patients and cells in which they were identified. These data suggest that FEZ1 is involved in that encode tumor suppressor proteins, and the universal cancer inactivation of several FEZ1 generation.

The nucleotide sequence of the cDNA generated from the mRNA expressed from FEZ1 in some of the tumors was determined. As shown in Table 3, Table 4 and Figure 3B, several internally shortened transcripts were identified. ORF of FEZ1 with one exception
The sequence of normal brain and prostate tissue (Clontech Inc.
h, Inc. ), Obtained from Palo Alto, Calif.), Normal esophageal tissue samples obtained from 7 individuals or matched normal cDNA obtained from patients E16, E26 and E41. It didn't differ from the type array. Testis cDNA sample (Clontech Inc.
h, Inc. ), Obtained from Palo Alto, Calif.), One of the 12 clones displayed a deletion of nucleotides 1441-1527 in the ORF.
The cDNAs obtained from the two esophageal cancer tissue samples displayed a frameshift, the c
With the result that the DNA encoded a protein of 76 amino acid residues.

[0285]

[Table 5]

[0286]

[Table 6]

The nucleotide sequence flanking the deleted region of the FEZ1 cDNA showed that the intron AG sequence was present at the 3 ′ boundary of the deleted region in the cDNA, and was deleted in the tumor. It has been suggested that the FEZ1 transcripts may be ascribed to physiologically inappropriate splicing events. 3'non-coding cd
The polymorphic site in the NA region, ie the 2134th nucleotide residue of the FEZ1 cDNA (numbered from the first nucleotide residue of the first codon) was used to analyze the expression status of the FEZ1 allele. . In four informative normal primary tissues, the FEZ1 gene was transcribed from both alleles (ie it was not imprinted). In contrast, FEZ1 expression in cancer cells expressing FEZ1 in each sample studied are from a single allele, can probably attributable to allelic loss.

[0288]   In 18 cancer cell linesFEZ1Using the ORF probeFEZ1Gene remains
Southern blot analysis of the loci shows that one breast cancer cell line
Single rearrangedFEZ1And expresses the normal allele
Showed that there was not. No homozygous deletions were detected in the other 17 cell lines tested.
I couldn't. Several tumor suppressor genes are associated with frequent allelic loss and then go
It is eventually involved in homozygous deletion (Weinberg, 1991, Science).
  254: 1138; Lasko et al., 1991, Ann. Rev. Genet.
25: 281; Knudson, 1993, Proc. Natl. Acad. S
ci. USA 90: 10914; Nowell, 1993, Adv. Canc
er Res. 62: 1; Bookstein et al., 1997, Br. J. Uro
l. 79 (Suppl. 1): 28; Bova et al., 1996, Genomics.
  35:46; MacGrogan et al., 1996, Genomics 35: 5.
5; Cher et al., 1994, Genes Chromosom. Cancer
11: 153; Bookstein et al., 1994, Genomics 24: 3.
17; Ohta et al., 1996, Cell 84: 587). These data are D8S261 Loci andFEZ1In the genomic region around the gene locus
LOH is a frequent abnormality, but homozygous deletions of this gene do not occur in tumors.
Suggest that it is. Therefore,FEZ1The main mechanism of inactivation is allele silencing.
"Two-hit" events such as loss and point mutations, and
Combined with easy-to-remain allele shutdown (ie null) transcription
It seems that allele loss can be attributed.

The experiments presented in this example demonstrate that loss of FEZ1 function enhances tumorigenesis in at least prostate, breast and esophageal cancers, and possibly other malignancies with chromosomal alterations at 8p22. Prove. Example 2 Effect of Fez1 Expression on Cell Growth of Breast Cancer Cell Line MCF7 The experiment described in this example induces FEZ1 expression in the absence of tetracycline and represses FEZ1 expression in the presence of tetracycline. A cell line of a breast cancer line called MCF7, which has been transfected with a vector (American Type Culture Collection).
ure Collection), available from Gatorsburg, MD; accession number HTB-22). Induction of FEZ1 expression inhibited cell growth in vitro and in vivo.

PTet-Off ™ in which at least the coding portion of the FEZ1 gene is operably linked to the tetracycline response element of the vector and the promoter.
MCF7 cells were stably transfected with the plasmid vector (ClonTech, Palo Alto, Calif .; GenBank Accession No. U89929). Cells are 2 micrograms of doxycycline per milliliter (Sigma Chemical Company (S
igma Chemical Co. ), St. Louis, Mo., Catalog No. D-9891) and 10% (v / v) certified fetal bovine serum (FBS;
Maintained in DMEM medium supplemented with ClonTech.
About 1 × 10 ⁵ cells were grown in 3.5 cm diameter culture dishes and according to the instruction manual according to the supplier's instructions (Gene Therapy Systems, San Diego, Calif.). , GenePORTER ™ reagent was used to transfect with approximately 4 nanograms of plasmid DNA.

Transfected cells were cultured in medium containing hygromycin (Gibco, Grand Island, NY) at a concentration of about 200 micrograms per milliliter, starting at 36 hours post transfection and ending at about 2 hours. By maintaining for a week, stable transfectants were created. Four
One well isolated transfectant clone was selected and designated clones 15, 18, 54 and 118. These clones were cultivated for 72 hours in tetracycline-free medium comprising 10% (v / v) serum medium in order to induce the expression of FEZ1 . In the experiments described in this application, tetracycline and doxycycline were used interchangeably as the tetracycline response element responds to tetracycline and doxycycline in a substantially equivalent manner.

Cellular proteins were extracted before and after induction of FEZ1 expression and SDS-
Separated by PAGE. Separated proteins were transferred to nitrocellulose membranes and immunoblot analysis was used to determine the presence of Fez1 protein or actin (as a control). A polyclonal antibody that specifically binds to Fez1 was used. The results of this immunoblot treatment are shown in Figure 6 and
It demonstrates that the Fez1 protein was produced by each of the selected clones when maintained in the absence of tetracycline. Fez1 protein is not produced by cells transfected with the vector alone,
It shows that there was no endogenous FEZ1 expression in MCF7 cells.

The effect of FEZ1 expression on in vitro cell growth of MCF7 cells was determined according to the manufacturer's instructions by Promega Corporation (Promega Corporation).
ation) (Madison, Wisconsin).
CellTiter 96] ™ aqueous (AQueous) non-radioactive cell proliferation assay was used for analysis. The absorbance of the assay system MTS compounds at 490 nanometers ranged between 10 ² and 10 ⁴ cells of MCF7 cells, as confirmed by cell counting where dead cells were excluded by trypan blue staining. There was a linear correlation between the numbers. The cells of clones 15, 18, 54 and 118 were
The wells of 96-well plates containing medium without tetracycline supplemented with 10, 5, 2.5, 1 or 0.5% (v / v) FBS were seeded. The medium was replaced daily with the corresponding fresh medium. The absorbance at 490 nanometers was evaluated to estimate the number of cells present in each well at selected times. The results of these experiments are presented in Figure 7, where the data were transfected relative to the number of control pseudo-MCF7 transfectants (ie, transfected with vector alone) cultured in the corresponding medium. Shown as a ratio of cell numbers. Data are calculated as the average of 4 independent experiments and the bars in Figure 7 show the standard deviation.

Figure 8 shows the results of cell cycle analysis of synchronized transfected MCF7 cells. Transfection of MCF7 in in the presence or absence of tetracycline (i.e., to induce expression of FEZ1 in a cell that is maintained in the absence of tetracycline) for 3 days, 1.5% (v / v) Cultured in growth medium supplemented with FBS. The cells were then maintained in medium containing thymidine to induce accumulation of cells in the G1 / S phase of the cell cycle. The medium containing thymidine was replaced with the same growth medium and the cells were then fixed at selected times. Cells were fixed in 70% ethanol and treated with propidium iodide and RNase A prior to flow cytometric analysis. As the ratio of the number of cells in the G2 / M phase of the cell cycle to the number of cells in the G1 phase of the cell cycle (FIG. 8A), or the number of cells in the S cycle of the cell cycle to the number of cells in the G1 phase of the cell cycle The ratio was calculated as a ratio (Fig. 8B). The results of this analysis show that expression of FEZ1 was found to be late S or G2 / M in the cell cycle.
We show that it appears to inhibit the proliferation of MCF7 cells in vitro by causing the accumulation of cells in phase.

About 5 × 10 ⁶ or about 2 × 10 ⁷ cells (MCF7 cells transfected with pTet-Off ™ vector alone, or MCF7 transfectant clone 15, 18, 56 or 118 clone cells). ), 6
A week-old female Balb / c nude mouse was subcutaneously inoculated into the left dorsal subclavian region. Four mice were used for each experimental group. By measuring in two directions using calipers to estimate tumor volume for each mouse, and was calculated as tumor volume = length × (width) ^2/2. These results show that expression of FEZ1 inhibited the growth of MCF7 cells in vivo, and that FEZ1 expression inhibited (or even reversed) proliferation of epithelial tumor cells in animals. Example 3 Construction of an adenovirus vector having an isolated nucleic acid encoding at least the influencing portion of the Fez1 protein incorporated therein Isolation of the FEZ1 cDNA To construct an adenovirus expression vector, a nucleotide sequence, 5'-CAG ATG GGC AGC GTC AGT AGC CTC A
TC-3 ′ (SEQ ID NO: 58) and 5′-TCA GAT CTC AGT GGC TAT GAT GTC-3
'(SEQ ID NO: 59), a full-length FEZ 1 cDNA was transferred to human normal placenta poly (A) by reverse transcription-polymerase chain reaction (RT-PCR) amplification using a pair of promoters. ⁺ Isolate from RNA. Of course, any other primer pair can be used to isolate the Fez1 cDNA, or the cDNA can be made synthetically. Because the sequence is now available (Figure 5B; SEQ ID NO: 2; GenBank (Ge
nBank) Accession number AF123659). When cDNA is isolated by RT-PCR, commercially available superscript-follow the supplier's instructions.
II [SuperScript-II] (trademark) system (Gibco-BRL (Gibc
(O-BRL), catalog number 18064-022, Rockville, MD).
Reverse transcription can be performed using PCR can be performed using, for example, Advantage Taq (Clontech, Catalog No. K1905-y) according to the supplier's instructions. For example, reverse-transcribed cDNA is added to a standard PCR reaction mixture at 94 ° C.
For 30 seconds and then the reaction mixture at 94 ° C. for 10 seconds at 58 ° C.
By carrying out 35 cycles comprising maintaining at 0 ° C for 10 seconds and at 72 ° C for 60 seconds, and then maintaining the reaction mixture at 72 ° C for 60 seconds.
It can be subjected to PCR amplification.

The amplified product was analyzed in Current Protocols in Molec.
ural Biology, Frederick M Ausubel, John Wiley & Sons, Inc. (John Wiley & Sons)
, Inc), 1987, 1.5% (W / v) agarose gel (Gibco-BRL, Catalog No. 15510-019).
Can be separated by electrophoresis in.

Poly (A) ⁺ RNA can be purchased from, for example, Clontech (catalog number 6518-1) and used to make cDNA. Clontech's poly (A) ⁺ RNA material is C
current Protocols in Molecular Biolog
y (John Wiley & Sons, Inc. (John Wiley &
Sons, Inc. ), 1987) and extracted and purified from normal placental tissue of Caucasian humans (ages 22-31 years).

Adenovirus shuttle vector DNA can be obtained, for example, from the Quantum company (Montreal, Quebec, Canada; eg pAdCMV-IRES-GFP, catalog number AES050M).

Amplified FEZ1 cDNA is isolated from agarose gel and purified using, for example, a Qiagen ™ PCR Purification Column (Stanford Valencia, Calif .; Catalog No. 28104) according to the supplier's instructions. . The DNA of the adenovirus shuttle vector was cloned into the restriction endonuclease BglII (Boehringer Mannheim-Roche).
r Mannheim-Roche); Indianapolis, Ind.). For example, T4 DNA polymerase (Promega)
, Madison, Wis.) And blunting the ends of the DNA using
0 nanogram of cDNA is ligated with 100 nanogram of vector DNA. The resulting construct was used to use electrocompetent Escherichia coli (Escherichi), such as strain DH5α (Gibco).
a. coli) strain and transform the transformed cells (eg Cu
current Protocol in Molecular Biology,
John Wiley & Sons Ink (John Wiley & So
ns, Inc. LB) supplemented with ampicillin (as described in 1987)
Transfer to a culture dish containing agarose medium.

Clones containing FEZ1 cDNA have been cloned, eg, (eg, Current)
Protocol in Molecular Biology, John Wiley & Sons, In
c. ). As described in 1987) and selected using the colony hybridization technique using full length FEZ1 cDNA as a DNA probe.
These "positive" clones are grown overnight in 5 ml LB medium and plasmid DNA is extracted from the positive clones using, for example, a Qiagen miniprep column. The sequence of the extracted plasmid DNA can be analyzed at this point to confirm recovery of the expected construct. For example, sequencing reactions and analyzes are performed using Applied Biosystems.
Stems) prism [Prism] (trademark) Big Die [BigDye] (
Trademark terminator reaction chemistry, as well as Perkin Elmer
Elmer's Gene Amp ™ PCR system 9600 and Applied Biosystems.
Can be performed using the Prism ™ 377 DNA Sequencing System (Norwalk, CT). CDNA in vector DNA
After confirming the strand orientation, the plasmid can be amplified in E. coli . Confirmation of Transient Expression Using the FEZ1 Adenovirus Shuttle Vector By assessing transient expression of FEZ1 in HeLaS3 cells (ATCC) maintained in F12 / MEM medium supplemented with 10% FBS, The promoter activity and validity of the plasmid vector can be confirmed. For example, 1
About 5 × 10 ⁵ cells per cubic centimeter are grown overnight in 6-well plates. Using 3 micrograms of plasmid, for example, the lipofection method (eg GenePORTER ™ reagent, Gene Therapy System Inc.).
Is used to transfect cells in each well. After maintaining the cells under culture conditions (eg, about 48 hours), the cells are harvested and, for example, Cur
rent Protocol in Molecular Biology (John Wiley & Son Inc.
s, Inc. ) FEZ1 expression is assessed by immunoblot analysis using an anti-Fez1 antibody as described in 1987).

An adenoviral vector (pQBI-AdCMV5-IRES-) capable of incorporating at least an isolated nucleic acid encoding an influencing portion of the Fez1 protein, and at least an isolated nucleic acid encoding a fluorescent portion of GFP.
The nucleotide sequence (designated GFP) (SEQ ID NO: 60) is listed in FIG.

Preparation of Recombinant Adenovirus Vectors Adenovirus vectors were prepared using the above-mentioned adenovirus shuttle vector and those described in (Miyake et al., 1996, Proc. Natl. Acad. S).
ci. USA 93: 1320; Kanegae et al. , 1994, J
pn. J. Med. Sci. Biol. 17: 157) by transfecting cells with adenoviral DNA (such as that obtained from Guantum) as described in Fetal Kidney 293 cells (Microbix Biosy).
stems Inc. , Toronto, Ontario, Canada). Microbix Biosystems Inc
． The 293 cells obtained from E.coli are low passage strains, and well homologous recombination efficiency could be obtained. 9 293 cells transfected
6-well plates are inoculated and well separated plaques are selected.

293 cells are transfected with the shuttle plasmid by the calcium phosphate precipitation method and can be grown in 100 mm diameter dishes. Twenty-four hours after transfection, transfectants are inoculated into individual wells of a 96-well plate (containing approximately 200 μl of culture medium per well). Cells in wells are untransfected 29
It is diluted about 10 to 100 times with 3 cells. After 2-3 weeks in culture, plaque-forming cells are harvested and viral particles are extracted, eg, by multiple freeze and thaw cycles. The number of wells forming plaques is calculated to be about 10-50 wells per 96-well plate.

The virus-containing supernatant from plaque forming wells was diluted to 29% in soft agar.
Subjected to serial infection of 3 cells. For example, approximately 5 × 10 ⁵ 293 cells are infected with 100 μl of virus-containing supernatant and cells are inoculated on a 1.25% (w / v) low melting gel (Gibco) in 60 mm diameter culture dishes. After 10 days, plaques formed in soft agar are isolated under a microscope. For example, in a vector encoding green fluorescent protein (GFP), GFP can be observed by fluorescence microscopy. The virus titer is, for example, 75 to 70 ml of liquid culture medium in a flask.
Expanded by serial infection of 293 cells grown in 175 ml.

Analysis of F EZ1 Expression in Cells Transfected with Adenovirus Vectors Adenovirus vectors containing an isolated nucleic acid encoding at least an operable portion of the Fez1 protein were used to transfect. Expression of FEZ1 in transfected cells can be detected by immunoblot analysis of proteins extracted from cells using, for example, rabbit anti-Fez1 polyclonal antibody. For example, the infectivity of a viral vector is
ATCC) together with the adenovirus vector-containing supernatant in a volume ratio of 1 / 40-1 /
Cultured at 10 (v / v), extracted protein from cells, and Fez1
It can be examined by examining whether the protein can be detected by immunoblot analysis. Alternatively, if the adenovirus vector encodes a detectable protein, such as GFP, the infectivity of the viral vector preparation is determined by expression of the detectable protein in cells cultured with virus-containing supernatant. You can find out by inspecting. By way of example, if the adenoviral vector encodes GFP, the infectivity of the viral vector can be examined by detecting fluorescence in the cells at the excitation / emission wavelength pair characteristic of GFP.

Example 4 Identification of Fez1 Binding Partner Protein Yeast Two-Hybrid Screening Yeast two-hybrid screening was performed according to the teachings of the supplier.
Performed in yeast strain Y190 using the HMAKER ^™ System 2 (Clontech). We have found that GAL4 fused to the full length Fez1 protein.
GAL using a fusion protein comprising a protein DNA binding domain
A large number of clones of the human testis cDNA expression library fused individually by the 4-protein transcription activation domain-fused pACT2 vector were screened. After initial screening with the β-galactosidase assay, DNA was extracted from positive clones and sequenced using vector primers to identify cDNA clones.

In Vitro Transcription / Translation, GST-Fusion Proteins and In Vitro Binding Assays In vitro transcription and translation is commercially available in rabbits by labeling with ³⁵ S-methionine according to the instructions of the supplier. Reticulocyte-based system (TNT ^™ T
7 Quick Coupled Transcription / Transla
The Tion System, Promega). The GST-fusion protein was isolated using a glutathione-agarose column (Pharmacia). The proteins are two types of binding buffers: buffer A (100 mMN
aCl, 0.5% NP-40, 0.75 mg bovine serum albumin per ml (
BSA), containing 20 mM Tris-HCl pH 8.0 and 1 mM EDTA)
And buffer B (150 mM NaCl, 0.1% (v / v) Tween 20)
0.75 mg BSA, 50 mM Tris-HCl pH 8.0, 5 per ml
Incubated in mMEDTA, 10% (v / v) glycerol). After glutathione-agarose beads were preincubated in a 10% (w / v) BSA suspension, the beads were mixed with the protein sample and washed 5 times, each wash with 10 volumes of beads and binding buffer. Including mixing. After the beads were washed, the bead-containing liquid was centrifuged to collect the bound protein. The sample was boiled for 3 minutes and then the proteins in the sample were separated by SDS-PAGE. The gel was dried and then at -80 ° C 4-
The film was exposed for 24 hours.

Approximately 100 clones were identified that encode proteins that exhibited binding to the Fez1 protein. When the DNAs corresponding to these clones were extracted and sequenced, many positive clones were found to overlap. Several independent clones were identified, including a clone encoding the peptide elongation factor 1-γ (EF1-γ; cDNA sequence deposited by other researchers under EMBL accession number X68142). EF1-γ is a member of the microtubule-associated protein family. To confirm the results, a β-galactosidase assay was performed and EF1-γ showed a strong interaction with Fez1. The reaction time is 15
<15 minutes compared to a reaction time of the positive control of -20 minutes and an unreacted negative control reaction time at> 48 hours.

The results of the in vitro binding assay demonstrating the binding between ³⁵ S-methionine-labeled EF1-γ and Fez1 protein are shown in FIGS. 11A, 11B and 11C. The in vitro binding assay mixture corresponding to lanes 1-8 contained the in vitro translated EF1-γ protein. The mixture corresponding to lane 2 is the glutathione S-fused with the full length (67 kilodalton) Fez1 protein.
The mixture containing transferase (GST) and corresponding to rows 4 and 7 was
GST fused to a truncated (40 kilodalton) Fez1 protein
Contained. The mixture corresponding to rows 5 and 8 is the in vitro translated EF1-
It contained only the γ protein. The reproducibility of binding was confirmed by performing binding assays in two different buffers, buffer A (lanes 1-5) and buffer B (lanes 6-8). The results of this experiment show that Fez1 protein and E
It illustrates that F1-γ binds to each other.

Other investigators report that peptide elongation factors form a protein family, which consists of at least EF-1α, EF-1β, EF-1γ and EF-1δ (J. Biol. Chem. 269: 31410-314.
17, 1994; Biol. Chem. 269: 2086-2092, 19
94). We analyzed the binding of ³⁵ S-methionine-labeled in vitro translated EF to the GST-fused Fez1 protein (lanes 9-14 in FIG. 11). No binding could be detected between Fez1 and either EF1-α or EF-1δ. The assay performed to detect the binding of EF1-β to Fez1 did not give information because EF1-β binds to GST.

Three ³⁵ S-methionine-labeled, in vitro translated, deletion mutants of the EF1-γ protein were generated: 15 amino-terminal but not all of EF1-γ.
A variant designated EF1-γ (N), in which three amino acid residues have been deleted, EF1-γ
Of carboxyterminal 126 amino acid residues but not all of EF1-γ
A variant designated (C) and a variant designated EF1-γ (M) in which not all but 149 amino acid residues in the central portion of EF-1γ were deleted (ie EF1-γ (M ) Is residue 154-measured from the amino terminus of EF1-γ.
It consisted of 302). The amino acid sequence of EF1-γ is GenBank Accession No. X.
68142. The in vitro binding of these deletion mutants with GST-fused Fez1 was analyzed. EF1-γ (N) bound to Fez1, but neither EF1-γ (C) nor EF1-γ (M) bound to Fez1.

[0312] EF1-gamma FEZ1 protein in-Vito b binding assays complementary binding assay to the amino terminal portion of the protein, ³⁵ S- methionine - full length translated in labeled in vitro 67KDaFez1 (columns in Fig. 12 1 And 2) or 4 trimmed
Buffer B with 0 kDa Fez1 protein (lanes 3 and 4 in FIG. 12)
Was carried out in. The assay mixture corresponding to columns 2 and 4 of FIG. 12 is EF1-γ (N
) Was fused to GST and the assay mixture corresponding to columns 1 and 3 of FIG. 12 contained GST protein (as a negative control). In vitro translated full length 67 kDa Fez1 protein (lane 5) or truncated 40 kDa Fez1 protein (lane 6) was added alone as a control. These results indicate that the amino-terminal 2/3 portion (40 kDa) of Fez1 protein is EF.
It shows in vitro binding to all or part of the 153 amino terminal amino acid residues of 1-γ.

In vitro dimerization of Fez1 protein The amino acid sequence of Fez1 contains a leucine-zipper-like region. The leucine zipper region is known to be involved in protein-protein and / or protein-nucleotide interactions in other proteins (Pro.
c. Natl. Acad. Sci. USA 96: 3928-3933,199.
9). The in vitro binding assay was performed in buffer B, where the assay mixture was ³⁵ S-methionine labeled in vitro translated full length (
67 kDa) Fez1 protein (lanes 1, 2 and 5 in FIG. 13) or in vitro translated truncated 40 kDa Fez1 labeled with ³⁵ S-methionine.
Contains any of the proteins (lanes 3, 4 and 6). The assay mixture also contained either the full length GST-fusion 67 kDaFez1 (lane 2), the truncated GST-fusion 40 kDaFez1 (lane 4), or the GST protein (lanes 1 and 3; negative control). . These results indicate that 67kDaFez1 and the truncated 40-kDaFez1 protein can dimerize.

Interaction of Fez1 with EF1-γ in Transfected Cells The full-length FEZ1 cDNA was engineered with pcDNAV5 vector (Invitrogen, Carlsbad, Calif.) To express the V5-labeled-fused Fez1 protein in cells transfected with the vector. It was connected. Full length EF
1-γ cDNA is expressed in EXP in cells transfected with the vector.
It was ligated with pcDNAHis vector (Invitrogen) to express the label-fusion EF1-γ protein.

HeLaS3 cells were transfected with these 2 cells using lipofection to analyze the in vitro interaction between Fez1 and EF1-γ.
Seed vectors were co-transfected. Immunoblot analysis with anti-labeled antibodies demonstrated that the transfected cells express the V5 / Fez1 fusion protein (lane 2 in FIG. 14) and the 50-kDaEXP / EF1-γ fusion protein (lane 8). Rows 1 and 7 in FIG. 14 represent vector control transfectant lysates, where no label was detectable. Anti-
A series of immunoprecipitation experiments using labeled antibody or control normal serum (NRS) (IP;
Rows 3-6 and 9-12) in Figure 14 were performed with co-transfected cell lysates. The interaction between Fez1 and EF1-γ is shown in columns 4, 6 of FIG.
, 10 and 12, as indicated by precipitation of apparently common bands by anti-Fez1, anti-EF1-γ and anti-V5 antibodies.

Example 5 Preparation of Antibodies that Specifically Bind Fez1 Protein A rabbit polyclonal antibody that specifically binds human Fez1 was developed.
The binding specificity of the polyclonal antibody for the Fez1 protein was demonstrated as follows. FEX1 cDNA is a GST-fusion expression vector (pGEX, Pharm
sia) and the protein is E. It was expressed in E. coli and purified. The Fez1-GST fusion protein was inoculated into rabbits to generate anti-Fez1 antibody, which was recovered according to standard methods.

FIG. 13A shows the results of immunoblot analysis performed with a polyclonal anti-Fez1 antibody. Human brain (rows 1 and 4), testis (rows 2 and 5) and spleen (row 3)
And about 100 (lanes 1-3) or 50 (lanes 4-6) μ obtained from 6 and 6)
g was blotted onto the surface. A relatively long exposure of the film showed faint expression of Fez1 in the testis and spleen. Row 7 contained the in vitro translated full length Fez1 protein and row 8 contained the in vitro translated truncated Fez1 protein (ie, lacking the C-terminal portion).

FIG. 13B shows the results of an immunoprecipitation assay performed with a polyclonal anti-Fez1 antibody. HeLaS3 cells that do not express FEZ1 were transfected with the FEZ1 cDNA in which the V5 labeling sequence was ligated into the expression vector pcDNA (Invitrogen) in frame. Cells were lysed and lysates were immunoprecipitated with polyclonal anti-Fez1 antibody (lane 1) or preimmune normal rabbit serum (lane 2). The precipitate was blotted and probed with anti-V5 labeled antibody.

Standard methods were used to construct one or more monoclonal antibodies that specifically bind the Fez1 protein.

Example 6 Post-Translationally Modified Fez1 Protein MCF7 clone 54 cells were transformed into aphidicolin.
And cultured in tetracycline-free medium with or without 10% (v / v) FBS to synchronize the cell cycle. At selected time points, the medium was replaced with aphidicolin-free medium containing 10% serum and the cells
Incubated for the time period indicated in A and 16B. After culturing, cell lysates were obtained and the lysates were subjected to immunoblot analysis with rabbit anti-Fez1 polyclonal antibody or anti-actin monoclonal antibody. The results of this experiment demonstrated that the cellular Fez1 protein is post-translationally modified in a manner that depends on cell cycle progression.

Fetal kidney 293 cells (expressing FEZ1 ) were maintained in serum-free medium containing aphidicolin and were cell cycle synchronized. At the selected time, the medium was 10% (
v / v) was replaced with aphidicolin-free medium containing FBS, and the cells were incubated for the times indicated in Figure 17, after which the proteins of the cultured cells were extracted.
The extracted proteins were subjected to immunoblot analysis with rabbit anti-Fez1 polyclonal antibody or anti-actin monoclonal antibody. The results of this experiment are shown in Figure 17, and the MCF7 / Fez1 transfectant lysates used in the experiment whose results are shown in Figure 16A were separated by SDS-PAGE in the presence of 6M urea. Under these separation conditions, the only band corresponding to the Fez1 protein was observed. Treatment of the same lysate with alkaline phosphatase (AP)
It resulted in the formation of the only band corresponding to FEZ1 in the SDS-PAGE separation. Treatment of lysates with AP inhibitor, β-glycerophosphate or control did not result in single band formation.

Cells of phosphorylated MCF7 clone 54 in vivo that are dependent on cell cycle progression of Fez1 were cultured for 2 days in medium containing 2% FBS and aphidicolin to synchronize the cell cycle in G1 / S. . At selected time points, the medium was replaced with aphidicolin-free medium containing 10% serum. Cells
Harvested at selected time points 0-8 hours after medium replacement and cells lysed,
The protein from it was extracted. The protein was immunoprecipitated using a rabbit anti-Fez1 polyclonal antibody, and the precipitated protein was SDS-PAGE.
Separated by Separated proteins were blotted onto the surface and labeled with anti-phosphoserine antibody (Sigma Chemical Co., St.
Louis, MO; lanes 1-5 in Figure 20 or labeled rabbit anti-Fez.
One of the polyclonal antibodies (lanes 6-10 in FIG. 20) was bound.
The results of this experiment demonstrate the cell cycle dependence of Fez1 phosphorylation.

Example 7 Subcellular Localization of Fez1 Protein Cytoplasmic and nuclear protein samples were prepared as follows. Cytosolic and nuclear proteins have been slightly modified as previously described (DNA 7: 47-55, 1998.
). Briefly, about 10 ⁷ 293 cells were harvested, P
It was washed with BS (10 mM NaPO ₄ pH 7.4, 150 mM NaCl). After sedimenting the cells, the packed cell volume (PCV) was measured and the cells were
CV freshly prepared hypotonic buffer (10 mM HEPES pH 7.9,0.
75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.1
resuspended in mMEGTA, 1 mM DTT, 10 mM KCl). 10 cells
Swelled at about 0 ° C for minutes and centrifuged at 300xg for 10 minutes at 4 ° C. The supernatant was collected as cytoplasmic extract I (C1).

The pellet was resuspended with 2.9 PCV of hypotonic buffer. The cells are Dou
It was disrupted by 10 strokes using an nce homogenizer (Kontes Glass Co.). One volume of sucrose recovery buffer (prepared by adding 9 volumes of 75% sucrose to 1 volume of 10x salt) was added and homogenized with an additional 10 sucrose in the homogenizer. The composition of 10x salt is as follows: 500 mM HEPES pH 7.9, 7.5 mM spermidine, 1.5 mM spermine, 100 mM KCl, 2 mM EDTA, 10 m.
MDTT. The homogenate was added to the Sorvall HB-4 rotor at 10,
It was centrifuged at 000 rpm (16,000 xg) for 30 seconds at 4 ° C. The supernatant was collected as cytoplasmic extract II (C2).

The pellet was resuspended in nuclear resuspension buffer with approximately 3 ml per 10 ⁹ cells. The nuclear resuspension buffer is 20 mM HEPES pH 7.9, 0.75
mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mMEG
Includes 9 volumes of TA, 2 mM DTT, 25% (v / v) glycerol and 1 volume of ammonium sulfate (4 ° C.) saturated solution. The resuspended pellet was incubated at 4 ° C for about 30 minutes with occasional rocking. The extract is 120 at 150,000 xg
Spun by centrifugation at 4 ° C for min. The supernatant was discarded, solid ammonium sulphate (0.33 g / ml of supernatant) was added, and the sample was incubated for 20 minutes with occasional rocking after dissolution of the ammonium sulphate. The sample was centrifuged at 85,000 xg for 20 minutes at 4 ° C. The pellet was lysed in nuclear dialysis buffer with 1 ml per 10 ⁹ cells and dialyzed overnight. Nuclear dialysis buffer was 20 mM HEPES pH 7.9, 20% (v / v) glycerol, 1
Contains 00 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 2 mM DTT. Nuclear extract (N) was stored at -80 ° C.

40 mg each of protein extracts C1, C2 and N by each method was SDS-P
Separated by AGE, transferred to membrane, and rabbit polyclonal anti-Fez1 antibody (
Columns 1-3 of FIG. 21) or anti-tubulin antibody (Santa Crutz Bi).
technology, Santa Crutz, CA; columns 4-6 in FIG. 21).
Was probed with either of. The results of these experiments demonstrate that the Fez1 protein fraction appears to reside in the nucleus, whereas the Fez1 protein is predominantly localized in the cytoplasm.

Example 8 Interaction of Fez1 with Microtubules The cytoplasmic protein fraction was incubated with paclitaxel to polymerize tubulin, Fez1-expressing 293 cells (“T” in FIG. 22).
ax ") and incubated for colchicine for depolymerization (ie, as a control) Fez1-expressing 293 cells (""in Figure 22).
Col "). 293 cells in 3 groups: unsynchronized cells (“untreated” in FIG. 22), G1 / S-synchronized cells (“0 hour” in FIG. 22) and S to G2 /
Selected from M-synch cells ("8 hours" in Figure 22). The protein fraction is
Centrifuged in the presence of a sucrose cushion as previously described (J. Cell Biol. 131: 1015-1024, 1995). The pelleted protein was subjected to immunoblot analysis using a rabbit polyclonal anti-Fez1 antibody. Proteins remaining in the supernatant (“Sup” in FIG. 22)
) Was also immunoblotted. The lower part of FIG. 22 illustrates the presence of tubulin in all tested samples. The results of these experiments demonstrate the in vivo interaction of Fez1 with microtubules and the deep involvement of Fez1 in tubulin polymerization.

[0328] GST- fusion FEZ1 proteins, in vitro tubulin polymerization tubulin and microtubules are implications deep purification of - associated protein MAP2 is,-GST, & GST- fusion FEZ1, & GST- fusion Mutant (29Ser → Pro) Fez1, PKA-phosphorylated GST-fused Fez1, and PKA-phosphorylated GST-fused mutant (29Ser → Pro) Fez1: were incubated at 37 ° C. for 0-40 minutes. Tubulin polymerization was examined by spectrophotometric measurement of absorbance increase at 350 nm, which is known to occur with polymerization. The results of this experiment are Fez1
It demonstrates that proteins can inhibit tubulin polymerization. The inhibitory effect of Fez1 protein on tubulin polymerization is modulated by the phosphorylation state of Fez1 as shown by the effect of PKA-mediated phosphorylation of Fez1 on tubulin polymerization in vitro.

Example 9 Proposed Biological Function for Fez1 Protein The properties described herein for the Fez1 protein and the nucleic acids that encode it, the physiological function of the Fez1 protein, are presented in this example. It is recognized that it does not depend on the accuracy and reliability of any theory. Thus, without being bound by any particular theory of operation, we have found that Fez1
We propose the following biological functions for proteins.

Immunoblot analysis of extracts obtained from cells expressing FEZ1 showed Fez
It illustrates that the 1 protein is significantly localized in the cytoplasm but is also found in the nucleus. A two-hybrid screen in yeast demonstrates that at least one peptide elongation factor (EF1-γ) is a potential binding partner for the Fez1 protein. Other investigators have found that the EF family of proteins not only functions as peptide chain extenders, but is also involved in interactions between microtubules and in the process of tubulin polymerization (eg, Eur. J. Bi
ochem. 171: 119, 1988; Proc. Natl. Acad. Sc
i. USA 90: 3028, 1993; Plant Cell 6: 893.
1994; Cell Motil. Cytoskel. 41: 168, 1998
,reference). Other researchers have shown that the EF protein can determine the susceptibility of cells to transformation (see, eg, Nature 359: 24, 1992).
. Overexpression of the EF protein has been observed in gastric, esophageal and colon cancers (eg Cancer 75: 1446, 1995; Gut, 38:66,
1996; Cancer 82: 816, 1998). The results of the experiments presented in this application demonstrate the interaction between the Fez1 protein and microtubules and their replacement proteins. For example, it was determined that Fez1 associates with tubulin precipitates when cell extracts from Fez1-expressing cells were incubated with paclitaxel to induce tubulin polymerization. However, it was determined that Fez1 does not associate with the depolymerized microtubule sediment in the presence of the tubulin polymerization inhibitor colchicine.

The data presented in this application indicate that the Fez1 protein acts to modulate microtubule polymerization and stability, and possibly other cytoskeletal structures. Thus, the Fez1 protein can be expected to be involved in cellular processes that are modulated by cytoskeletal stability and alterations. Examples of such cellular processes include initiation of mitosis, modulation of rate and stage of mitosis, modulation of initiation and rate of cell proliferation and growth, modulation of cell shape and hardness, modulation of cell motility, cell D
Modulation of NA replication rate and stage, modulation of intracellular distribution of organelles (eg, mitochondria, endoplasmic reticulum, Golgi apparatus, chloroplasts, etc.),
It includes modulation of the metastatic potential of cells, as well as modulation of transformation of cells from a non-cancerous to a cancerous phenotype.

For example, higher eukaryotic cell division is initiated according to a kinetic process that requires the so-called mitotic apparatus (organic complex of proteins) to distribute replicated chromosomes to daughter cells, And is known to be regulated (eg Nur
Se, 1990, Nature 344: 503-508). The elongated microtubule cytoskeleton of interphase cells is broken down into tubulin subunits and, where appropriate points in the cell cycle occur, the tubulin subunit functions as a central part of the mitotic machinery. Reassembled into two sets of polarized spindle tubes. When spindle tube nucleation occurred, the growing tubules attached to the ends of fused chromosomes. At the other end, tubules bind or adhere to a collection of proteins called the centrosome or organelle center of microtubules. Centrosome complexes have been isolated by other investigators (see, for example, Telzer, 1979, J. Cell Bio.
l. 81: 484-497; Mitchison, 1984, Nature 3
12: 232-237), and earlier reports characterized soluble protein precursors in centrosomes. Centrosomes contain α-, β- and γ-tubulin, heat shock protein 70 and elongation factor protein (Eur. J. Bioc.
hem. 171: 119, 1988; Proc. Natl. Acad. Sci.
USA 90: 3028, 1993; Plant Cell 6: 893, 19
94; Cell Motil. Cytoskel. 41: 168, 1998).

[0333] As the normal cycle of cell division progresses, both microtubule disassembly and reassortment occur. Thus, certain gene products or agents that target microtubules or their subunits can be used to modulate progression through the cell cycle. Tubulin is a known anticancer drug such as paclitaxel (which can induce tubulin polymerization) and vinca ( Vinca , Periwinkle) alkaloid (which can inhibit the polymerization process; Med. Res. Rev. 18: 259-296, 1998). It is the target of sex drugs. Other known tumor suppressor genes have been shown to be involved in microtubule assembly and degradation kinetics. For example, APC can promote microtubule assembly (Eur. J. Biochem. 253: 591,199.
8; Cancer Res. 54: 3672, 1994). Fhit can induce microtubule assembly (J. Biol. Chem. 274: 34, 1999). As illustrated herein, Fez1 can inhibit tubulin polymerization.
As illustrated here, Fez1 binds to at least one EF protein, and since these proteins have been identified as soluble protein components from centrosomes, Fez1 is a process of cell division or centrosome dynamics. It can be expected to have a role in late events. This is consistent with the finding herein that the Fez1 protein induces cell accumulation in the late S-G2 / M stages of the cell cycle. At these stages, centrosomes are undergoing assembly in daughter cells.

The experiments described herein demonstrate that at least two ways in which the activity of Fez1 can be affected, namely by phosphorylation of the Fez1 protein and by binding a polypeptide or polypeptide-like molecule to the Fez1 protein. It illustrates the method by. The results presented here demonstrate that phosphorylation of Fez1 by PKA can reduce the ability of Fez1 to inhibit tubulin polymerization.

Agents that directly phosphorylate Fez1 or that induce its phosphorylation by other proteins or inhibit its dephosphorylation include tubulin polymerization, as well as tubulin or tubulin-like proteins. It is useful for reducing the ability of Fez1 to inhibit the corresponding growth / contraction and maintenance of cytoskeletal structures (eg microtubules). Agents that directly dephosphorylate Fez1, or agents that induce its dephosphorylation or inhibit its phosphorylation by other proteins, are associated with polymerization and cytoskeletal structures containing tubulin or tubulin-like proteins. Fez1 inhibits growth / contraction and maintenance
It is useful for enhancing the ability of.

Agents that can specifically bind to Fez1 protein can also modulate its physiological activity. Examples of such agents are antibodies raised against Fez1 protein, tubulin and EF1-γ. Such proteins, such as the Fc portion of an antibody or the EF1-γ (N) fragment described herein, have F similar to the effects of whole protein on the Fez1 protein.
The effect on ez1 protein can be demonstrated. Similarly, a peptide or peptidomimetic compound that mimics the structure of a portion of a protein that specifically binds to Fez1 protein can exhibit an effect on Fez1 protein that is similar to that of the corresponding whole protein on Fez1 protein. We have found that a number of methods known in the art include libraries of compounds that are structurally similar to proteins that bind specifically to Fez1 protein (eg, Fez1).
It is recognized that peptides or peptidomimetic compounds) that are structurally similar to one or more portions of tubulin, EF1-γ or antibodies that specifically bind proteins can be constructed and used to screen. Furthermore, Fez
The observation in the present inventors that one protein appears to form a dimer or a multimer shows that a compound which is the same as the structure of a part of Fez1 involved in the dimerization or the multimerization, or a compound which mimics the same is observed. , Also show that it can be used to modulate the physiological activity of Fez1. Thus, methods for constructing and screening libraries of compounds that are identical or structurally similar to dimerization / multimerization domains (eg, libraries containing random fragments of the Fez1 protein) are described in Fez1. It can be used to identify compounds that modulate physiological activity.

The disclosures of all patents, patent specifications and publications cited herein are fully incorporated by reference.

Although the present invention has been disclosed in terms of particular embodiments, other embodiments and variations of the invention are known to those skilled in the art without departing from the true spirit and scope of the invention. It is clear that a person can devise it. The accompanying claims include all such embodiments and equivalent variations.

[Sequence list]

[Brief description of drawings]

1 comprises an invention, FIGS. 1A, 1B and 1C, and each of these figures relates to loss of heterozygosity (LOH) at human chromosome 8p in a primary esophageal cancer tissue sample. FIG. 1A, which comprises FIGS. 1Ai-1Aviii, shows a series of representative LOs obtained using tissue samples obtained from two patients designated E26 and E46.
It is a result of H analysis. 1Ai, 1Aiii, 1Av and 1Avii are patient E
26 depicts results from tissues obtained from 26. Figures 1Aii, 1Aiv, 1Avi and 1Aviii depict results from tissues obtained from patient E46. In each figure, D obtained from normal (N) and tumor (T) tissue samples from corresponding patients
Amplification of NA generated fluorescent PCR products and the products were separated by size. For each tracing, the horizontal axis represents the size of the DNA fragment and the vertical axis (ie peak height) represents the relative amount of each fragment. 1Ai and 1Aii correspond to D8S264; FIGS. 1Aiii and 1Ai
v corresponds to LPL; FIGS. 1Av and 1Avi correspond to D8S136; and FIGS. 1Avii and 1Aviii correspond to FGFR1. The size of some fragments (base pairs) are shown. FIG. 1B is a diagram depicting a summary of the LOH analysis described herein. Results for each patient displaying LOH at least one locus are shown. Black circles represent loss of allele. Circles containing × are not information based on the heterozygosity of the corresponding locus (
non-informative) result is shown. Open circles represent the retention of both alleles. The shaded area in the figure represents the area of allelic loss. Areas with fine parallel lines represent the non-informative resulting areas within the allelic loss region. The numbers at the top of each column refer to individual patients. The designation beside each row refers to the polymorphic marker. Surrounding the region near the marker D8S261 locus described herein. FIG. 1C is a diagram depicting the approximate position of the 8p22 genomic contig constructed as described herein. The top line depicts the position of the polymorphic locus on 8p. YAC contig (white square) and BAC
The corresponding position of the contig (horizontal line) is shown below the 8p map. CDNA selection and shotgun sequencing was performed on YACs and BACs identified by *. 87 potentially expressed sequences were isolated and located within the contig; the approximate position of these sequences is indicated by the designation under the double-headed arrow. Underlined letters indicate sequences expressed in normal tissue. Following expression analysis in tumor and normal tissues, the 9 cDNAs (circled designations) were subjected to further analysis. Candidate fragment e37 is F3 as described herein.
Corresponds to 7 cDNA.

FIG. 2 comprises FIGS. 2A, 2B, 2C and 2D according to the invention. Predicted Fe
The amino acid sequence of z1 (SEQ ID NO: 4) is depicted in Figure 2A. Figure 2A shows the FEZ1 cD
The predicted amino acid sequence of the FEZ1 protein as it arises from NA is listed. The underlined amino acid residues represent the region of homology to the DNA binding domain of the ATF-5 protein. The double underlined amino acid residue represents the leucine zipper motif, indicating the repeated leucine residues. The heavily underlined amino acid residues are cAMP / cGMP-dependent kinases (serine residue 2
9) or a residue that can be phosphorylated by either a tyrosine kinase-dependent kinase (tyrosine residue 67). The area under the dotted line represents the area with the relevant amino acid sequence motif. Serine and threonine residues in the thick or thin dotted lines represent potential casein kinase II and protein kinase C phosphorylation sites, respectively. Triangles indicate exon boundaries. * Indicates a missense or nonsense mutation site. In FIG. 2B, the predicted amino acid sequence of a region of Fez1 (amino acid residues 301-369; SEQ ID NO: 6) corresponding to the predicted DNA binding region and leucine zipper region is shown in FIG. The regions are compared (SEQ ID NOS: 7 and 8, respectively). Identical amino acid residues are shown by the dotted box, and similar amino acid residues are shown by the solid box. Gaps introduced by the FASTA program are represented by "-". Bullets are used to indicate repeated leucine residues. FIG. 2C is an image of SDS-PAGE results as described elsewhere herein. FIG. 2D is an image showing the results of Northern blot analysis showing FEZ1 gene expression in normal tissues. In the above figure, the FEZ1 ORF probe (SEQ ID NO: 3) was used to detect FEZ1 expression. In the figure below, β-actin probe was used as a control to detect β-actin gene expression. The arrow on the left in the upper figure indicates the approximate position of the 6.8 kilobase FEZ1 transcript. Poly (A) ⁺ RNA (5 micrograms) was obtained from normal (ie non-cancerous) tissue and loaded as follows: lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; Lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas; lane 9, spleen; lane 10, thymus; lane 11, prostate; lane 12, testis; lane 13, ovary; lane 14, small intestine. Lane 15, colon; and Lane 16, peripheral blood leukocytes.

[Figure 3]   3A, 3B and 3C according to the invention and in a tumor cellF EZ1 Regarding gene changes.   FIG. 3A shows that in cancer cellsFEZ1Results of Northern blot analysis of gene expression
Is an image of.FEZ1  cDNA probe (upper figure) and β-actin pro
Probe (below) was used to detect expression of the corresponding gene. The arrow on the left side of the above figure FEZ1 The approximate position of the 6.8 kilobase transcript of is shown. Poly (A)⁺  RNA
(5 micrograms) were obtained from tumor cell lines and loaded as follows: esophagus
Cancer cell lines KYSE170 (lane 1), TE12 (lane 2), TE8 (lane
3) and TE3 (lane 4); prostate cancer cell line DU145 (lane 5), LN
CaP (lane 6), PC3 (lane 7); normal prostate (lane 8); breast cancer cells
Cell line MB231 (lane 9), SKBr3 (lane 10), BT549 (lane
11), HBL100 (lane 12), MB436S (lane 13), BT20
(Lane 14), MB543 (lane 15), MB175 (lane 16), MC
F7 (lane 17) and T47B (lane 18); normal breast (lane 19)
Normal breast total RNA (lane 20); cervical cancer cell line HeLa S3 (lane
22); Chronic myelogenous leukemia cell line K562 (lane 23); lymphoblastic leukemia
Cell line MOLT4 (lane 24); Burkitt lymphoma cell line Raji (lane
25); colorectal adenocarcinoma cell line SW480 (lane 26); lung cancer cell line A549 (
Lane 27); as well as the melanoma cell line G361 (lane 28). Total RNA (5
Was obtained from the promyelocytic leukemia cell line HL60 and in lane 21
Loaded   FIG. 3B comprising FIGS. 3Bi-3Bvi was mutated.FEZ1Gene
Obtained from 3 individuals withFEZ1Is a series of sequence chromatograms of genes
It As shown in FIG. 3Bii, a point mutation in FEZ1 at codon 29 (T
CC / Ser → CCC / Pro) is the primary esophageal cancer tissue support obtained from patient E44.
Identified in the sample. Nucleotide sequence from normal DNA from patient E44 (
N) and the nucleotide sequences from BAC contig (B) are shown for comparison.
The bold line overlaps the changed codon. Primary esophagus obtained from patient E50
In the cancer tissue sample, as shown in FIG. 3Biv,FEZ1Point mutation (
AAG / Lys → GAG / Glu) was detected at codon 119 and was found. Positive
The sequence chromatogram of the normal BAC is shown in Figure 3Biii. Figure 3Bvi (array black
The matogram is 3'to 5 ') and FEZ at codon 501
The third point mutation in 1 (CAG / Gln → TAG / termination) is a prostate cancer cell line P
It was identified by C3. Repeated sequencing shows that the first nucleotide of codon 501
One weak system corresponding to guanine (G) in the large adenine (A) signal at
Indicating that gnal is present and a small portion of the cancer cells carry the normal FEZ1 allele.
Was suggested.   Figure 3CFEZ1Draw the results of Southern blot analysis using gene loci
It is a statue. Restriction endonucleases from high molecular weight DNA from cancer cellsEcoR Cleavage using I, electrophoretic separation, transfer to nylon membrane, and 1
． 7 kilobasesFEZ1Probed with the ORF probe (SEQ ID NO: 3).
The DNA applied to each lane (10 micrograms per lane) is the following cells
Obtained from: Lane 1, cell line MB436S; Lane 2, obtained from first healthy individual
Normal placental cells; lane 3, cell line MB231; lane 4, cell line MB3
61; lane 5, cell line TE8; and lane 6, cell line TE3. Suitable for lane 7
The DNA used was isolated from normal placental cells obtained from a second healthy individual.

[Figure 4]   According to the invention, it comprises FIGS. 4A and 4B. FIG. 4A is described herein.
Shortened observed in such cancer cellsFEZ1It is a figure which draws a transcription material. normal
The exon / intron structure is shown on the top line of the figure and
In non-cancerous) brain, prostate and esophageal cDNA sequencing and BAC FEZ1 It was determined by sequencing the gene. Boxes represent exons; fine parallel
The lined region represents the open reading frame (1788 base pairs; SEQ ID NO: 3). Horizontal line
Represents the intron, and the filled circles are the observed bumps as described herein.
Represents a mutation. The enclosed notation "LZ" refers to the leucine zipper described herein.
Represents the approximate position of the motif. "FS" is the frame shift described herein
Represents the approximate position of. The abnormal transcripts observed in the tumor are shown below the top line in the figure.
Draw with a thick line on the line.   FIG. 4B shows a frame-shifted FE with a molecular weight of approximately 8.6 kilodaltons.
Putative amino acid sequence encoded by the Z1 transcript (SEQ ID NO: 6). The transfer
The amino acid residues encoded by the frame-shifted parts of the feature are underlined.
Has been.

Figure 5 is a series of nucleotide and amino acid sequences comprising Figures 5A-5Q according to the invention. Figure 5A lists the nucleotide sequence (SEQ ID NO: 1) of the portion of the human genome that comprises Figures 5Ai-5Avi and that comprises the FEZ1 gene. FIG. 5B lists the nucleotide sequence of the cDNA (SEQ ID NO: 2) comprising FIGS. 5Bi-5Biv and reflecting the nucleotide sequence of the full-length mRNA transcript of wild type FEZ1 . FIG. 5C lists the nucleotide sequence of the cDNA (SEQ ID NO: 9) that reflects the nucleotide sequence of the ORF region of the truncated (E16T8) FEZ1 mRNA transcribed by tumor cells. FIG. 5D is a nucleotide sequence of a cDNA (SEQ ID NO: 10) that reflects the nucleotide sequence of the ORF region of the truncated (E264162) FEZ1 mRNA transcribed by tumor cells.
To enumerate. FIG. 5E comprises ORFs of truncated (T8D145M4) FEZ1 mRNA comprising FIGS. 5Ei and 5Eii and transcribed by tumor cells.
The nucleotide sequence of the cDNA (SEQ ID NO: 11) that reflects the nucleotide sequence of the region
) Are listed. Figure 5F comprises Figures 5Fi and 5Fii and lists the nucleotide sequence of the cDNA (SEQ ID NO: 12) that reflects the nucleotide sequence of the ORF region of the truncated (D14) FEZ1 mRNA transcribed by tumor cells. . FIG. 5G, which comprises FIGS. 5Gi and 5Gii, and lists the nucleotide sequence of the cDNA (SEQ ID NO: 13) that reflects the nucleotide sequence of the ORF region of the truncated (G3611) FEZ1 mRNA transcribed by tumor cells. . Figure 5H lists the nucleotide sequence of the cDNA (SEQ ID NO: 14) that comprises Figures 5Hi and 5Hii and reflects the nucleotide sequence of the ORF region of the truncated (G3612) FEZ1 mRNA transcribed by tumor cells. . FIG. 5I comprises FIGS. 5Ii and 5Iii and is the wild-type FEZ1 mR.
Nucleotide sequence of cDNA reflecting the nucleotide sequence of the ORF region of NA (
SEQ ID NO: 3) is listed. Figure 5J comprises Figures 5Ji-5Jv and lists the amino acid sequence of the full-length human wild-type Fez1 protein (SEQ ID NO: 4). FIG. 5K lists the amino acid sequence of the truncated (E16T8) Fez1 protein expressed by tumor cells (SEQ ID NO: 15). FIG. 5L comprises FIGS. 5Li and 5Lii and is truncated by tumor cells (E26
4162) Lists the amino acid sequence of Fez1 protein (SEQ ID NO: 16). FIG. 5M comprises the amino acid sequence of the truncated (T8D145M4) Fez1 protein comprising FIG. 5Mi-5Miv and expressed by tumor cells (SEQ ID NO: 1).
List 7). FIG. 5N lists the amino acid sequence of the truncated (D14) Fez1 protein (SEQ ID NO: 18) comprising FIGS. 5Ni-5Niv and expressed by tumor cells. FIG. 5O lists the amino acid sequence (SEQ ID NO: 19) of the truncated (G3611) Fez1 protein comprising FIGS. 5Oi-5Ov and expressed by tumor cells. Figure 5P lists the amino acid sequence of the truncated (G3612) Fez1 protein (SEQ ID NO: 20) comprising Figure 5Pi-5Pv and expressed by tumor cells. Figure 5Q lists the nucleotide sequence of the F37 probe described herein (SEQ ID NO: 21).

FIG. 6 shows an inventive MCF7 cell line that has been transfected with a pTet-Off ™ vector alone (“control”) or a vector having at least a coding portion of the FEZ1 gene operably linked to its promoter. Image of immunoblots of proteins isolated from clones (clones 118, 54, 18 and 15). The protein is the presence of tetracycline (“+”)
) Or from cells that were maintained in the absence ("-").

FIG. 7 comprises the inventions of FIGS. 7A (clone 15), 7B (clone 54), 7C (clone 18) and 7D (clone 118), 10% (◯), 5% (
●), 2.5% (□), 1% (■) or 0.5% (▲) (v / v) fetal bovine serum-containing control cells of cells maintained in tetracycline-free medium. FIG. 6 is a set of four graphs showing the time dependence of the ratio of the number of transfected MCF7 clone cells to the number.

FIG. 8 is a ratio of the number of transfected MCF7 clonal cells according to the invention, comprising FIGS. 8A and 8B, which were in the G2 cell cycle with respect to the number of M phases (FIG. 8A).
; G2 / M), or the ratio of the number of cells in S phase to the number in G1 phase (FIG. 8).
B; a pair of graphs showing S / G1). In these figures, the solid line corresponds to clone 18 and the dashed line corresponds to clone 54. Open circles correspond to the ratio in the presence of tetracycline (ie FEZ1 non-expression) and open circles correspond to the ratio in the absence of tetracycline (ie FEZ1 expression).

FIG. 9 is a representation of the invention comprising FIGS. 9A and 9B, wherein about 5 × 10 ⁶ (FIG. 9A) or about 2 × 10 ⁷ (FIG. 9B) MCF7 cells transfected with vector alone ( ○), transfected MCF7 clone 15 cells (●), transfected MCF7 clone 18 cells (■), transfected MCF7 clone 56 cells (□) or transfected MCF7 clone 118 cells (⋄). 3 is a pair of graphs showing the time dependence of tumor volume in nude mice transplanted with.

10 includes pQBI-AdCMV5-I, comprising FIGS. 10A-10F, in accordance with the invention.
The nucleotide sequence of RES-GFP (SEQ ID NO: 60) is listed.

FIG. 11 is a triplicate image of the results of an in vitro binding assay demonstrating binding between ³⁵ S-methionine labeled EF1-γ and Fez1 proteins, comprising FIGS. 11A, 11B and 11C according to the invention. is there.

FIG. 12: In vitro evidence of binding between ³⁵ S-methionine labeled EF1-γ (N) and Fez1 protein and between EF1-γ (N) and truncated Fez1 protein according to the invention. It is an image of the result of the binding assay.

FIG. 13 is an image of the results of an in vitro binding assay demonstrating Fez1 protein dimerization and truncated Fez1 protein dimerization according to the invention.

Figure 14 comprises an invention, comprising Figures 14A, 14B, 14C and 14D, V5 /
FIG. 4 is a series of four images depicting the results of immunoblotting experiments involving HeLaS3 cells cotransfected with a vector encoding a Fez1 fusion protein and a vector encoding an EXP / EF1-γ fusion protein.

FIG. 15 is a pair of images of the results of an immunoblotting experiment, comprising FIGS. 15A and 15B, according to the invention.

FIG. 16: Concurrency of the invention, comprising FIGS. 16A and 16B, using an antibody that specifically binds Fez1 (“Fez1”) and an antibody that specifically binds actin (“Actin”). 2A is a pair of images of the results of an immunoblotting experiment on transfected MCF7 cells that had been challenged. The numbers above the columns indicate the time elapsed after aphidicolin treatment. Proteins immunoblotted in the experiment corresponding to Figure 16A were obtained from transfected MCF7 cells maintained in the presence of 10% (v / v) FBS, and immunoblotted in the experiment corresponding to Figure 16B. Proteins were obtained from transfected MCF7 cells maintained in the absence of FBS.

FIG. 17 is an image of the results of an immunoblotting experiment involving a protein extracted from fetal kidney 293 cells subjected to cell cycle synchrony according to the invention.

FIG. 18: SDS-P of proteins obtained from cells corresponding to FIG. 16A according to the invention.
It is an image of the result of AGE separation. The cells corresponding to lanes 1, 2, 3 and 4 in Figure 18 correspond to the lanes designated 0, 1.5, 5 and 9 in Figure 16A.

FIG. 19 shows the results of an immunoblotting experiment in which the cell lysate used in the experiment corresponding to FIG. 16A according to the invention was contacted with alkaline phosphatase (lane 1), β-glycerophosphate (lane 2) or a control. It is a statue.

FIG. 20: 0 (lanes 1 and 6) to 8 hours after arrest of cell cycle inhibition according to the invention (
Lanes 5 and 10) were obtained from transfected MCF7 cells that were synchronized with increasing time and anti-Fez1 polyclonal antibody (lanes 6-10) or anti-phosphoserine antibody (lanes 1-5). 3) is an image of Fez1 protein separated by SDS-PAGE, which was immunoblotted with any of the above.

FIG. 21: Cytoplasmic (“C1” and “C2”) and nuclear (“N”) protein extracts obtained from 293 cells according to the invention were labeled with a polyclonal anti-Fez1 antibody (“F”).
ez1 ”) or an anti-tubulin antibody (“ tubulin ”).

FIG. 22: Paclitaxel (“Tax”) or colchicine (“Col” according to the invention).
Of the results of an experiment using a polyclonal antibody to detect the Fez1 protein "Fez1" in extracts obtained from spun down cell structures in synchronized cells incubated with It is a statue.

FIG. 23 is a graph showing the effect of Fez1 protein on the inhibition of tubulin multimerization in the presence of MAP2 protein according to the invention. The reaction mixture is in addition to the reaction buffer: none (open circles); tubulin (open diamonds): tubulin and MAP2 (open squares); tubulin, MAP2 and GST (diamonds including x); tubulin, MAP2 And Fez1 fused to GST (filled circles); tubulin, MAP2, mutated fused to GST (29 Ser Pr
o) Fez1 (filled squares); Fuz1 fused to GST phosphorylated with tubulin, MAP2 and PKA (circles containing x); and tubulin, M
Mutated fused to GST phosphorylated at AP2 and PKA (29
Ser Pro) Fez1 (square containing x) was included. "PKA" is protein kinase A, a 3 ': 5'-monophosphate dependent protein kinase.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 C07K 14/82 4C087 A61P 35/00 16/32 4H045 C07K 14/82 C12Q 1/02 16 / 32 1/68 A C12N 5/10 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 33/574 Z 37/00 102 33/566 C12N 15/00 ZNAA 33/574 5/00 B 37/00 102 A61K 37/02 F term (reference) 2G045 AA40 DA12 DA13 DA36 FB02 FB03 4B024 AA01 AA12 BA36 CA03 CA09 EA02 FA02 HA14 HA17 4B063 QA01 QA08 QA18 QA19 QQ08 QQ43 QQ60 QQ79 QR32 QR48 QR55 QR62 QR77 QR82 QS25 QS34 4B065 AA93X AA93Y AB01 CA24 CA44 CA46 4C084 AA02 AA06 AA07 AA13 AA17 BA22 BA35 CA53 CA56 MA52 MA55 MA66 NA14 ZB261 A4A0A AA93A52 55 MA66 NA14 ZB26 ZC78 4H045 AA10 AA11 AA30 BA10 CA41 DA75 DA86 EA28 EA51 FA74

Claims (98)

[Claims] 1. An isolated polynucleotide comprising a portion that anneals with high stringency to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. 2. The human FEZ1 gene has the nucleotide sequence of SEQ ID NO: 1.
The isolated polynucleotide of claim 1. 3. A portion of at least 30 of at least one strand of the human FEZ1 gene.
The isolated polynucleotide of claim 1, which anneals with high stringency to a contiguous number of nucleotide residues. 4. The isolated polynucleotide of claim 1, wherein the portion is substantially homologous to at least 20 consecutive nucleotide residues of the human FEZ1 gene. 5. A portion of at least 20 of at least one strand of the human FEZ1 gene.
The isolated polynucleotide of claim 4, which is completely homologous to 4 consecutive nucleotide residues. 6. A portion of at least 20 contiguous FEZ1 exon regions selected from the group consisting of nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550 of the chain of SEQ ID NO: 1. 5. The isolated polynucleotide of claim 4, which is substantially homologous to the nucleotide residue that 7. The isolated polynucleotide of claim 6, wherein the isolated polynucleotide comprises a portion having the nucleotide sequence of the strand of SEQ ID NO: 3. 8. The isolated polynucleotide further comprises a promoter.
The isolated polynucleotide according to claim 6. 9. The isolated polynucleotide of claim 8, wherein the promoter is a constitutive promoter. 10. The isolated polynucleotide of claim 8, wherein the promoter is an inducible promoter. 11. The isolated polynucleotide of claim 8, wherein the promoter is a tissue-specific promoter. 12. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide is incorporated into a nucleic acid vector. 13. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide is encoded by a nucleic acid incorporated into a nucleic acid vector. 14. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide has sequence homology with the strand of SEQ ID NO: 1. 15. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide is detectably labeled. 16. An isolated polynucleotide that is detectably labeled is an immobilized polynucleotide, a polynucleotide linked to a protein of a protein-ligand pair, a polynucleotide linked to a ligand of a protein-ligand pair, a biotinylated polynucleotide. 16. The isolated polynucleotide of claim 15, selected from the group consisting of a fluorophore-linked polynucleotide, a chromophore-linked polynucleotide, an enzyme-linked polynucleotide and a radio-labeled polynucleotide. 17. The isolated polynucleotide according to claim 16, wherein the immobilized polynucleotide is immobilized on the surface of the gene chip. 18. The isolated polynucleotide of claim 1, wherein the isolated polynucleotide is substantially purified. 19. The isolated polynucleotide of claim 1, wherein at least two nucleotide residues of the isolated polynucleotide are linked by a non-naturally occurring bond other than a phosphodiester bond. 20. Non-naturally occurring bonds are phosphonates, phosphorothioates, phosphorodithioates, phosphoramidates, methoxyethyl phosphoramidates, formacetals, thioformacetals, diisopropylsilyl,
Acetamidate, carbamate, dimethylene - sulfide _{(-CH 2 -S-CH 2)} , dimethylene - sulfoxide _{(-CH 2 -SO-CH 2)} , dimethylene - sulfone _{(-CH 2 -SO 2 -CH 2)} , 2 '-O-alkyl and 2'-deoxy-2'-fluorophosphorothioate, phosphorotriester, siloxane, carbonate, carboxymethyl ester, acetamidate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate 20. A single of claim 19 selected from the group consisting of: a bridged phosphoramidate, a bridged methylene phosphonate, a phosphorothioate, a methylphosphonate, a phosphorodithioate, a bridged phosphorothioate bond, a bridged sulfone bond and combinations of such bonds. Isolated polynucleotide. 21. The isolated polynucleotide of claim 1, wherein the end of the isolated polynucleotide is nucleolytically blocked. 22. An isolated polynucleotide comprising a portion having a sequence that anneals with high stringency to at least 20 consecutive nucleotide residues of the strand of SEQ ID NO: 3. 23. A kit for amplifying a portion of the human FEZ1 gene, which comprises:
The kit comprises a first isolated polynucleotide and a second isolated polynucleotide, wherein the first isolated polynucleotide anneals with high stringency to at least 20 contiguous nucleotide residues of the coding strand of SEQ ID NO: 1. Wherein the second isolated polynucleotide comprises at least 20 of the non-coding strand of SEQ ID NO: 1.
A kit as set forth above comprising a portion that anneals at high stringency to consecutive nucleotide residues. 24. A kit for amplifying a portion of a cDNA produced from a transcript of the human FEZ1 gene, the kit comprising a first isolated polynucleotide and a second isolated polynucleotide, wherein The portion of the first isolated polynucleotide anneals with high stringency to at least 20 contiguous nucleotide residues of the coding strand of SEQ ID NO: 1, and wherein the portion of the second isolated polynucleotide is the non-coding portion of SEQ ID NO: 1. A kit as described above, which anneals with high stringency to at least 20 consecutive nucleotide residues of a chain. 25. At least nucleotide residues 112 to 456 of the chain of SEQ ID NO: 1,
An animal cell comprising a foreign DNA molecule having a portion substantially homologous to nucleotide residues 1707-2510 and nucleotide residues 4912-5550. 26. The foreign DNA molecule further comprises a promoter operably linked to said portion, whereby the foreign DNA molecule is expressed in an animal cell.
The animal cell according to claim 25. 27. A genetically modified cell comprising a cell into which a foreign DNA molecule has been artificially introduced, the foreign DNA molecule having a portion substantially homologous to at least the coding region of the chain of the human FEZ1 gene. Animals. 28. The foreign DNA molecule comprises at least nucleotide residues 112-456, nucleotide residues 1707-2510 and nucleotide residues 4912-5550 of the strand of SEQ ID NO: 1.
28. The genetically modified animal of claim 27 having a portion substantially homologous thereto. 29. The genetically modified animal of claim 28, wherein the foreign DNA molecule comprises a portion having a sequence that is substantially homologous to the strand of SEQ ID NO: 2. 30. An isolated human Fez1 protein. 31. The isolated human Fez1 protein of claim 30, wherein the protein has an amino acid sequence that is substantially homologous to SEQ ID NO: 4. 32. The isolated human Fez1 protein of claim 30, wherein the protein has an amino acid sequence that is completely homologous to SEQ ID NO: 4. 33. The isolated human Fez1 protein of claim 30, wherein the protein is substantially purified. 34. An isolated antibody that specifically binds to human Fez1 protein. 35. A hybridoma cell producing an antibody that specifically binds to human Fez1 protein. 36. A method of determining a cancerous state in a sample tissue, comprising comparing FEZ1 expression in the sample tissue with FEZ1 expression in a control tissue of the same type,
The method as described above, wherein the decrease in the expression of FEZ1 in the sample tissue relative to the expression of FEZ1 in the control tissue is used as an indicator that the sample tissue has cancer. 37. The method of claim 36, wherein the sample tissue is a phenotypically abnormal portion of human body tissue and the control tissue is a phenotypically normal portion of body tissue. 38. The method of claim 37, wherein the body tissue is epithelial tissue. 39. The body tissue is selected from the group consisting of gastrointestinal tract tissue, esophageal tissue, gastric tissue, colon tissue, prostate tissue, breast tissue, hematopoietic tissue, lung tissue, melanoma tissue, cervical tissue and ovarian tissue. 38. The method of claim 37. By 40. comparing the relative amounts of the index of the sample tissue and control tissue, the FEZ1 expression sample tissue compared to FEZ1 expression in control tissue,
Index cDNA prepared using FEZ1 mRNA, FEZ1 mRNA, selected from the group consisting of the DNA and Fez1 protein preparation by amplification of any of these, according to claim 36
The method described in. 41. A method of determining the cancerous state of a sample tissue, comprising comparing the nucleotide sequence of a FEZ1 -associating polynucleotide obtained from the sample tissue and a nucleotide sequence of a control FEZ1 -associating polynucleotide. , Wherein the difference between the nucleotide sequence of the FEZ1 -associated polynucleotide obtained from the sample tissue and the nucleotide sequence of the control FEZ1 -associated polynucleotide is used as an indicator that the sample tissue is cancerous. 42. A method of determining the cancerous state of a human sample tissue, the method comprising:
The length of the FEZ1 -transcript-associated polynucleotide obtained from the sample tissue was compared to the control FEZ1-
Comprising comparing the length of the transcript-association polynucleotide, whereby the length of the FEZ1 -transcript-association polynucleotide obtained from the sample tissue is shorter than the length of the control FEZ1 -transcript-association polynucleotide. Then, the above method using this as an indicator that the sample tissue is cancerous. 43. A method of determining the cancerous state of a sample tissue, the method comprising:
In the organizationFEZ1Comprising assessing expression, which results in FEZ1 One in which the substantial absence of expression is an indicator that the sample tissue is cancerous
Law. 44. FEZ1 expression, FEZ1 mRNA, cDNA prepared using FEZ1 mRNA
, DNA prepared by amplification of any of these, and assessed by the presence or absence of an indicator selected from the group consisting of the Fez1 protein,
The method of claim 43. 45. A method of determining a cancerous state of a sample tissue, comprising detecting aberrant splicing of FEZ1 transcripts sample tissue, thereby sample organization aberrant splicing of FEZ1 transcript The above method using as an indicator that cancer is cancer. 46. aberrant splicing of FEZ1 transcripts, exon boundaries polynucleotide probe FEZ1 - transcript - is detected by assessing the ability to annealing at a meeting polynucleotide high stringency, where exon boundaries 46. The method of claim 45, wherein the polynucleotide probe is capable of annealing with high stringency to the end portions of two consecutive FEZ1 exons when the end portions are adjacent, but not when the end portions are not adjacent. 47. The use of an exogenous Fez1 protein to make an agent for modulating abnormal growth of human cells having a modified FEZ1 gene, the cell being provided when the agent is provided to the cell. The above uses in which abnormal growth of is suppressed, slowed or prevented. 48. The use according to claim 47, wherein the exogenous Fez1 protein is a composition comprising isolated human Fez1 protein. 49. The use according to claim 48, wherein the human Fez1 protein has the amino acid sequence of SEQ ID NO: 4. 50. An Fez1 protein of exogenous origin comprises an expression vector comprising a polynucleotide having a coding region encoding a functional Fez1 protein, whereby the polynucleotide is expressed in a cell. Use according to 47. 51. The use according to claim 50, wherein the polynucleotide comprises the human FEZ1 gene. 52. The use according to claim 50, wherein the coding region comprises a portion having the nucleotide sequence of the strand of SEQ ID NO: 3. 53. The use of claim 47, wherein the polynucleotide further comprises a constitutive promoter operably linked to the coding region. 54. The method of claim 47, wherein the polynucleotide further comprises an inducible promoter operably linked to the coding region, and the method further comprises administering to the cell an inducer of the inducible promoter. Use as described in. 55. The use of claim 47, wherein the polynucleotide further comprises a tissue-specific promoter operably linked to the coding region. 56. The use of claim 47, wherein the polynucleotide further comprises a wild type FEZ1 promoter region. 57. A functional Fez1 protein is encoded to produce an agent for suppressing tumorigenesis in human cells, which suppresses tumorigenesis in cells when the vector is provided to the cells. Use of an expression vector comprising a polynucleotide having a coding region. 58. Use of an inhibitor of Fez1 expression for the preparation of a medicament for inducing cell proliferation, which induces cell proliferation when the inhibitor is provided inside the cell. 59. The use according to claim 58, wherein the inhibitor is an isolated polynucleotide comprising a portion that anneals with high stringency to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. 60. The use according to claim 59, wherein the isolated polynucleotide is delivered intracellularly by administering to the cell a gene vector comprising a promoter operably linked to the isolated polynucleotide. . 61. The use according to claim 58, wherein the cells are located in the body of an animal. 62. The use according to claim 61, wherein the cells are human. 63. A method of determining whether a test compound is an inducer of cell proliferation, comprising incubating cells comprising a functional FEZ1 gene in the presence of the test compound and Comprising assessing FEZ1 expression, whereby FEZ1 expression in the cell is reduced when compared to FEZ1 expression in the same type of cells incubated in the absence of the test compound, The above method, wherein the method is an inducer of cell proliferation. 64. A method of determining whether a test compound is effective for slowing aberrant growth of cells having a modified FEZ1 gene, comprising incubating the cells in the presence of the test compound, And comprising assessing FEZ1 expression in the cell, whereby FEZ1 expression in the cell is increased relative to FEZ1 expression in the same type of cell incubated in the absence of the test compound. ,
The above method wherein the test compound is effective for slowing the abnormal growth of cells. 65. A method of determining whether a Fez1 protein binds to a polynucleotide having a test nucleotide sequence, wherein the Fez1 protein and the test polynucleotide having the test nucleotide sequence are contacted, wherein at least one Fez1 protein. And the test polynucleotide is detectably labeled, and subsequently forms a detectably labeled Fez1-polynucleotide complex.
The above method in which the Fez1 protein is used as an index for binding to the polynucleotide having the test nucleotide sequence by evaluating whether or not the complex is formed. 66. A method of identifying an inducer of cell proliferation, comprising contacting a Fez1 protein and a polynucleotide to which the Fez1 protein binds in the presence and absence of a test compound and forming a Fez1-polynucleotide complex. And the formation of the complex in the presence of the test compound is reduced relative to the formation of the Fez1-polynucleotide complex in the absence of the test compound, the test compound is The above method as an indicator of being an inducer. 67. A kit for selecting an anti-cancer therapeutic compound for administration to a human suffering from cancer, for assessing FEZ1 expression in a plurality of candidate anti-cancer therapeutic compounds and cells. The kit comprising the reagents of. 68. A method of inducing cell proliferation, comprising inhibiting FE Z1 expression in the cell, whereby cell proliferation is induced. 69. The method of claim 68, wherein the cells are removed from a human. 70. The method of claim 69, wherein the cell is reintroduced to the human after suppressing FEZ1 expression in the cell. 71. Expression of FEZ1 in a cell is at least that of a chain of the human FEZ1 gene.
7. Inhibited by providing to the interior of the cell an isolated polynucleotide comprising a moiety that anneals with high stringency to 20 consecutive nucleotide residues.
The method according to 8. When 72.] drug was administered to humans, tumorigenesis is inhibited in humans, to create a medicament for inhibiting tumorigenesis in a human, the FEZ1 gene expression inducer, the FEZ1 gene expression Selected from the group consisting of enhancers, inhibitors of Fez1 phosphorylation, enhancers of phosphorylation-Fez1 dephosphorylation, agents that inhibit the binding of Fez1 to EF1-γ and agents that inhibit the binding of Fez1 to tubulin. Use of compounds. 73. An enhanced human cell culture technique, which comprises incubating a human cell and suppressing FEZ1 expression in the cell according to known human cell culture techniques. 74. A method of detecting FEZ1 expression in a sample tissue, which comprises labeling an isolated antibody that specifically binds to human Fez1 protein, and contacting a preparation of the isolated antibody with the sample tissue, Rinse the tissue sample, thereby rinsing non-specifically bound antibody from the tissue sample, and assess the presence of labeled antibody in the tissue sample, thereby determining the presence of labeled antibody in the tissue sample in the tissue sample. The above method using FEZ1 as an indicator of expression. 75. A method for determining whether a test compound is useful for ameliorating a disorder associated with aberrant tubulin polymerization, the method comprising tubulin, Fez1 and a test compound. Comprising comparing tubulin polymerization in one assay mixture with tubulin polymerization in a second assay mixture comprising tubulin and Fez1 but no test compound, wherein the first and second assays The above method, wherein the difference between the tubulin polymerizations in the mixture is an indication that the test compound is useful in ameliorating the disorder. 76. The method of claim 75, wherein the disorder is a tubulin overpolymerization disorder. 77. The method of claim 75, wherein the disorder is a tubulin hypopolymerization disorder. 78. A disorder in which the disorder is associated with aberrant initiation of mitosis, a disorder associated with aberrant modulation of rates and stages of mitosis, a disorder associated with aberrant modulation of initiation and rate of cell proliferation, Disorders associated with aberrant modulation of cell growth initiation and velocity, disorders associated with aberrant modulation of cell shape, disorders associated with aberrant modulation of cell hardness, disorders associated with aberrant modulation of cell motility, cells Disorders associated with aberrant modulation of the rate of replicative DNA replication, disorders associated with aberrant modulation of the replication stage of cellular DNA, disorders associated with aberrant modulation of organelle intracellular distribution, and aberrant modulation of cell metastatic capacity. -Related disorders and cell shape from non-cancerous to cancerous phenotypes 77. The method of claim 75, selected from the group consisting of disorders associated with aberrant modulation of quality conversion. 79. The method of claim 75, wherein the disorder is selected from the group consisting of tumorigenesis, tumor survival, tumor growth and tumor metastasis. 80. The method of claim 75, wherein the difference is a difference in the rate of tubulin polymerization between the first and second assay mixtures. 81. The method of claim 75, wherein the difference is a difference in the degree of tubulin polymerization between the first and second assay mixtures. 82. The test compound is a fragment of Fez1, a peptide mimic of a fragment of Fez1, a fragment of tubulin, a peptide mimic of a fragment of tubulin, a fragment of EF1-γ and a fragment of EF1-γ. 76. The method of claim 75, selected from the group consisting of: 83. The first and second assay mixtures are substantially identical,
76. The method of claim 75, wherein the presence or absence of the test compound is not the same. 84. A method of determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1, comprising: Fez1, at least one kinase, a phosphate source and a test compound. Comprising comparing the phosphorylation of Fez1 in a first assay mixture comprising Fez1 with the phosphorylation of Fez1 in a second assay mixture comprising Fez1, a kinase and a phosphate source but no test compound. Wherein the difference in Fez1 phosphorylation between the first and second assay mixtures is an indicator that the test compound is useful in ameliorating the disorder. 85. The method of claim 84, wherein the disorder is selected from the group consisting of tumorigenesis, tumor survival, tumor growth and tumor metastasis. 86. A method of determining whether a test compound is useful for ameliorating a disorder associated with aberrant phosphorylation of Fez1, comprising phosphorylated Fez1, at least one phosphatase and a test compound. Phosphorylation of Fez1 in a first assay mixture consisting of and a second comprising phosphorylated Fez1 and a phosphatase but no test compound
Comprising comparing the phosphorylation of Fez1 in the assay mixture, wherein the difference in phosphorylation of Fez1 between the first and second assay mixtures serves as an indicator that the test compound is useful in ameliorating the disorder. The above method. 87. A method of determining whether a test compound is useful for ameliorating a disorder associated with aberrant binding of Fez1 and a protein to which Fez1 normally binds, comprising Fez1, the protein and the test. Fez1 in a first assay mixture comprising a compound
And binding between the proteins and binding between Fez1 and the protein in a second assay mixture comprising Fez1 and the protein but not the test compound, wherein the first and second assays are performed. The above method, wherein the difference in Fez1 and protein binding between the mixtures is an indicator that the test compound is useful in ameliorating the disorder. 88. The method of claim 87, wherein the protein is selected from the group consisting of tubulin and EF1-γ. 89. The method of claim 87, wherein the disorder is selected from the group consisting of tumorigenesis, tumor survival, tumor growth and tumor metastasis. 90. The nucleic acid vector is an adenovirus vector.
2. The isolated polynucleotide according to 2. 91. The isolated polynucleotide of claim 91, wherein the polynucleotide is incorporated into a vector polynucleotide having the nucleotide sequence of SEQ ID NO: 60. 92. A method of determining whether a test compound is an inhibitor of cell proliferation, comprising incubating a cell comprising a functional FEZ1 gene in the presence of the test compound, and FEZ1 in the cell. Comprising assessing expression, whereby FEZ1 expression in a cell is increased if FEZ1 expression is increased relative to FEZ1 expression in the same type of cell incubated in the absence of the test compound, The above method, which is used as an indicator that it is an inhibitor of cell proliferation. 93. An isolated polynucleotide comprising a portion that is substantially homologous to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. 94. The isolated polynucleotide of claim 93, wherein the human FEZ1 gene has the nucleotide sequence of SEQ ID NO: 1. 95. The isolated polynucleotide of claim 93, wherein the portion is at least 90% homologous to at least 20 consecutive nucleotide residues of the chain of the human FEZ1 gene. 96. The isolated polynucleotide of claim 93, wherein the portion substantially comprises at least 50 consecutive nucleotide residues of the chain of the human FEZ1 gene. 97. The isolated human Fez1 protein of claim 30, wherein the protein has an amino acid sequence that is substantially homologous to SEQ ID NO: 4. 98. An isolated polypeptide having an amino acid sequence substantially homologous to at least 20 consecutive residues of SEQ ID NO: 4.