AU5645200A - Diagnostic methods and gene therapy using reagents derived from the human metastasis suppressor gene KAI1 - Google Patents
Diagnostic methods and gene therapy using reagents derived from the human metastasis suppressor gene KAI1 Download PDFInfo
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- AU5645200A AU5645200A AU56452/00A AU5645200A AU5645200A AU 5645200 A AU5645200 A AU 5645200A AU 56452/00 A AU56452/00 A AU 56452/00A AU 5645200 A AU5645200 A AU 5645200A AU 5645200 A AU5645200 A AU 5645200A
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Description
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): THE GOVERNMENT OF THE UNITED STATES OF AMERICA, REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES and JOHN HOPKINS UNIVERSITY Invention Title: DIAGNOSTIC METHODS AND GENE THERAPY USING REAGENTS DERIVED FROM THE HUMAN METASTASIS SUPPRESSOR GENE KAI1 The following statement is a full description of this invention, including the best method of performing it known to me/us: la 0 Title of Invention DIAGNOSTIC METHODS AND GENE THERAPY USING REAGENTS DERIVED FROM THE HUMAN METASTASIS SUPPRESSOR GENE KAI Field of Invention This invention is in the field of cancer diagnostics and therapeutics. In particular, this invention relates to detection of alterations of wild-type KAII gene sequence, KAIl mRNA and KAI protein useful in determining the presence of malignant cancer in a subject or a genetic predisposition to malignancy in a subject.
The invention further relates to the use of gene therapy to restore the wild-type KAII gene product.
15 Background of Invention It has been widely accepted that carcinogenesis is a multistep process involving genetic and epigenetic changes that dysregulate molecular control of cell proliferation and differentiation. The genetic changes 20 can include activation of proto-oncogenes and/or the inactivation of tumor suppressor genes that can initiate tumorigenesis as well as lead to the progression of tumors. For example, the tumor suppressor gene p53 may be involved in late stages of colorectal carcinomas (Baker, S.J. et al., (1989) Science, 244: 217-221) and a putative metastasis suppressor gene, nm23, was found down-regulated in metastatic tumors versus nonmetastatic tumors (Steeg, P.S. et al., (1988) J. Natl. Canc. Inst., 80:200-204). In addition, the activation of ras oncogene and the amplification of N-myc have been associated with progression of human tumors such as breast carcinomas (Liu, E. et al., (1988) Oncogene 3:323-327); and neuroblastomas (Brodeur, G.M. et al., (1984) Science, 224:1121-1124; Schwab, M. et al., (1984) Proc. Natl. Acad.
Sci. 81:4940-4944) but they are unlikely to be 2 0 universal determinants of tumor progression (Nicolson, G.L. Bio Essays, 13:337-342 (1991).
However despite these advances in understanding the genetic changes underlying carcinogenesis, metastasis, which is the main cause of death for most cancer patients (Rosenberg, Surgical Treatment of Metastasis Cancer (Lippincott, Philadelphia PA 1987)), remains one of the most important but least understood aspects of cancer (Liotta, L.A. et al. (1991) Cell, 64:327-336; Nicolson, G.L. (1991) BioEssavs, 13:337-342 and Steeg, P.S. (1992) Curr. Opin. Oncol., 4:134-141). Accordingly, the isolation of metastasis tumor suppressor genes is of great importance for the diagnosis and therapy of cancers.
Cell fusion studies by Ramshaw et al. ((1983) Int. J. Cancer, 32:471-478) in which hybridization of non- 15 metastatic and metastatic tumor cells produced cell hybrids which are tumorigenic but no longer metastatic demonstrated the existence of metastasis suppressor genes.
More recently, Ichikawa et al. (1991) Cancer Res., 51:3788-3792) demonstrated that the metastatic ability of S 20 rat prostatic cancer cells was suppressed when fused to non-metastatic cancer cells and that the reexpression of metastasis was associated with the consistent loss of a normal rat chromosome. A subsequent study using microcell-mediated chromosome transfer further mapped a putative human metastasis suppressor gene to the llpll.2- 13 region of human chromosome 11. (Ichikawa et al. (1992) Cancer Res., 52:3486-3490) In this study, these researchers demonstrated that a hybrid retaining human chromosome llcent-pl3 showed a suppression of metastasis while hybrids retaining 11cent-pll.2 did not.
In sum, the data presented in the Ichikawa et al. papers suggested that a putative suppressor gene in the p11.2-13 region of human chromosome 11 may play a role in metastasis. However to date, no gene has been identified in this region which is a candidate metastasis 3 suppressor gene. Thus, there is a need in the art to identify such gene(s) in this chromosome region and to determine if any such gene(s) is associated with metastasis.
It will be clearly understood that. although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.
Summary of the Invention The present invention relates to methods for detecting alterations of the wild-type KAII gene where detection of such alterations is useful in determining the presence of a malignant cancer in a subject or a genetic predisposition to malignancy in a subject. A first method for detecting alterations of the wild-type KAII1 gene comprises analysing the DNA of a subject for mutations of 2 the KA11 gene. A second method for detecting alterations of the KAIl gene comprises analysing the RNA of a subject for mutations and altered expression of the mRNA product of the KAIl gene.
The present invention therefore provides nucleic acid probes for detection of alterations of the wild-type 25 KAII gene.
The present invention further provides a diagnostic kit containing purified and isolated nucleic oo i acid sequences useful as PCR primers in analysing RNA or DNA of a subject for alterations of the wild-type KAII gene. These PCR primers can also be used to determine the nucleotide sequence of KAIl alleles.
A third method for detecting alterations of the wild-type KAI1 gene comprises analysing protein of a subject for alterations in the expression of KAIl protein.
The invention therefore relates to antibodies to the KAII protein and to a diagnostic kit containing antibodies to KAI1 protein useful for detecting alterations \\mlb_fils\homS\cinta\Kop\spci\55790.96 div.doc 1/09/00 4 in KAI1 protein expression in a subject.
The present invention further provides a method for supplying the wild-type KAI1 gene to a cell having altered expression of the KAI1 protein, the method comprising: introducing a wild-type KAI1 gene into a cell having altered expression of KAI1 protein such that the wild-type gene is expressed in the cell.
For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.
Description of Figures Figure 1 shows the results of a Northern blot of mRNA from normal tissue (human prostate) and from both metastatic (AT6.1, AT6.1-11-2 and AT6.1-11-3) and nonmetastatic (AT6.1-11-1*) tumor cells. 2gg of poly A RNA per sample was loaded in each lane and the blots were hybridised sequentially with KAII cDNA and rat P-actin probes. The asterisk identifies the hybrid AT6.1-11-1 that contained the llpcen-pl3 region and was suppressed in metastatic ability.
Figure 2 shows the results of Southern blot analysis of DNA isolated from human placenta, rodent cells 25 (A9 and AT6.1) and human-rodent microcell hybrids (A9llneo, AT6.1-11-1*, AT6.1-11-2 and AT6.1-11-3). For each sample, 15 gg of DNA was digested with Hind III, separated S" on a 1.2% agarose gel and hybridised to a KAI1 cDNA probe.
As in Figure 1, the asterisk identifies the hybrid AT6.1-11-1 that contained the llpcen-pl3 region and was suppressed in metastatic ability.
Figure 3 shows the nucleotide (upper line) and deduced amino acid (lower line) sequences of the KAI1 cDNA where the abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. The four \\melb.flles\hou\cinta.\Keep\speci\55790.96 djv.doc 1/09/00 4a putative transmembrane domains are noted by a dotted underline and the potential N-linked glycosylation sites are doubly underlined.
Figure 4 shows the results of Northern blot analysis of 15 ig of total RNA isolated from human normal prostate tissue and from cell lines derived from human 5 S metastatic prostate cancers. The blot was hybridized sequentially with KAII and human 3-actin probes.
Figure 5 shows the results of Northern blot analysis of 2 ,g of poly A RNA prepared from the various human tissues indicated at the top of Figure 5. The blot was hybridized sequentially with KAI1 and human 3 -actin probes.
Figure 6 shows the results of a "zoo" blot of EcoRI-digested genomic DNA of the various species indicated at the top of Figure 6. The blot was hybridized with KAII probe.
Detailed Description of Invention The present invention relates to the cloning and characterization of a metastasis suppressor gene on human 15 chromosome 11. The nucleotide and deduced amino acid sequences of this gene, designated KAII herein, are shown in Figure 3. The nucleotide sequence shown in Figure 3 was cloned from a metastasis suppressed cell hybrid clone AT6.1-11-1* and represents the wild-type KAII sequence.
20 A search of the KAII cDNA sequence in GenBank and EMBL databases revealed that the KAII cDNA sequence is identical to three cDNA clones from human lymphocytes, designated C33, R2 and IA4 by different laboratories (Imai, T. et al. (1992) J. Immunol., 149, 2879-2886 (1992); Fukudome, K. et al. (1992) J. Virol., 66, 1394-1401 (1992); Gaugitsch, H. et al. (1991) Eur. J.
I' Immunol., 21, 377-383 (1991); Gil, M. L. et al. (1992) J.
Immunol., 148, 2826-33 (1992)). C33 is associated with the inhibition of virus-induced syncytium formation (Imai, T. et al. (1992); Fukudome, K. et al. (1992)); R2 is strongly up-regulated in mitogen-activated human T cells (Gaugitsch, H. et al. (1991)), and IA4 is highly expressed in several B lymphocyte lines (Gil, M. L. et al.
(1992)). However, none of these three clones were suggested to function in metastasis and the function of 6 the protein encoded by these clones was not known prior to the present invention.
The present invention further relates to association of alterations of the wild-type KAII gene with metastasis. Accordingly, the present invenoian relates to methods for detecting alterations of the wild-type KAII gene in a subject where such methods can provide diagnostic and prognostic information. For example, since loss of expression of the KAII gene has been observed in metastatic prostate tumors, these are tumors in which KAII has a role in metastasis. Thus, detection of alterations of the wild-type KAII gene in a subject may effect the course of treatment chosen by a clinician. In addition, since KAII is expressed in all tissues tested including spleen, thymus, prostate, testes, ovary, small intestine, 15 colon, blood leukocyte, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, alterations of the wild-type KAII gene may contribute to metastasis in these tissues.
It is further understood by those of ordinary skill in the art that the methods of the present invention are applicable to any tumor in which alterations of wildtype KAI occur. Moreover, the methods of detection disclosed in the present invention can be used prenatally to screen a fetus or presymptomatically to screen a subject at risk of having cancer based on his/her family history. For purposes of the present invention, subject means a mammal.
According to the diagnostic methods of the present invention, alterations of the wild-type KAII gene are detected. "Alterations of the wild-type KAII gene" as used throughout the specification and claims encompasses mutations of the wild-type KAII gene where such mutations include deletions, inversions, insertions, transversions or point mutations of the wild-type KAII gene. It is 1q believed that many mutations found in tumor tissues will 7 0 be those leading to decreased expression of KAIJ protein.
However, mutations leading to non-functional gene products can also lead to malignancy. It is further understood that point mutations can occur in regulatory.regions (e.g.
promoter) or can disrupt proper RNA processing.thus leading to loss of expression of the KAIl gene products respectively.
"Alterations of the wild-type KAII gene" as used throughout the specification and claims can also be detected on the basis of altered expression of the wildtype KAll-specific mRNA and KAII protein. The altered expression of these KA1 gene products may be detected as a loss or reduction in the levels of KAII mRNA and protein relative to wild-type levels. Alternatively, the altered expression of KAII protein can encompass a loss of 15 function of the KAII protein. Those of ordinary skill in the art would therefore understand that altered expression of the KAII gene products may be caused by a variety of events, including but not limited to, mutations of the wild-type KA1 gene, changes in the posttranslational 20 modification of the KAI protein glycosylation) or loss of a trans-acting factor necessary for transcription of the KAII gene.
Provided with the KAII cDNA and deduced amino acid sequences shown in Figure 3, design of particular probes useful in detecting alterations of the wild-type KAII gene is well within the skill of the ordinary artisan.
In one embodiment of the invention, the method for detecting alterations of the KAIl gene comprises analyzing the DNA of a subject for mutations of the wildtype KAIl gene. For analysis of DNA, a biological specimen is obtained from the subject. Examples of biological specimens that can be obtained for use in the present methods include, but are not limited to, tissue ie biopsies, whole blood, lymphocytes and tumors. Means for 8 S enriching a tissue preparation for tumor cells are known in the art. For example, the tissue may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry and other techniques well known in the art. Alternatively, primary cell cultures can be established from tumor biopsies using methods known to those of ordinary skill in the art.
The DNA isolated from the biological specimen can be analyzed for mutations of the wild-type KAII gene by a variety of methods including, but not limited to, Southern blotting after digestion with the appropriate restriction enzymes (restriction fragment length polymorphism, RFLP) (Botstein, D. (1980) Amer. J. Hum.
Genet., 69:201-205, denaturing gradient electrophoresis technique (Myers, (1985) Nature, 313:495-498), 15 oligonucleotide hybridization (Conner, R. et al., (1984) EMBO 3:13321-1326), RNase digestion of a duplex between a probe RNA and the target DNA (Winter, E. et al., (1985) Proc. Natl. Acad. Sci. 82:7575-7579), polymerase chain reaction (PCR) (Saiki, P.K. et al., 20 (1988) Science, 239:487-491; U.S. Patents 4,683,195 and 4,683,202), ligase chain reaction (LCR) (European Patent Application Nos. 0,320,308 and 0,439,182), and PCR-single S: stranded conformation analysis (PCR-SSCP) (Orita, M. et al. (1989) Genomics, 5:874-879; Dean, M. et al. (1990) Cell, 61:863-871).
In one preferred embodiment, Southern blot analysis can be used to examine DNA isolated from a subject for gross rearrangement of the KAII gene. The DNA to be analyzed via Southern analysis is digested with one or more restriction enzymes. Following restriction digestion, resultant DNA fragments are separated by gel electrophoresis and the fragments are detected by hybridization with a labelled nucleic acid probe (Southern, E.M. (1975) J. Mol. Biol., 98:503-517).
2, The nucleic acid sequence used as a probe in 9 0 Southern analysis can be labeled in single-stranded or double-stranded form. Labelling of the nucleic acid sequence can be carried out by techniques known to one skilled in the art. Such labelling techniques can include radiolabels and enzymes (Sambrook, J. et ai.: (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, New York). In addition, there are known non-radioactive techniques for signal amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al. (1973) Proc. Natl. Acad. Sci., 70:2238-2242; Heck, R.F. 1968) S. Am. Chem. Soc., 90:5518-5523), methods which allow detection by chemiluminescence (Barton, S.K. et al.
(1992) J. Am. Chem. Soc., 114:8736-8740) and methods utilizing biotinylated nucleic acid probes (Johnson, T. K.
15 et al. (1983) Anal. Biochem., 133:126-131; Erickson, P.F.
et al. (1982) J. of Immunology Methods, 51:241-249; Matthaei, F.S. et al. (1986) Anal. Biochem., 157:123-128) and methods which allow detection by fluorescence using commercially available products. Each of the nucleic acid 20 sequences used as a probe in Southern analysis is derived from the wild-type KAII gene. Preferred probes are derived from having the cDNA sequence shown in Figure 3.
Once the separated DNA fragments are hybridized to the labelled nucleic acid probes, the restriction digest pattern can be visualized by autoradiography and compared with the restriction digest pattern of the wildtype KAII gene. The presence or absence of a restriction fragment length polymorphism (RFLP) in the restriction pattern of the subject's DNA relative to the wild-type restriction pattern indicates an alteration of the wildtype KAII gene.
In another preferred embodiment, genomic DNA may be analyzed for mutations in the wild-type KAI1 gene via PCR-SSCP. In this method, each of the pair of primers 3 selected for use in PCR are designed to hybridize with 10 0 sequences in the wild-type KAI1 gene to permit amplification and subsequent detection of mutations in the denatured amplification product via non-denaturing polyacrylamide gel electrophoresis. In one embodiment, primer pairs are derived from the KAII cDNA sequence shown in Figure 3.
In another embodiment, primer pairs useful in the analysis of genomic DNA mutations of the wild-type KAII gene may be derived from intronic sequences which border the 5' and 3' ends of a given exon of the KAIl gene. Examples of primer pairs permitting specific amplification of specific exons of the KAII gene include: SEQ ID NO:1: AGAAGATCAAGTTGAAGAGG SEQ ID NO:2: GGGACCTCATTTCCTAGCTG SEQ ID NO:3: ATGAAACTGCTCTTGTCGG 15 SEQ ID NO:4: TCAGCTCTTGGCTCCCCATT SEQ ID NO:5: TGGGCACGGGTTTCAGGAAAT SEQ ID NO:6: TGCAGAGAGCCCCAAATGCA i. SEQ ID NO:7: AGGGTGAGCCGTGAGCACAA SEQ ID NO:8: TGCTGAGAGTACCCAGATGC 20 SEQ ID NO:9: GATGGCCACACCCACGCCC O*O* SEQ ID NO:10: TGCATGGAGAAGGTGCAGGC SEQ ID NO:11: CCTCTTGCCCACCCTGACTGA SEQ ID NO:12: TTCACACCATTCTCCTGCCT where SEQ ID NOS:1 and 2 bound exon 3; SEQ ID NOS:3 and 4 bound exon 4; SEQ ID NOS: 5 and 6 bound exon 6; SEQ ID NOS:7 and 8 bound exon 7; SEQ ID NOS:9 and 10 bound exon 8; and SEQ ID NOS:11 and 12 bound exon 9.
Each primer of a pair is a single-stranded oligonucleotide of about 15 to about 50 base pairs in length which is complementary to a sequence at the 3' end of one of the strands of a double-stranded target sequence. Optimization of the amplification reaction to obtain sufficiently specific hybridization to the KAII gene sequence is well within the skill in the art and is IF preferably achieved by adjusting the annealing 11 temperature. In yet another embodiment, RNA may be analyzed for mutations in the KAII gene by RT-PCR-SSCP.
In this method, single stranded cDNA is prepared from either total RNA or polyA+-enriched RNA using.reverse transcriptase. In this method, each of the pairs of primers selected for use in PCR of the resultant singlestranded cDNA are designed to hybridize with sequences in the KAII cDNA which are an appropriate distance apart (at least about 100-300 nucleotides) in the gene to permit amplification and subsequent detection of mutations in the denatured amplification product via non-denaturing polyacrylamide gel electrophoresis. Such primer pairs can be derived from the KAII cDNA sequence set forth in Figure 3. Each pair comprises two such primers, complementary to sequences on each strand separated by generally about 100 15 to about 300 base pairs.
"The primers of this invention can be synthesized using any of the known methods of oligonucleotide synthesis the phosphodiester method of Agarwal et al. (1972) Agnew. Chem. Int. Ed. EnQl., 11:451, the phosphotriester method of Hsiung et al. (1979). Nucleic Acids Res., 6:1371, or the automated diethylphosphoramidite method of Beuacage et al. (1981).
Tetrahedron Letters, 22:1859-1862), or they can be isolated fragments of naturally occurring or cloned DNA.
In addition, those skilled in the art would be aware that oligonucleotides can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially custom ordered and prepared. In one embodiment, the primers can be derivatized to include a detectable label suitable for detecting and/or identifying the primer extension products biotin, avidin, or radiolabelled dNTP's), or with a substance which aids in the isolation of the products of amplification (e.g.
biotin or avidin).
Ie The present invention therefore provides a 12 S diagnostic kit for detecting mutations of the KAIl gene.
This diagnostic kit comprises purified and isolated nucleic acid sequences useful as hybridization probes or as PCR primers in analyzing DNA or RNA for alterations of the wild-type KAII gene.
In an alternative embodiment, nucleic acid probes can be selected to hybridize to mutant alleles of the KAIl gene. These allele-specific probes are useful to detect similar mutations in other subjects on the basis of hybridization rather than mismatches. Where such nucleic acid probes are primer pairs which hybridize to mutations in the KAII gene sequence, these primer pairs can be used to amplify specific mutant gene sequences present in a biological sample via PCR.
The amplification products of PCR can be 15 detected either directly or indirectly. Direct detection of the amplification products is carried out via labelling of primer pairs. Labels suitable for labelling the primers of the present invention are known to one skilled in the art and include radioactive labels, biotin, avidin, enzymes and fluorescent molecules. The desired labels can be incorporated into the primers prior to performing the amplification reaction. A preferred labelling procedure utilizes radiolabelled ATP and T4 polynucleotide kinase (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY). Alternatively, the desired label can be incorporated into the primer extension products during the amplification reaction in the form of one or more labelled dNTPs. In the present invention, the labelled amplified PCR products can be analyzed for mutations of the KAII gene via separating the PCR products by non-denaturing polyacrylamide gel electrophoresis, denaturing polyacrylamide gel electrophoresis (PCR-SSCP) or via direct sequencing of the PCR-products.
,2 In yet another embodiment, unlabelled 13 S amplification products can be analyzed for mutations in the KAII disease gene via hybridization with nucleic acid probes radioactively labelled or, labelled with biotin, in Southern blots or dot blots. Nucleic acid probes useful in this embodiment are those described earlier for Southern analysis. In a further embodiment, detection of point mutations may be accomplished by molecular cloning of the allele present in the tumor tissue using the cDNA sequence set forth in Figure 3 and sequencing that allele using techniques well known in the art.
A second method for detecting alterations of the wild-type KAII gene comprises analyzing the RNA of a subject for mutations and altered expression of KA1Ispecific mRNA.
For the analysis of RNA by this method, RNA can 15 be isolated from, for example, a tumor biopsy sample obtained from said subject where said tumors include, but are not limited to, prostate tumors.
The RNA to be analyzed can be isolated from blood or tumor biopsy samples as whole cell RNA or as poly(A) RNA. Whole cell RNA can be isolated by methods known to those skilled in the art. Such methods include extraction of RNA by differential precipitation (Birnbiom, H.C. (1988) Nucleic Acids Res., 16:1487-1497), extraction of RNA by organic solvents (Chomczynski, P. et al. (1987) Anal. Biochem., 162:156-159) and extraction of RNA with strong denaturants (Chirgwin, J.M. et al. (1979) •Biochemistry, 18:5294-5299). Poly(A) RNA can be selected from whole cell RNA by affinity chromatography on oligod(T) columns (Aviv, H. et al. (1972) Proc. Natl. Acad.
Sci., 69:1408-1412).
The methods for analyzing RNA for mutations and altered expression of KAll-specific mRNA include Northern blotting (Alwine, J.C. et al. (1977) Proc. Natl. Acad.
Sci., 74:5350-5354), dot and slot hybridization (Kafatos, F.C. et al. (1979) Nucleic Acids Res., 7:1541-1522), 14 0 filter hybridization (Hollander, M.C. et al. (1990) Biotechniques; 9:174-179), S, analysis (Sharp, P.A. et al., (1980) Meth. Enzymol., 65:750-768), RNase protection (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY), reverse-transcription polymerase chain reaction (RT- PCR) (Watson, J.D. et al. (1992) in "Recombinant DNA" Second Edition, W.H. Freeman and Company, New York) and
RT-PCR-SSCP.
Where expression of KAII mRNA is measured, diminished KAII mRNA expression is indicative of alteration of the wild-type KAII gene. One preferred method for measuring alterations in the level of KAIIspecific mRNA expression is Northern blotting where the nucleic acid sequence used as a probe for detecting KAI1- 15 specific mRNA expression is complementary to all or part of the KAII cDNA sequence shown in Figure 3.
A second preferred method for measuring, alterations in the level of KAI-specific mRNA expression is detection of KAII mRNA expression via hybridization of a nucleic acid probe derived from KAIl cDNA sequence to RT-PCR products generated from RNA isolated from a biological sample.
A third method for detecting alterations of the wild-type KAII gene comprises analyzing the protein of a 25 subject for alteration of wild-type KAII protein. In one embodiment, alteration of wild-type KAII protein encompasses a loss or reduction in the level of expression of KAII protein in a biological sample.
Examples of immunoassays useful in determining the level of expression of KAII protein include, but are not limited to, immunoprecipitation, radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, immunohistochemical assay and enzyme-linked immunosorbent assay (ELISA). In 1e addition, the above immunoassays may be used in 15 0 combination such as immunoprecipitation followed by Western blot. The above methods are described in Principles and Practice of Immunoassay, Price and Newman, eds., Stochton Press, 1991. Such assays may be a direct, indirect, competitive or noncompetitive immunoassay as described in the art (Oelbrick, N. (1984) J. Clin. Chem.
Clin. Biochem., 22:895-904). The protein to be analyzed by such methods may be obtained from biological samples such as tumor biopsies and the protein can be obtained as a crude lysate or it can be further purified by methods known to those of ordinary skill in the art including immunoaffinity chromatography using antibodies to the KAII protein (Sambrook, J. et al (1989) in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY). Alternatively, levels of KAII protein may be 15 detected by immunohistochemistry of fixed or frozen tumor sections.
For detection of KAII protein by immunoassay, the present invention provides anti-KAll antibodies where such antibodies may be polyclonal or monoclonal. If polyclonal antibodies are desired, serum containing polyclonal antibodies to KAII protein can be used or the polyclonal antibodies can be purified from other antigens present in the serum by immunoaffinity chromatography.
Alternatively, monoclonal antibodies directed against KAII 060* 25 can readily be produced by one of ordinary skilled in the art. Methods of producing monoclonal or polyclonal antibodies are known to one of ordinary skilled in the art (Goding, J.W. (1983) monoclonal antibodies: Principles and Practice, Plodermic Press, Inc., NY, NY, pp. 56-97; Hurn, B.A.L. et al. (1980) Meth. Enzvmol., 70:104-141).
Suitable immunogens which may be used to produce the polyclonal or monoclonal antibodies of the present invention include cell lysate prepared from cells transfected with a recombinant KAI1 protein, partially or substantially purified recombinant or native KAII protein, 16 0 or peptides derived from the KAII amino acid sequence shown in Figure 3. When purification of the recombinant or native KAII protein is desired, it can be accomplished by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis, affinity, and immunoaffinity chromatography and the like.
In the case of immunoaffinity chromatography, the recombinant protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the KAII protein.
In a preferred embodiment, the immunogen is a recombinantly produced KAII protein or fragments thereof.
Production of recombinant KAI1 protein or a fragment 15 thereof may be directed by a natural or synthetic nucleic acid sequence inserted into a suitable expression vector.
A preferred nucleic acid sequence is the KAII cDNA sequence shown in Figure 3. In one embodiment, restriction digest fragments containing the full-length cDNA or fragments thereof containing a coding sequence for JKAII can be inserted into a suitable expression vector.
By suitable expression vector is meant a vector that can function in eukaryotic or prokaryotic cells and is capable of carrying and expressing a nucleic acid sequence 25 encoding the KAII protein or a fragment thereof. Such vectors and their use in producing recombinant proteins •are known to those of ordinary skill in the art (Sambrook, J. et al. (1989) in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Plainview, NY).
The immunogen of the present invention can be used in a suitable diluent such as saline or water, or in complete or incomplete adjuvants. Further, the immunogen may or may not be bound to a carrier to make the protein immunogenic. Examples of such carrier molecules include 2r but are not limited to bovine serum albumin (BSA), keyhole 17 S limpet hemocyanin (KLH), tetanus toxoid, and the like.
The immunogen can be administered by any route appropriate for antibody production such as intravenous, intraperitoneal, intramuscular, subcutaneous, and the like. The immunogen may be administered once: r at periodic intervals until a significant titer of anti-KAll antibody is produced. The antibody may be detected in the serum using an immunoassay.
The antibodies or antigen binding fragments may also be produced by genetic engineering. The technology for expression of both heavy and light chain genes in E.
coli is the subject of PCT patent applications; publication number WO 901443, WO 901443, and WO 9014424 and in Huse et al. (1989) Science, 246:1275-1281.
Alternatively, anti-KAll antibodies can be 15 induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-KAll antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent.
Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed.
25 Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-KAll antibodies, or by affinity chromatography using anti-KAll antibodies bound to the affinity matrix.
In an alternative embodiment, the antibodies of the present invention can be used in situ to detect KAII protein in cells or tissues. In one embodiment, the antibodies are used in direct or indirect immunofluorescence. In the direct method, anti-KA1 18 S antibody labelled with a fluorescent reagent such as fluorescein isothiocyanate, rhodamine B isothiocyanate and the like is reacted directly with the KAII present in cells or tissues. In the indirect method, unlabelled anti-KA1 antibody is reacted with the KAII prQtein present in cells or tissue. The unlabelled anti-KAll antibody is then reacted with a labelled second antibody.
The second antibody can be labelled with a fluorescent tag as described above. The fluorescently labelled cells or tissues can then be detected using techniques known to one skilled in the art such as a fluorescence-activated cell sorter, light microscopy using a fluorescent light lamp and the like. Alternatively, KAII protein can be detected in situ via the use of radiolabelled anti-KAll antibody or via the use of an unlabelled anti-KA1 antibody followed 15 by a radiolabelled second antibody reactive to the anti- KA1 antibody.
The antibodies of the present invention may also be used to immunoprecipitate the KAII protein from a mixture of proteins. The use of immunoprecipitation as a sensitive and specific technique to detect and quantitate target antigen in mixtures of proteins is well known to those of ordinary skill in the art (see Molecular Cloning, A Laboratory Manual, 2d Edition, Maniatis, T. et al. eds.
(1989) Cold Spring Harbor Press).
25 The antibodies of the present invention may also be affixed to solid supports for use in the isolation of KAII protein by immunoaffinity chromatography. Techniques for immunoaffinity chromatography are known in the art (Harlow, E. and Lane, D. (1888) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) including techniques for affixing antibodies to solid supports so that they retain their immunoselective activity; the techniques used may be those in which the antibodies are adsorbed to the support as well as those in which the antibodies are covalently 19 0 linked to the support. Generally, the techniques are similar to those used in covalent linking of antigen to a solid support; however, spacer groups may be included in the bifunctional coupling agents so that the. antigen binding site of the antibody remains accessible.
The above described antibodies and antigen binding fragments thereof may be supplied in a diagnostic kit useful for the detection of alterations in the expression of KAII protein.
In a second embodiment, alteration of wild-type KAII protein encompasses loss of function of the KAI1 protein. The present method therefore includes assays useful in determining the functional status of the KAI protein For example, since an association between processing of N-linked oligosaccharides and metastatic 15 phenotype has been well-documented (Hakomori, (1989) Advanc. Cancer Res., 52:257-331; Dennis, J. et al.
(1987) Science, 236:582-585; Ishikawa, M. et al. (1988) Cancer Res., 48:665-670) it is believed that glycosylation of the KAII protein is required for the protein to function as a metastasis suppressor. Thus, detection of a loss of function of KAII protein as evidenced by an absence of glycosylation is indicative of the presence of metastatic cancer in a subject.
The present invention also relates to a gene 25 therapy method in which an expression vector containing a nucleic acid sequence representing the wild-type KAII gene is administered to a subject having a mutation of the KAIl gene. A nucleic acid sequence representing wild-type KAI1 gene is that shown in Figure 3. Such nucleic acid sequence may be inserted into a suitable expression vector by methods known to those of ordinary skill in the art.
Expression vectors suitable for producing high efficiency gene transfer in vivo include retroviral, adenoviral and vaccinia viral vectors.
Expression vectors containing a nucleic acid 20 S sequence representing wild-type KAI1 gene can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or orally. A preferred route of administration is intraperitioneally.
Any articles or patents referenced'herein are incorporated by reference. The following examples are presented to illustrate various aspects of the invention but are in no way intended to limit the scope thereof.
Examples Materials and Methods Cell lines: AT6.1 is a highly metastatic Dunning rat prostatic cancer cell line. AT6.1-11 clones are microcell hybrids that have a portion of human chromosomes 11 as the sole human genetic materials in 15 AT6.1 cells. Microcell hybrid AT6.1-11-1* contains a fragment of human chromosome 11, cen-pl3, from the centromere to region p13, and was suppressed for metastatic ability. Microcell hybrids AT6.1-11-2 and -3 have smaller fragments of human chromosome 11 from the centromere to P11.2 and were not suppressed for metastatic ability. The characteristics and growth condition for these cell lines have been previously described in detail (Ichikawa et al, (1992) Cancer Res., 52:3486-3490). A9llneo is a mouse A9 cell line containing human chromosome 2* 25 llpter-q23 (Koi et al, (1989) Mol Carcinoq., 2:12-21).
Isolation and sequencing of KAII cDNA clone: Poly (A) RNA was isolated from exponentially growing AT6.1-11-1* cells, using a FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). Oligo (dT) was used to prime the first strand cDNA synthesis from 5 fg of poly (A) RNA. Double-stranded cDNA was cloned into plasmid pSPORT 1 vector by procedures recommended by the vendor (GIBCO BRL, Grand Island, NY). Human Alu sequence primer Alu 559 21 S (Nelson, D.L. et al (189) Proc. Natl. Acad Sci U.S.A.
86:6686-6690.) was used to amplify genomic DNA from suppressed hybrid AT6.1-11-1* and the nonsuppressed clone AT6.1-11-2 by PCR. The multiple Alu-PCR fragments of AT6.1-11-1* were cloned into a T-tailed vector-pCR1000 (Invitrogen, San Diego, CA). Individual clones corresponding to each fragment of Alu-PCR products were isolated after comparing the size of these Alu-PCR products to molecular weight markers in a agarose gel stained with ethidium bromide. Eleven fragments unique to AT6.1-11-1* were labeled by random priming (GIBCO BRL, Bethesda, MD) and used to screen 5 x 10 4 recombinants of the cDNA library under stringent wash conditions (65 0 C in 0.1 x SSC 0.1% SDS for 30 min.). Five independent clones were obtained and their inserts were sequenced using the 15 Sequenase kit (US Biochemical, Cleveland, OH). DNA sequences were analyzed with the GCG package (version 7.3, 1993, Madison, WI).
RNA analysis: Cytoplasmic RNA from AT6.1, AT6.1-11-1*, -2 and -3 were prepared from exponentially growing cells, using FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). Other poly (A)'RNA and human multiple tissue Northern blots were purchased from Clontech (Palo Alto, CA). 2 gg of poly (A)+RNA was denatured with formamide and fractionated on a 1.2% agarose gel in formaldehyde buffer. The RNA was then transferred onto nylon membrane, baked in an oven at 80 0
C
for 90 min, and then hybridized with a labeled probe in QuickHyb hybridization solution (Stratagene, La Jolla, CA) at 68 0 C for 1.5 hours and washed at 68 0 C for 30 minutes in 0.1% x SSC, 0.1% SDS and autoradiographed.
DNA analysis: 15 Ag of genomic DNA was digested with BamHI and separated on a 1.2% agarose gel. Following denaturation and neutralization, the DNA in the gel was transferred onto nylon membrane. The "zoo" blot containing EcoRl-digested genomic DNA from human, rat, 22 0 mouse, dog, cow, rabbit, chicken and yeast (Zoo-blot) was purchased from Clontech (Palo Alto, CA). The Southern blots were further hybridized and washed under the same conditions as described above for the Northern blots.
PCR: All PCRs in this study were carried out in 50 pl with 25 pmol of each primer, 10 mM Tris.HCI, 500 mM KCI, 1.5 mM MgCl 2 0.01% gelatin, 250 mM of each dNTP and units of Taq DNA polymerase (Perkin-Elmer, Norwalk, CT). The initial DNA denaturation was performed at 95 0
C
for 5 min, followed by 35 cycles of 94 0 C denaturation for 1 min, 55 0 C annealing for 1 min and 72 0 C extension of 4 min, with a final extension of 72 0 C for 8 min.
Probes and OliQonucleotides seauences: The KAII probe (nucleotides 64-1094 of the KAII cDNA) used in Southern and Northern blot analyses was generated by PCR with 15 primers shown as SEQ ID NO: 13 AGTCCTCCCTGCTGCTGTGTG and SEQ ID NO:14 TCAGTCAGGGTGGGCAAGAGG. Human and rat -actin probes were PCR products generated by templates and primers purchased from Clontech (Palo Alto, CA). The primer sequences for human f-actin are shown as SEQ ID NO:15 GAGGAGCACCCCGTGCTGCTGA and SEQ ID NO:16 CTAGA AGCATTTGCGGTGGACGATGGAGGGGCC and the primer sequences for rat f-actin are shown as SEQ ID NO: 17 TTGTAACCAACTGGGACGATATGG and SEQ ID NO:18
GTCTTGATCTTCATGGTGCTAGG.
Example 1 Clonina of the KAII Gene To clone the gene on human chromosome 11 responsible for the metastasis suppression of AT6.1 prostatic cancer cells, genomic DNA fragments from the p11.2-13 region were isolated using human-specific Alu element-mediated PCR (Alu-PCR) (Nelson, D.L. et al Proc.
Natl. Acad. Sci. 86:6686-6690) with DNAs from the metastasis suppressed microcell hybrid AT6.1-11-1* and the ?a non-suppressed hybrids AT6.1-11-2 and AT6.1-11-3. The 23 S Alu-PCR fragments found only in the AT6.1-11-1 DNA were then used as probes to screen a cDNA library prepared from the suppressed cell hybrid clone AT6.1-11-1* that contains human chromosomal region llcen-pl3. Of five cDNA clones obtained, all were expressed in the suppressed hybrid but not in the nonsuppressed hybrids as detected by reverse transcription-polymerase chain reaction (RT-PCR) using primers derived from these cDNA sequences. Northern analysis of RNA isolated from human prostate and cell lines AT6.1, AT6.1-11-1*, -2 and -3 revealed that two of the cDNA clones detected a 2.4 hb and 4.0 kb transcript respectively in human tissue and the suppressed AT6.1-11-1* cells. The results of a Northern blot for one such clone, designated KAII for Kang Ai (Chinese for anticancer), are shown in Figure 1 and clearly demonstrate 15 that KAII mRNA was abundant in the metastatic suppressed AT6.1-11-1* cells but absent from the parental AT6.1 cells and the nonsuppressed hybrids. Therefore, the KAII clone was analyzed further.
To confirm that the KAII gene was isolated from the p11.2-13 region of human chromosome 11 involved in metastasis suppression, Southern blot analysis was conducted on 15 Ag of genomic DNA from human placenta, rodent cells (A9 and AT6.1) and human-rodent microcell "hybrids (AT6.1-11-1*, AT6,1-11-2 and AT6.1-11-3), digested 25 with Hind III, separated on a 1.2% agarose gel and hybridized with KAII probe. The results shown in Figure 2 demonstrate that only the cell hybrids that have the p11.2-13 region involved in metastasis suppression (AT6.1- 11-1*) have the pattern observed with normal human DNA when hybridized to KAII probe. Fluorescence in situ hybridization of a KAII probe to metaphase chromosomes further localized KAII to the p11.2 region.
24 Example 2 Nucleotide And Deduced Amino Acid Sequences of the KAII cDNA The nucleotide and deduced amino acid sequences of the KAII cDNA are shown in Figure 3. The KAII cDNA has a single open reading frame from nucleotide positions 166 to 966, predicting a protein of 267 amino acids with a calculated molecular weight of 29,610 daltons. An Alu element was present in the 3'-untranslated region of the 1 cDNA. The predicted protein had four hydrophobic and presumably transmembrane domains and one large extracellular hydrophilic domain with three potential N-glycosylation sites. As noted earlier, the KAII cDNA sequence is identical to three cDNA clones from human Slymphocytes, C33, R2 and IA4.
Example 3 Determination That KAII Is A Metastasis Suppressor Gene To investigate if KAII is the gene responsible for metastasis suppression in AT6.1-11-1*, KA1 cDNA was subcloned into a constitutive expression vector and transfected into parental AT6.1 cells as follows. In brief, KAII cDNA was cloned into pCMVneo, in which transcription is driven by the constitutive human cytomegalovirus promoter (Eliyahu, D. et al Proc Natl Acad Sci (1989) 86:8763-8767). The resultant plasmid pCMV-KAII was transfected into AT6.1 cells by calcium phosphate precipitate method and the vector alone was also transfected as a negative control. Individual transfectants were isolated in selection medium (RPMI-1640 plus 10% fetal calf serum, 2units/ml pen-strep and 500 ug/ml neomycin). Exponentially growing untransfected AT6.1, AT6.1-11-1* and AT6.1-11-2 cells and exponentially growing vector (AT6.1VEC-1, AT6.1VEC-2 and AT6.1VEC-3) and KAII (AT6.1KAI-1, AT6.1KAI-2 and AT6.1KAI-3) transfectants 25 0 were collected by scraping and cell clumps were broken up by gentle pipetting. The cell suspension was placed in a tube and allowed to stand at room temperature for 30 min.
Cells from the supernatant suspension were collected, washed, and resuspended in cold PBS at 106 cells/ml.
Four-to-five-week-old male Ncr nu/nu nude mice (National Cancer Institute Animal Program, Bethesda, MD) were injected with 0.1 ml of the indicated cell suspension (105 cells) (the column designated "Clone" in Table 1) subcutaneously at sites on both the right and left midlateral, about 1/4 of the distance from the base of the skull to the base of the tail. About 6 weeks after injection, the tumors were weighed and the lungs were inflated with Bouin's solution. Tumor foci on the surface of lungs were scored under a dissecting microscope.
15 Individual transfectants were analyzed for KAII expression and for their ability to suppress lung metastases and the results of one experiment are shown in Table 1.
**0 26 TABLE 1 Mean Number* KAI1' Tumor Tumor Number of Metastasis mRNA Latency* Age Weight(g) Mice with Per. Mouse Mean Clones Level (days) (Days) Excision Metastases (#mice)- P AT6.1 0 4.3 27 2.58 19/19 58(32-135) AT6.1-11-1* 10 3.7 37 2.79 6/7 7(0-9) <0.005' AT6.1-11-1-2 0 4.2 37 2.78 6/6 26(20-40) AT6.1VEC-1 0 4.9 43 2.32 17/17 30(16-57) AT6.1VEC-2 0 4.0 43 3.26 17/17 30(12-71) AT6.1VEC-3 0 5.5 43 2.57 18/18 47(15-183) AT6.1KAI-1 10 4.2 43 3.99 18/20 6(0-14) <0.001 15 AT6.1KAI-2 7 4.5 41 1.79 17/19 7(0-17) <0.001 AT6.1KAI-3 1 4.5 43 2.56 18/19 23(0-36) <0.021 The data shown in this table are from a large, age-matched cohort of "side-by-side" nude mice, with cells inoculated at the same time.
KAI expression was determined by Northern blot analysis. The KAII signals on the Northern blot 20 were scored by a densitometer. The value for AT6.1KAI-1 was standardized to 10 and the values for other clones were adjusted accordingly.
Latency is the time following injection for a palpable tumor to appear.
The numbers in parentheses indicate the range of metastases in individual mice.
Compared.to the number of metastases with AT6.1-11-2 cells.
25 Compared to the mean number of metastases with all of the three vector transfectants.
The results presented show that expression of KAII resulted in a significant suppression of the number of lung metastases per mouse but did not affect the growth 3 rate of the primary tumor. Further, whereas the parental AT6.1 cells yielded 58 metastasis per mouse when injected subcutaneously into nude mice, two transfectants with levels of KAII mRNA expression similar to the high level of expression observed in AT6.1-11-1* cells gave only 6 or 7 lung metastases per animal. In contrast, the three 27 S vector control transfectants produced 30-47 lung metastases per mouse, which is on average 5.5 times the number of metastases observed with the 2 KAII transfectants with high KAII mRNA expression.(AT6.1KAI-1 and AT6.1KAI-2). In addition, while the AT6.1KAI-3 clone which had low KAII expression produced 23 lung metastases, this was still significantly less than the mean number of lung metastases for control transfectants. Finally, Northern analysis showed that KAII expression was undetectable or very low in 28 lung metastases from KAIl transfectants suggesting that selection for cells with absent or reduced KAI1 expression resulted in metastasis formation. These results indicate that the metastatic ability of AT6.1 cells is suppressed by KAII expression.
5 Example 4 KAII mRNA Expression In Cell Lines Derived From Metastatic Human Prostate Tumors To determine whether KAII mRNA expression was i reduced in human metastatic prostate tumors relative to expression in normal human prostate, 15 jig total RNA from human normal prostate tissue and from cell lines derived from metastatic prostate cancers (Kaighn, M.E. et al (1979) Invest. Urol.:17:16; Horoszewicz, J.J. et al. in Models for Prostate Cancer, G.P. Murphy. Ed. (Alan R.
25 Liss, Inc., New York, 1980). pp 115-132: T. lizumi. et al. (1987) J. Urol. 137:1304, D.D. Mickey et al., in Models for Prostate Cancer. G. P. Murphy. Ed. (Alan R.
Liss, Inc. New York, 1980), pp. 67-84) were denatured with formamide, electrophoresed fractionated on a 1.2% agarose gel and hybridized sequentially to KAII and human S-actin probes. The results of this Northern blot analysis are shown in Figure 4 and clearly demonstrate that KAII expression was significantly reduced in the human cell lines derived from metastatic prostate tumors (PC-3, c LNCaP, TSU-Prl and DU145) when compared to normal prostate 28 S (prostate). In addition, while longer exposures (4 days at -80 C) of the autoradiogram shown in Figure 4 (overnite at -80 C) revealed expression of KAIl mRNA in all of the tumor cells, the level of expression was still much lower than in normal prostate.
To rule out the possibility that the metastasis suppression by KAII was due to an indirect immune mechanism, two other experiments were performed. First, parental AT6.1 cells, cell hybrid clone AT6.1-11-1*, or a KAI1 transfectant (AT6.1 KAI-1) were inoculated into the leg of severe combined immune deficient (SCID) mice at 5 x 105 cells/mouse. When tumors reached 3-5 cm', the leg with tumor was surgically removed and animals were followed until 50 to 60 days post inoculation. Lung metastases for each mouse were analyzed as described for Table 1. For 15 AT6.1, 9/9 mice had lung metastases with an average number of 83 per mouse. For AT6.1-11-1*, 4/9 mice had lung metastases with an average number of 6 per mouse. For AT6.1KAI-1, 2/7 mice had lung metastases with an average number of 2 per mouse. These studies demonstrated that even in SCID mice, which are more immune compromised than nude mice, metastasis suppression was observed.
Second, highly metastatic rat mammary cancer cells into which the KAII gene was introduced via microcell-mediated chromosome transfer, retained their 25 ability to metastasize (Rinker-Schaefer, C.W. et al.
(1994) Cancer Res., 54:6249-6256) even though the hybrids expressed similar level of KAII mRNA. Based upon these data, a more direct mechanism appears to be responsible for the metastasis suppression by KAII. Consistent with this possibility, high KAII expressing AT6.1-11-1* hybrid cells have about 50% reduction in their invasive ability as compared to parental AT6.1 cells or nonsuppressed AT6.1-11-2 hybrid cells in Boyden chamber assay. In brief, Boyden chamber invasion assays were performed as Ae described by J. Vukanovic et al. (1993) Cancer Res., 53: 29 S 1833), using matrigel coated filters and 5% fetal bovine serum as chemoattractant in the lower well. During the 12 hours of the assay, 19±3 parental AT6.1 cells per high power field invaded through the matrigel filters versus 10±2 for the metastasis suppressed AT6.1-11i-1 hybrid cells and 18±2 for the nonsuppressed AT6.1-11-2 hybrid cells.
Example Expression of KAII Gene In Human Tissues To evaluate the expression level of KAII gene in various human tissues, Northern analysis was performed on RNA isolated from multiple human tissues. In brief, a human multiple tissue Northern blot purchased from Clontech (Palo Alto, CA) was hybridized sequentially with 15 KAII and human f-actin probes under conditions described in the Methods section. The results presented in Figure show that the 2.4 kb KAI1 transcript was detected in all the human tissues tested, with high abundance in prostate, lung, liver, kidney, bone marrow and placenta; moderate abundance in mammary gland, pancreas, skeletal muscle and thymus; and low expression in brain, heart, ovary, stomach and uterus.
Example 6 Conservation Of The KAII Gene Across Species To determine if the KAII gene is evolutionarily conserved across species, a zoo blot containing EcoRIdigested genomic DNA from various species was purchased from Clontech (Palo Alto, CA) and hybridized with KAII probe. The results presented in Figure 6 show that the evolutionary conservation of KAI1 coding sequence is high in human, monkey, dog and rabbit and moderate in cow, rat, mouse. The evolutionary conservation and wide tissue distribution for KAII suggest that the gene may have an essential biological function.
30 Example 7 Correlation Of Altered KAII Expression In Human Tissue Samples With Metastasis Tumor biopsies of liver metastases from prostate cancer patients and liver biopsies from healthy patients are analyzed for KAII mRNA expression by Northern blotting. KAII mRNA expression is lost in the tumor samples indicating that the presence of liver metastases in the prostate cancer patients is correlated with altered KAII expression.
The entire disclosure in the complete specification of our Australian Patent Application No.
55790/96 is by this cross-reference incorporated into the present specification.
1 b** Sooee
C.
31 SEQUENCE LISTING GENERAL INFORMATION: APPLICANTS: THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES AND JOHN HOPKINS UNIVERSITY (ii) TITLE OF INVENTION: DIAGNOSTIC METHODS AND GENE THERAPY USING REAGENTS DERIVED FROM THE HUMAN METASTASIS SUPPRESSOR GENE KAII (iii) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: MORGAN FINNEGAN, L.L.P.
STREET: 345 PARK AVENUE CITY: NEW YORK COUNTRY: USA ZIP: 10154 COMPUTER READABLE FORM: MEDIUM TYPE: FLOPPY DISK COMPUTER: IBM PC COMPATIBLE OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: WORDPERFECT 5.1 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: TO BE ASSIGNED FILING DATE: 25-APR-1996
CLASSIFICATION:
(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/430,225 FILING DATE: 28-APR-1995
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: BLUM, ISRAEL REGISTRATION NUMBER: 26,710 REFERENCE/DOCKET NUMBER: 2026-4172PCT (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (212) 758-4800 TELEFAX: (212) 751-6849 TELEX: 421792 32 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGAAGATCAA GTTGAAGAGG INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 15 GGGACCTCAT TTCCTAGCTG INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGAAACTGC TCTTGTCGG 19 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TCAGCTCTTG GCTCCCCATT 33 0 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID TGGGCACGGG TTTCAGGAAA T 21 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 15 TGCAGAGAGC CCCAAATGCA INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AGGGTGAGCC GTGAGCACAA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGCTGAGAGT ACCCAGATGC 34 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GATGGCCACA CCCACGCCC 19 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs S. TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 9** 5 (xi) SEQUENCE DESCRIPTION: SEQ ID 15 TGCATGGAGA AGGTGCAGGC INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CCTCTTGCCC ACCCTGACTGA 21 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TTCACACCAT TCTCCTGCCT 35 0 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: AGTCCTCCCT GCTGCTGTGT G 21 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 15 TCAGTCAGGG TGGGCAAGAG G 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GAGGAGCACC CCGTGCTGCT GA 22 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CTAGAAGCAT TTGCGGTGGA CGATGGAGGG GCC 33 36 0 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TTGTAACCAA CTGGGACGAT ATGG 24 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear o..
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 15 GTCTTGATCT TCATGGTGCT AGG 23
Claims (9)
1. A method of detecting the ability of a cell to metastasise, comprising: comparing the level of KAI1 mRNA or KAI1 protein in the cell to the level of wild-type KAI1 mRNA or protein in a control cell to determine the presence of a reduction of expression of KAI1 mRNA or protein, wherein the reduction of expression indicates an ability of the cell to metastasise.
2. The method of claim 1, wherein said reduction of expression of KAI1 protein is detected by Western blotting or immunohistochemistry.
3. The method of claim 1, wherein the wild-type 15 KAI1 protein has an amino acid sequence as set forth as SEQ ID
4. A method for detecting the ability of a cell to metastasise, comprising: comparing the glycosylation of the KAI1 protein 20 of the cell to the glycosylation of the wild-type KAIl protein to determine the presence of a change in glycosylation, wherein the change in glycosylation indicates the ability of the cell to metastasise.
A method for determining the prognosis of a tumor in a subject, comprising: comparing the level of KAI1 mRNA or KAI1 protein in the cell to the level of wild-type KAI1 mRNA or protein in a control cell to determine the presence of a reduction of expression of KAI1 mRNA or protein, wherein the reduction of expression indicates the prognosis of the tumor in the subject.
6. The method of claim 5, wherein the tumor is a breast, prostate, or ovarian tumor.
7. The method of claim 5, wherein said reduction of expression of KAI1 protein is detected by Western blotting or immunohistochemistry.
8. A method for determining the prognosis of a \\molbjfiles\home$\cintae\Keep\speci\55790.96 div.doc 1/09/00 38 tumor in a subject, comprising: comparing the glycosylation of the KAIl protein of the cell to the glycosylation of the wild-type KAIl protein to determine the presence of a change in glycosylation, wherein the change in glycosylation indicates the prognosis of the tumor in the subject.
9. The method of claim 8, wherein the tumor is a breast, prostate, or ovarian tumor. A method according to any one of claims 1, 4, 5 or 8 substantially as herein described with reference to the Examples. Dated this 1st day of September 2000 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, REPRESENTED S: 15 BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES AND JOHN HOPKINS UNIVERSITY By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and 20 Trade Mark Attorneys of Australia ***Oo \\melb.fils\home$\cinta.\Keep\speci\5579og6 div.doc 1/09/00
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