WO2000009526A2 - Methode de localisation nucleaire de dpc4 (smad4) - Google Patents

Methode de localisation nucleaire de dpc4 (smad4) Download PDF

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WO2000009526A2
WO2000009526A2 PCT/US1999/018540 US9918540W WO0009526A2 WO 2000009526 A2 WO2000009526 A2 WO 2000009526A2 US 9918540 W US9918540 W US 9918540W WO 0009526 A2 WO0009526 A2 WO 0009526A2
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dpc4
protein
cell
binding
compound
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PCT/US1999/018540
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WO2000009526A3 (fr
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Scott E. Kern
Jia Le Dai
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The Johns Hopkins University School Of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to gene expression in normal and neoplastic cells, and more specifically to a tumor suppressor gene, Dpc4 (Smad4), and its role in tumor suppression.
  • Dpc4 tumor suppressor gene
  • Cancer genes are broadly classified into “oncogenes” which, when activated, promote tumorigenesis, and “tumor suppressor genes” which, when nonfunctional, fail to suppress tumorigenesis. While these classifications provide a useful method for conceptualizing tumorigenesis. it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
  • Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation.
  • the gene coding for a tumor suppressor protein is mutated, deleted or transcriptionally nonfunctional, the resulting mutant protein or the complete lack of tumor suppressor protein expression may fail to correctly regulate cellular proliferation, and abnormal cellular proliferation may take place, particularly if there is already existing damage to the cellular regulatory mechanism.
  • a number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes.
  • tumor suppression genes include, but are not limited to, the retinoblastoma susceptibility gene or RB gene, the p53 gene, the deleted in colon carcinoma (DCC) gene and the neurofibromatosis type 1 (NF-1) tumor suppressor gene (Weinberg, R. A. Science, 1991 , 254: 1 138).
  • Loss of function or inactivation of tumor suppressor genes may play a central role in the initiation and/or progression of a significant number of human cancers.
  • the transforming growth factor- ⁇ (TGF ⁇ ) ligand initiates signals crucial for tumor suppression.
  • Dpc4 (deleted in pancreatic carcinoma, locus 4) or Smad4 is implicated in mediation of signals from TGF ⁇ and related ligands.
  • Wild-type Dpc4 mediates TGF ⁇ -stimulated gene transcription at specific DNA sequences bound by Dpc4, called Smad-Binding Element or SBE.
  • Cancer cells harboring Dpc4 alterations have a universal impairment of the TGF ⁇ -Dpc4-SBE signaling pathway. Defects in the TGF ⁇ pathway are highly prevalent in tumors and occur at multiple levels, involving mutations and aberrant expression of TGF ⁇ receptors and genetic alterations of Smad genes " .
  • Dpc4 activity is a major final determinant of the TGF ⁇ tumor-suppressor function.
  • the Dpc4 gene (Smad4, MADH4) was cloned as a target of homozygous deletion in pancreatic and occasional other cancers .
  • Dpc4 protein is structurally similar to a family of related (Smad) proteins conserved among diverse species " . These similarities are greatest in an N-terminal region termed MH1 (Mad homology 1) and a C-terminal region, MH2 (Fig. 1). During human tumorigenesis, point mutations cause premature truncation and amino acid substitutions of the C-terminal MH2 region and, at lesser frequency, substitutions in the N-terminal MH1 region of Dpc4 " ' " (Fig. 1). Presumably, each of these mutations inactivates a function required for tumor suppression. A mechanistic understanding of the natural defects in Dpc4 function found in tumors might provide a means to restore the downstream transcriptional output of the TGF ⁇ tumor-suppressive signal.
  • Dpc4 A number of properties of the Dpc4 protein have been identified. Smad proteins can mediate or mimic signaling that is initiated by ligands of the TGF ⁇ superfamily, including bone morphogenetic proteins, activin, and TGF ⁇ ,5 . Dpc4 binds other Smads, perhaps aiding signal transduetion events that originate from various ligands Nuclear localization of Dpc4 is demonstrable and may be under regulatory control More direct evidence for a role in nuclear events is provided by the ability of the C- terminus of Dpc4 to activate transcription when fused to a modular DNA-binding domain None of these properties have been demonstrated to be uniformly deficient among cells harboring natural Dpc4 alterations.
  • Dpc4 was found to bind to sequences found in the TGF ⁇ -responsive construct p3TP-lux 22 , and, with greater avidity and specificity, to an unrelated palindromic 8-basepair sequence termed the SBE (Smad- Binding Element) that can confer Dpc4-dependent transcriptional activation to a minimal promoter .
  • SBE Smad- Binding Element
  • the invention provides a method of treating a cell proliferative disorder in a subject by administering a composition comprising a chimeric polypeptide wherein the polypeptide comprises Dpc4 coupled to a factor or a Dpc4-binding moiety coupled to a factor.
  • the factor is effective in localizing Dpc4 to the nucleus of the cell.
  • the invention provides a method of identifying a TGF- ⁇ inducible gene, comprising contacting a polynucleotide with a Dpc4 polypeptide and detecting binding of the polypeptide to the polynucleotide where Dpc4 is made to be active by localizing it to the nucleus as described herein.
  • the invention provides a method for identifying a compound which modulates the nuclear localization of Dpc4 and binding to a Smad- binding element (SBE) by incubating components comprising the compound and a SBE operably linked to a reporter gene in a cell under conditions sufficient to allow the components to interact and measuring the expression of the reporter gene, wherein expression is indicative of the nuclear localization of Dpc4 and binding to the SBE.
  • SBE Smad- binding element
  • the invention provides a method for identifying a Dpc4- interacting peptide, comprising contacting a Dpc4 polypeptide or fragment thereof with a compound that localizes Dpc4 to the nucleus and detecting binding of the compound to Dpc4 through the use of a Smad-binding element (SBE) operably linked to a reporter molecule.
  • SBE Smad-binding element
  • the invention further provides a method for inducing a biological process in a cell comprising by contacting a cell with at least one genetic construct encoding a chimeric protein comprising at least one ligand-binding domain and a heterologous protein domain which induces the biological process, under conditions which allow for the expression of the protein and exposing the cell to an agent which binds with the Hgand in an amount effective to activate the heterologous protein and thereby induce the biological process.
  • Figure 1 shows diagrams of reporter and expressor constructs used in transfections and translations.
  • nSBE n tandem copies of the SBE palindromic sequence.
  • 6MBE six tandem copies of a modified SBE having two nucleotide substitutions and lacking Smad-binding ability.
  • SV40 the SV40 minimal promoter.
  • CMV the cytomegalovirus enhancer/promoter.
  • MER the modified murine estrogen receptor- binding core domain.
  • WT wild-type.
  • pDPC4-mut a mutation series comprising the individual mutations at the given codons: DPC4-100T, DPC4-130S, etc. T, mutation to threonine.
  • S mutation to serine.
  • H mutation to histidine.
  • fs frameshift mutation, st, stop (nonsense) mutation.
  • Figure 2 shows a bar graph of Dpc4-dependent, SBE-mediated transcriptional activation.
  • A) Sensitivities of pnSBE-Luc reporter series and p6MBE-Luc control reporter for Dpc4 function.
  • the DPC4-n ll breast cancer cells MDA-MB-468 were cotransfected 0.3 mg of a reporter construct, with or without 0.3 mg pDPC4-WT, as indicated.
  • Transfected cells were treated with 1 ng/ml TGF ⁇ or untreated. Luciferase activities were normalized to levels of b-galactosidase, which remained relatively constant. Mean + SEM is determined from three independent experiments.
  • 0.2 mg p6SBE-luc was cotransfected with 0.2 mg SMAD2, SMAD3, and DPC4 expression constructs into MDA-MB-468 cells either individually or in combination. Luciferase activities are normalized to b-galactosidase activity. Mean + SEM was determined from four independent experiments. Results are given relative to cells transfected with the parental expression vector (arbitrarily set at a value of 1). Figure 3 shows a bar graph of impaired function among the mutant Dpc4 proteins and expression of such proteins. A) SBE-mediated transactivation upon transient transfection of a panel of DPC4 expression constructs.
  • MDA-MB-468 cells were cotransfected with 0.3 mg p6SBE-Luc reporter and 0.3 mg wild-type or mutant DPC4 expression constructs as indicated. Luciferase activities were normalized to b-galactosidase and plotted relative to cells transfected with the parental (pcDNA3.1) expression vector (value of 1). Mean + SEM was determined from three to seven independent experiments. Similar results were obtained in another £> C -null cell line, BxPC3 .
  • Figure 4 is a bar graph showing nuclear redistribution of Dpc4 restores defective TGF-b-stimulated signal transduetion.
  • the DPC4-null MDA-MB-468 cells were transfected with 0.3 mg of the p6SBE-Luc reporter, with or without 0.3 mg expression vector, as indicated.
  • Transfected cells were treated with 1 ng/ml TGF- ⁇ and/or 100 nM 4-OHT (4-hydroxytamoxifen) or untreated. Luciferase activities were normalized to levels of b-galactosidase. Mean + SEM is determined from two to seven independent experiments. In TGF- ⁇ type II receptor-negative HCT116 cells, 4-OHT caused a 4-5-fold induction .
  • Figure 5 is a bar graph showing defects in the TGF-b-Dpc4-SBE pathway in genetically defined cancer cell lines.
  • A) A panel of pancreatic, breast, and colorectal cancer cell lines having different DPC4 genetic status transfected with a reporter construct. Luciferase activity was normalized to b-galactosidase activity and expressed as a ratio of p6SBE-Luc to p6MBE-Luc values. Mean + SEM was determined from three independent experiments.
  • Cell transfectants were treated with 1 ng/ml TGF ⁇ or untreated.
  • the luciferase activities of the p6SBE-Luc reporter were normalized to b- galactosidase activity and were expressed relative to those of the parental reporter vector. Mean + SEM was determined from three independent experiments.
  • Figure 6 is a Western blot showing stable expression of functional Smad4-MER in transfected clones of the breast cancer line MDA-MB-468. Immunoblots confirm the expression of full-length Smad4-MER, MER, and 100T-Smad4-MER.
  • Figure 7 is a bar graph showing that fusion proteins exhibit the expected activity with SBE-containing transcriptional reporters.
  • Stable clones were transfected with SXBE-Luc (S) or 3TP-lux (T) before being treated for 20 h with (+) or without (-) 1 OOnM 4-OHT.
  • pCMN ⁇ was cotransfered to normalize for transfection efficiencies. Luciferase and ⁇ -galactosidase activities were determined. The luciferase activities in the absence of 4-OHT is arbitrarily set at 1. The means of three independent experiments are shown. Bars represent S.E.
  • Figure 8 shows growth curves for growth suppression following Smad4 nuclear translocation.
  • Clones that stably expressed Smad4-MER (Panel A and Panel B), MER (Panel C), or 100T-Smad4-MER (Panel D) were treated with (square and dashed line) or without (circle and solid line) 100 nM 4-OHT for 0, 1, 2, or 4 days before harvesting for cell counting.
  • Data are analyzed from three independent experiments, and presented as mean ⁇ S.E.
  • Figure 9 is a bar graph showing G, cell cycle arrest upon nuclear translocation of Smad4. Clones that stably expressed Smad4-MER, MER, or 100T-Smad4-MER were treated for 1 day with (+) or without (-) 100 nM 4-OHT. Data from three to four independent experiments are shown, (mean ⁇ S.E., *P ⁇ 0.05, ** ⁇ 0.01 , comparison to 4- OHT-untreated groups, using paired t-test.)
  • Figure 10 shows the results of TU ⁇ EL assays performed to identify apoptosis upon nuclear translocation of Smad4.
  • MDA-MB-468 cells that stably expressed Smad4- MER (Panel A), MER (Panel B), and 100T-smad4-MER (Panel C) were treated with (right column (+)) or without (left column (-)) 100 nM 4-OHT for 2 days. Both attached and unattached cells were harvested and processed for TU ⁇ EL assays. A representative set from three independent experiments is shown for each clone.
  • Figure 11 is a bar graph showing the data analysis of the TU ⁇ EL assays of cells in Figure 10. The induction was determined using percentages of apoptotic cells in the presence (+) of 4-OHT divided by those in the absence (-). Paired t-test was used. *P ⁇ 0.05 vs in the absence of 4-OHT mean ⁇ S.E.
  • Figure 12 is a bar graph showing that caspase inhibitors interfere with the Smad4- induced apoptosis in Smad4-MER cells.
  • Cells were pretreated with 50 ⁇ M caspase inhibitor I (I), 50 ⁇ M caspase-3 inhibitor II (II) or vehicle (V) or 1 h before 100 nM 4- OHT was added. An average of two independent experiments is presented. DETAILED DESCRIPTION
  • DPC4 can be viewed as a switch, in an "on" or "off state.
  • the present invention is based upon the discovery that a major factor governing the switch is Dpc4's nuclear localization. TGF- ⁇ can cause Dpc4 to go to the nucleus.
  • the inventors have made an estrogen-receptor fusion that drives Dpc4 to the nucleus. Even in cells lacking TGF- ⁇ receptors, Dpc4 localized to the nucleus turned on downstream reporters (based on the SBE binding element that allows an assay of Dpc4 function) when estrogen was added. In other words, Dpc4 was driven into the nucleus. This indicates that a key aspect of the upstream TGF-b defects of cancer cells can be bypassed by artificially driving Dpc4 to the nucleus by a moiety unrelated to the natural Smad binding partners of Dpc4.
  • the invention provides a chimeric drug that includes a Dpc4-interacting peptide, linked to a nuclear-localizing sequence.
  • the drug could be from about 10-20 amino acids. It is possible to screen drug libraries to find a random drug that does the same, and then optimize the structure. The examples below demonstrate the principle that the simple direction of Dpc4 to the nucleus is adequate to activate gene expression through the DNA-binding site bound by Dpc4. Modifications expected in the development of a therapeutic would include:
  • the one capability is biologically similar to the other.
  • the reporter used in the current invention comprises 8-basepair repeats termed the SBE, each of which constitutes two 4-basepair SBE units arranged head-to-head. Since each Dpc4 molecule binds only a
  • 4-basepair sequence of the SBE in the reporter 4-basepair sequences in the genome can also serve as a site of binding and cause the expression of nearby genes.
  • the repeated sequence GTCT in a number of orientations or spacings is expected to provide for Dpc4 what is in essence the same biological activity.
  • GTCT sequences are the near-equivalent in DPC4 binding to GCCT sequences
  • GTCTAGAC is near-equivalent to GTCTAGGC.
  • Exemplary peptides include GTCT, GNCT, GTCTAGAC (SEQ ID NO:2) and GNCTAGNC (SEQ ID NO:3), wherein N is any nucleotide. 4) Nuclear localization capabilities provided in trans or in cis.
  • the current invention provides the basic principle, using a fusion protein that provides a nuclear localization motif in cis (meaning, in the same molecule).
  • An interacting moiety such as an artificial compound or a polypeptide, such as PKKKRKVEDP (SEQ ID NO: 18), could also provide the nuclear localization provided that it had a region that bound to Dpc4 protein; this is the provision of nuclear localization in trans (meaning, by an interacting molecule).
  • Other short sequences enriched in basic amino acid residues that constitute a nuclear localization property will be known in the art or can be readily determined.
  • the modeling of a drug that would provide the nuclear localization in trans was the reason for the current work.
  • Other nuclear localization sequences are known in the art.
  • Nuclear localization capabilities enabled in trans whether they are provided by polypeptides expressed within the cell or by molecules (either polypeptides or chemical compounds) provided outside the cell and allowed to enter the cell, as in drug administration. It is not necessary to specify the exact mode of nuclear translocation, since it is known in the art that the nuclear localization of proteins can be accomplished by increased delivery of a protein to the nucleus or by increased retention of the active protein within the nucleus and that multiple means are known in the art by which these properties can be affected by interactions in trans.
  • Drugs that act to directly activate Dpc4 function by redistributing Dpc4 to the nucleus, whether identified by the screening of random compounds or by rational drug design. Any selection of compounds can be screened for particular compounds that activate the expression of a gene linked in cis to 4-basepair SBE units or 8-basepair palindromic SBE units. It is not necessary to determine the nuclear localization by a direct means in order to identify an appropriate drug activity, since the SBE-binding and gene transcriptional activities of Dpc4 can be measured by elevations in the level of expression of genes linked in cis.
  • the invention provides a method of treating mutation or other defect in the TGF- ⁇ regulatory pathway. For example, mutations in TGF- ⁇ receptors, Smad molecules and Dcp4 molecules that effect their normal operations and/or expression of genes could be treated by the methods of the present invention.
  • “Mutation” is the process whereby changes occur in the quantity or structure of the genetic material of an organism. Mutations are permanent alterations in the genetic material which may lead to changes in phenotype. Mutation can involve modifications of the nucleotide sequence of a single gene, blocks of genes or whole chromosomes. Changes in single genes may be the consequence of point mutations, which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence, or they can be the consequence of changes involving the insertion or deletion of large numbers of nucleotides. Modifications of whole chromosomes include both changes in number or structural changes involving chromosome abnormalities. Numerical chromosome mutations can involve multiples of the complete karyotype, termed
  • polyploidy or they may involve deviations from the normal number of chromosomes, termed “aneuploidy”. Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements within genomes. They are also induced following exposure to chemical or physical mutagens. Such mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse range of chemicals such as the alkylating agents, and polycyclic aromatic hydrocarbons, all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids. The DNA lesions induced by such environmental agents may lead to modifications of the base sequence when the affected DNA is replicated or repaired and thus to a mutation.
  • somatic mutations as causally important in the induction of human cancers. These somatic mutations may accumulate in the genomes of previously normal cells, some of which may then demonstrate the phenotypes associated with malignant growth.
  • Such oncogenic mutations may include a number of different types of alterations in DNA structure, including deletions, translocations and single nucleotide alterations. The latter, also known as point mutations, may frequently intervene in carcinogenesis, since a variety of mutagenic chemicals induce such mutations. In addition, such mutations may occur spontaneously as a result of mistakes in DNA replication.
  • mutant or mutated as applied to a target neoplastic nucleotide sequence shall be understood to encompass a mutation, a restriction fragment length polymorphism, a nucleic acid deletion, or a nucleic acid substitution.
  • a point mutation constitutes a single base change in a DNA strand, for example a G residue altered to a T. Such a mutation may alter the identity of the codon in which it lies thereby creating a missense mutation or nonsense mutation.
  • Transition mutations involve the substitution of one purine in the DNA by another purine or one pyrimidine by another pyrimidine, that is A by G and vice versa, or T by C and vice versa.
  • a missense mutation is a point mutation in which a codon is changed into one encoding amino acid other than that normally found at a particular position.
  • a nonsense mutation is any mutation that converts a codon specifying an amino acid into one coding for termination of translation.
  • a splicing mutation is any mutation affecting gene expression by affecting correct RNA splicing. Splicing mutations may be due to mutations at intron-exon boundaries which alter splice sites.
  • a polyadenylation site mutant is a mutation of the consensus sequence required for addition of poly(A) to the 3' end of mature mRNA and which results in premature mRNA degradation.
  • An insertion is any mutation caused by the insertion of a nucleotide or stretch of nucleotides into a gene. For example, naturally occurring insertion mutations can be the result of the transposition of transposable genetic elements.
  • Mutations that occur in somatic cells are not transmitted to the sexually produced offspring. However, such somatic mutations may be transferred to descendant daughter cells and mutations in some specific genes have been implicated in cancer. It is now clear that mutations may lead to the induction of cancer when they occur in one or more of a battery of normal genes referred to as the proto-oncogenes.
  • Proto-oncogenes may be modified by a variety of mutational changes to produce the cancer-causing oncogenes of which more than forty have been identified.
  • Proto-oncogenes play an essential part in the control of cell growth and differentiation and disruption of their normal activity by mutational events may lead to the aberrant growth characteristics observed in cancer cells.
  • Such genes were identified because most tumors that lack functional copies of a suppressor gene (i.e., p53, Dcp4 or Rb) display two identically mutated (i.e., homozygous) alleles, while the unaffected tissues can be shown to carry one mutant tumor suppressor allele and one wild-type allele. Therefore, the loss of heterozygosity (LOH) at particular chromosome regions in tumor cells (compared with somatic cells from the same individual) is generally regarded as evidence for the unmasking of mutations in tumor suppressor genes located in these regions.
  • LHO heterozygosity
  • siRNA mutations changes in DNA sequence which do not result in a detectable phenotypic change, termed "silent mutations", also occur in structural genes. Mutations in structural genes are silent if they do not change the amino acid inserted or if they result in substitution of a residue that does not affect protein function. Silent mutations can also be located outside protein-coding regions. A neutral mutation constitutes a genetic change which results in a phenotype which has no effect on an organism's fitness. Thus, even when the substitution results in the insertion of a different amino acid into a polypeptide, a missense mutation, the amino acid may be an acceptable substitute and may not lead to any significant change in the activity of the polypeptide.
  • missense amino-acid changes can have drastic effects upon the folding of polypeptide chains or upon the configuration of the active site of an enzyme.
  • Other base substitutions may have drastic effects because they convert a triplet coding for an amino acid into one of the three termination signals which lead to the premature termination of polypeptide synthesis.
  • Such nonsense changes are usually accompanied by the loss of function of the gene product.
  • Dpc4 activity may include peptides, peptidomimetics, polypeptides, chemical compounds and biologic agents. Dpc4 activity can be assayed using methodology as described in the present Examples. Incubating includes conditions which allow contact between the test compound and Dpc4 or a test compound, Dpc4 and an SBE. Contacting includes in solution and in solid phase, or in a cell. The test compound may optionally be a combinatorial library for screening a plurality of compounds.
  • Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al, Bio/Technology, 3: 1008-1012, 1985), allele-specific ohgonucleotide (ASO) probe analysis (Conner, et al, Proc. Natl. Acad. Sci. USA, 80:278, 1983), ohgonucleotide ligation assays (OLAs) (Landegren, et al, Science, 241 : 1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al, Science, 242:229-237. 1988).
  • agonists that influence Dpc4 activity may be used to provide a therapeutic for such cell proliferative disorders.
  • mutations that affect Dpc4 binding to SBE sites and subsequent regulation of TGF- ⁇ inducible genes may be overcome by administering wild-type Dpc4 to the cell or subject.
  • localization of Dpc4 to the nucleus may also provide therapeutic benefit.
  • factors either delivered along with Dpc4 or alternatively attached to Dpc4 may effect Dpc4 nuclear localization and thus effect Dpc4 activity.
  • polynucleotides encoding a chimeric polypeptide, wherein the polypeptide comprises Dpc4 coupled to a nuclear localization sequence or estrogen-receptor Hgand may be delivered to the subject or cell requiring such therapy.
  • "Chimeric" as used herein means a polynucleotide or polypeptide that comprise two or more individual units that are linked, for example a chimeric polypeptide may comprise a unit such as an estrogen-receptor Hgand linked to a second unit such as a Dpc4 polypeptide or a Dpc4-binding polypeptide.
  • a chimeric polynucleotide would be a polynucleotide sequence encoding an estrogen-receptor Hgand unit linked to a polynucleotide encoding a Dpc4 polypeptide unit or a Dpc4-binding polypeptide.
  • a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • viral vectors which can be utilized for gene therapy as taught herein include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the antisense polynucleotide.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include but are not limited to ⁇ 2, PA317 and PA 12, for example. These cell lines produce empty virions, since no genome is packaged.
  • a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al, Trends Blochem. Sci., 6:77, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al, Biotechniques, 6:682, 1988).
  • the composition of the liposome is usually a combination of phosphohpids, particularly high-phase-transition-temperature phosphohpids, usually in combination with steroids, especially cholesterol. Other phosphohpids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the Hpid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phosphohpids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes has been classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system
  • RES in organs which contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific Hgand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • Hpid groups can be incorporated into the Hpid bilayer of the liposome in order to maintain the targeting Hgand in stable association with the Hposomal bilayer.
  • Various linking groups can be used for joining the Hpid chains to the targeting Hgand.
  • the compounds bound to the surface of the targeted delivery system will be ligands and receptors which will allow the targeted delivery system to find and "home in" on the desired cells.
  • a Hgand may be any compound of interest which will bind to another compound, such as a receptor.
  • the therapeutic agents useful in the method of the invention can be administered parenterally by injection or by gradual perfusion over time. Administration may be intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride
  • lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents and inert gases and the like.
  • the invention includes polypeptides having an amino acids sequence substantially the same as the amino acid sequence set forth in SEQ ID NO: 1 or functional fragments thereof, or amino acid sequences that are substantially identical to SEQ ID NO:2.
  • substantially the same or substantially identical is meant a polypeptide or nucleic acid exhibiting at least 80%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
  • substantially identical is also meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein assayed, (e.g., as described herein).
  • such a sequence is at least 85%, more preferably identical at the amino acid level to SEQ ID NO:2.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705.
  • Minor modifications of the Dpc4 primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the Dpc4 polypeptide described herein.
  • Such proteins include those as defined by the term having essentially the amino acid sequence of the polypeptide of SEQ ID NO: 1.
  • modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the biological activity of Dpc4 still exists.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would have broader utility. For example, one can remove amino or carboxy terminal amino acids which are not required for Dpc4 biological activity. For example, the amino terminal end of Dpc4 is not necessary for SBE binding.
  • the Dpc4 polypeptides of the invention encoded by the polynucleotide of the invention includes the disclosed sequence (SEQ ID NOs: 1 and 2) and conservative variations thereof.
  • DNA sequences of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, 2) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest, and 3) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features.
  • hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, 2) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest, and 3) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features.
  • the Dpc4 polynucleotide of the invention is derived from a mammalian organism, and most preferably from human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available.
  • Ohgonucleotide probes which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known.
  • the DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • DNA sequences encoding Dpc4 can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double- stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • genomic DNA isolates are the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
  • the synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression.
  • phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al, Nucl Acid Res., JI:2325, 1983).
  • a cDNA expression library, such as lambda gtl 1 can be screened indirectly for
  • Dpc4 peptides having at least one epitope using antibodies specific for Dpc4.
  • Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of Dpc4 cDNA.
  • DNA sequences encoding Dpc4 or a chimeric Dpc4-interacting nuclear localization factor can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the polynucleotide sequences of Dpc4 or a chimeric Dpc4-interacting nuclear localization factor may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the Dpc4 genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al, Gene, 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. , 263:3521 , 1988) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • Polynucleotide sequences encoding Dpc4 or a chimeric factor can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaC method using procedures well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the Dpc4 of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • the invention includes a method of identifying a TGF-b- inducible gene by the detection of genes whose expression is induced upon the nuclear localization of Dpc4 as described herein.
  • differential gene expression screening by methods known in the art are applied to identify genes whose expression is induced. Augmented expression of a gene constitutes evidence of a TGF-b-inducible property.
  • additional assays for TGF-b-inducibility are well known in the art and can be used to further evaluate this particular property of the gene identified by application of the method of the invention.
  • the present invention provides a method for identifying a target gene transcriptionally-modulated by a chimeric protein, such as, for example, Dpc4-MER, by contacting a cell with at least one genetic construct encoding a chimeric protein having at least one ligand-binding domain and a heterologous protein domain which induces activation of a target gene; exposing the cell to an agent which binds with the Hgand in an amount effective to activate the heterologous protein, wherein the activated protein effects the activation of transcription of a target gene under the transcriptional control of a transcriptional control element responsive to said protein; and detecting the effect of the protein on the target gene transcription, thereby identifying the gene transcriptionally- modulated by the protein.
  • a chimeric protein such as, for example, Dpc4-MER
  • an “agent” is any molecule which effects the ligand-binding domain attached to the heterologous protein such that the protein is functional.
  • a “ligand-binding domain”, as used herein, is any peptide or other molecule which effects the activity of the protein to which it is attached.
  • An “agent”, as used herein, is any molecule which modulates the effect of the ligand-binding domain on the protein.
  • a fusion gene consisting of Dpc4 linked to a modified Hgand binding domain of the murine estrogen receptor (MER) has been constructed.
  • An agent such as 4-hydroxytamoxifen (4-OHT), can modulate the cytoplasmic sequestration effect of MER on Dpc4 (Smad4).
  • heterologous protein domain is any nucleic acid sequence encoding a protein useful in the method of the present invention.
  • the domain encodes a protein which localizes to the nucleus of a cell. More preferably, the domain encodes a protein involved in cell cycle control, such as , for example
  • the method of the invention is useful for studying genes controlled by Dpc4 in a variety of experimental cell systems.
  • the present method can be used to identify those genes which are modulated by the nuclear localization of Dpc4. Detection of such modulation can be by any means know in the art including modulation of transcription and protein expression.
  • transcription modulation techniques such as subtractive hybridization or SAGE can be used to detect increased or decreased levels of a particular transcript.
  • techniques such as Western blots are useful in the present invention.
  • the method of the invention is useful for identifying the effect of, for example, Dpc4 nuclear localization, on hyperplasia (oncogenic function) or involution (apoptotic function) in a particular cell or tissue.
  • Biochips or arrays of binding agents, are used in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
  • arrays find use by serving as a substrate to which is bound polynucleotide "probe” fragments.
  • probe polynucleotide fragments.
  • the targets are then hybridized to the immobilized set of polynucleotide "probe” fragments.
  • the present invention provides nucleic acid and amino acid sequences useful, for example, for screening for differential expression of genes in the presence of Dpc4 nuclear translocation.
  • COMPOUNDS WHICH MODULA TE DPC4 NUCLEAR LOCALIZA TION Also included is a method of identifying compounds that regulates Dpc4 activation by modulating Dpc4 nuclear localization.
  • the method includes identifying a compound which binds to Dpc4 polypeptide, or fragments thereof, by incubating a test compound and Dpc4 polypeptide under conditions sufficient to allow the compound and Dpc4 polypeptide to form a complex.
  • modulate includes any compound which increases or decreases the activity of Dpc4 by affecting the nuclear translocation of Dpc4.
  • hydrophobic molecules such as long chain fatty acids may require buffers including DMSO or glycerol.
  • Compounds can thus be any number of molecules including polypeptides, carbohydrates, fatty acids, and/or steroids.
  • the molecules may be labeled.
  • the Dpc4 or the test compound may be chemically labeled with a fluorescence compound, a radioactive element or a metal chelating agent.
  • the method also involves separating a complex of Dpc4 polypeptide and the binding compound from unbound Dpc4 polypeptide and measuring the binding or effect of binding of the compound to Dpc4 polypeptide. Separation of the compounds and Dpc4 can be accomplished by any number of means including chromatography, gel electrophoresis and other well known to those skilled in the art.
  • Dpc4 nuclear localization involves the interaction of a nuclear-localization inhibiting molecule with Dpc4, thereby preventing the activation of Dpc4.
  • the present invention also includes a method for identifying a compound which affects the nuclear localization of Dpc4 by modulating the binding of Dpc4 with an inhibitor of Dpc4 nuclear localization.
  • the murine estrogen receptor (MER) fused to Dpc4 functions, in part, to prevent nuclear localization of Dpc4 by interacting with heat shock proteins.
  • the binding of 4-hydroxytamoxifen (4-OHT) blocks the ability of heat shock proteins in the cytoplasm to bind the estrogen receptor, and thus the Dpc4-estrogen receptor chimera is no longer tethered to the cytoplasm. This then permits the nuclear localizing sequence of Dpc4 to promote nuclear translocation of Dpc4.
  • the present invention provides a method to identify a compound which promotes Dpc4 activation, indirectly, by interacting with molecules which prevent Dpc4 nuclear translocation.
  • the method of the invention encompasses those molecules, such as proteins, peptides or small molecules, which activate Dpc4 but do not interact directly with Dpc4.
  • Dpc4 nucleic acids, proteins, and derivatives of the present invention also have uses in screening assays to detect molecules that specifically bind to Dpc4 nucleic acids, proteins, or derivatives and thus have potential use as agonists or antagonists of Dpc4, in particular, molecules that affect cell proliferation.
  • such assays are performed to screen for molecules with potential utility as anti-cancer drugs or lead compounds for drug development.
  • the invention provides assays to detect molecules that specifically bind to Dpc4 nucleic acids, proteins, or derivatives.
  • recombinant cells expressing Dpc4 nucleic acids can be used to recombinantly produce Dpc4 proteins in these assays, to screen for molecules that bind to a Dpc4 protein.
  • Molecules e.g., putative binding partners of Dpc4 are contacted with the Dpc4 protein (or fragment thereof) under conditions conducive to binding, and then molecules that specifically bind to the Dpc4 protein are identified. Similar methods can be used to screen for molecules that bind to Dpc4 derivatives or nucleic acids. Methods that can be used to carry out the foregoing are commonly known in the art. By way of example, diversity libraries, such as random or combinatorial peptide or nonpeptide libraries can be screened for molecules that specifically bind to Dpc4.
  • libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries.
  • agonists and antagonists of Dpc4 can be identified using "biochip” technology.
  • Biochips or arrays of binding agents, such as oligonucleotides and peptides, have become an increasingly important tool in the biotechnology industry and related fields.
  • binding agent arrays in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like.
  • One important use of biochips is in the analysis of differential gene expression, where the expression of genes in different cells, normally a cell of interest and a control, is compared and any discrepancies in expression are identified. In such assays, the presence of discrepancies indicates a difference in the classes of genes expressed in the cells being compared.
  • arrays find use by serving as a substrate to which is bound polynucleotide "probe” fragments.
  • probe polynucleotide fragments.
  • the targets are then hybridized to the immobilized set of polynucleotide "probe” fragments. Differences between the resultant hybridization patterns are then detected and related to differences in gene expression in the two sources.
  • the present invention provides nucleic acid and amino acid sequences useful for screening for differential expression of Dpc4 in a cell.
  • kits for identifying chemical compounds that modulate Dpc4 (Smad4) nuclear localization are also included in the screening method of the invention.
  • Dpc4 Dpc4 nuclear localization
  • the term "agent” as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of modulating the physiological function of a Dpc4 protein.
  • agent concentrations are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • Drug screening identifies agents that provide enhancement or regulation of Dpc4 function in affected cells.
  • screening assays for agents that have a low toxicity for human cells.
  • a wide variety of assays may be used for this pu ⁇ ose, including labeled in vitro protein-protein binding assays, protein-DNA binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification or amidification to produce structural analogs.
  • the screening assay is a binding assay
  • the label can directly or indirectly provide a detectable signal.
  • Various labels include radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin.
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • a variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g.
  • albumin that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • Reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors or anti-microbial agents may be used.
  • the mixture of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hours will be sufficient.
  • the invention provides a method of treating a subject having or at risk of having a cell proliferative disorder associated with expression of Dpc4 comprising administering to a cancerous cell of the subject, an agent that augments Dpc4 activity or expression.
  • the method includes those treatments which promote nuclear localization of Dpc4.
  • Administration of the reagent can be in vivo or ex vivo.
  • Such an agent includes a polynucleotide the encodes a polypeptide or compound that interacts with Dpc4 and localizes Dpc4 to the nucleus, a polypeptide that has Dpc4 activity or a Dpc4 polypeptide or fragments thereof.
  • the present invention further provides a method for enhancing nuclear localization of wild-type Dpc4 in a neoplastic cell by introducing in to the cell a agent which inhibits wild-type Dpc4 cytoplasmic sequestration and promotes Dpc4 nuclear localization.
  • the invention also provides a method for inhibiting mutant Dpc4 nuclear translocation by introducing in to the cell an agent which promotes mutant Dpc4 cytoplasmic sequestration.
  • agent as used herein, describes any molecule, e.g. protein, small molecule or pharmaceutical, with the capability of modulating the physiological function of a Dpc4 protein.
  • a Dpc4-modulating agent encompasses molecules such as proteins, peptides, small molecules or any compound which modulates Dpc4 activity.
  • the agent is an expressed protein or peptide
  • any of the methods known to the art for the insertion of DNA fragments into a vector as described, for example, in Maniatis, T, et al, (1989): Molecular Cloning (A Laboratory manual), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and Ausubel, F. M., Brent, R., guitarist, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.
  • Dpc4 (Smad4) encoding gene expression vectors consisting of appropriate transcriptional/translational control signals and the desired Dpc4 (Smad4) cDNA sequence downstream from the first in-frame AUG codon.
  • These methods may include in vitro DNA recombinant and synthetic techniques and in vivo genetic recombination.
  • Expression of a nucleic acid sequence encoding Dpc4 may be regulated by a second nucleic acid sequence so that Dpc4 (Smad4) is expressed in a host infected or transfected with the recombinant DNA molecule.
  • expression of Dpc4 (Smad4) may be controlled by any promoter/enhancer element known in the art.
  • the promoter activation may be tissue specific or inducible by a metabolic product or administered substance.
  • Promoters/enhancers which may be used to control gene expression of a Dpc4- modulating polypeptide include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama, H., et al., 1989, J. Exp. Med., 169: 13), the human .beta.-actin promoter (Gunning, P., et al. , 1987, Proc. Natl Acad. Sci. USA, 84:4831), the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (HHTV LTR) (Klessig, D. F., et al, 1984, Mol. Cell Biol.
  • CMV cytomegalovirus
  • HHTV LTR mouse mammary tumor virus long terminal repeat
  • MULV LTR Moloney murine leukemia virus
  • MULV LTR Moloney murine leukemia virus
  • SV40 early region promoter Bernoist and Chambon, 1981, Nature 290:304
  • RSV Rous sarcoma virus
  • HSV he ⁇ es simplex virus
  • HSV thymidine kinase promoter/enhancer
  • Expression vectors compatible with mammalian host cells for use in genetic therapy of tumor or cancer cells include, but are not limited to: plasmids, retroviral vectors, adenovirus vectors, he ⁇ es viral vectors, and non-replicative avipox viruses, as disclosed, for example, by U.S. Pat. No. 5,174,993, inco ⁇ orated herein by reference. Methods of administering viral vectors are well known.
  • a retroviral vector, an adenovirus vector, a plasmid vector, or any other appropriate vector capable of expressing the Dpc4 (Smad4)-modulating protein can be administered in vivo to a neoplastic cell by a wide variety of manipulations. All such manipulations have in common the goal of placing the vector in sufficient contact with the target tumor to permit the vector to transduce or transfect the tumor cells.
  • Neoplastic cells present in the epithelial linings of hollow organs may be treated by infusing the vector suspension into a hollow fluid filled organ, or by spraying or misting into a hollow air filled organ.
  • the tumor cell may be present in or among the epithelial tissue in the lining of pulmonary bronchial tree, the lining of the gastrointestinal tract, the lining of the female reproductive tract, genitourinary tract, bladder, the gall bladder and any other organ tissue accessible to contact with the vector.
  • a viral expression vector may be introduced into a target cell in an expressible form by infection or transduetion.
  • a viral vector includes, but is not limited to: a retrovirus, an adenovirus, a he ⁇ es virus and an avipox virus.
  • Dpc4 (Smad4)- modulating polypeptide When Dpc4 (Smad4)- modulating polypeptide is expressed in any abnormally proliferating cell, the cell replication cycle is arrested, thereby resulting in senescence and cell death and ultimately, reduction in the mass of the abnormal tissue, i.e., the tumor or cancer.
  • a vector able to introduce the gene construct into a target cell and able to express Dpc4 (Smad4)- modulating polypeptide therein in cell proliferation-suppressing amounts can be administered by any effective method.
  • a cancer or tumor present in a body cavity such as in the eye, gastrointestinal tract, genitourinary tract (e.g., the urinary bladder), pulmonary and bronchial system and the like can receive a physiologically appropriate composition (e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector) containing an effective concentration of active vectors via direct injection with a needle or via a catheter or other delivery tube placed into the cancer or tumor afflicted hollow organ.
  • a physiologically appropriate composition e.g., a solution such as a saline or phosphate buffer, a suspension, or an emulsion, which is sterile except for the vector
  • Any effective imaging device such as X-ray, sonogram, or fiberoptic visualization system may be used to locate the target tissue and guide the needle or catheter tube.
  • target tumor or cancer cells can be treated by introducing Dpc4 (Smad4)-modulating protein into the cells by any known method.
  • Dpc4 (Smad4)-modulating protein for example, liposomes are artificial membrane vesicles that are available to deliver drugs, proteins and plasmid vectors both in vitro or in vivo (Mannino, R. J. et al, 1988, Biotechniques, 6:682) into target cells (Newton, A. C. and Huestis, W. H., Biochemistry, 1988, 27:4655; Tanswell, A. K. et al, 1990, Blochmica et Biophysica Acta, 1044:269: and Ceccoll, J. et al. Journal of Investigative Dermatology, 1989, 93:190).
  • Dpc4 (Smad4) protein can be encapsulated at high efficiency with liposome vesicles and delivered into mammalian cells in vitro or in vivo.
  • Liposome-encapsulated Dpc4 (Smad4)-modulating protein may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, intramuscularly, subcutaneously or by any other effective means at a dose efficacious to treat the abnormally proliferating cells of the target tissue.
  • the liposomes may be administered in any physiologically appropriate composition containing an effective concentration of encapsulated Dpc4 (Smad4) protein.
  • a tumor cell is transduced with a retrovirus vector, an adenovirus vector, a plasmid vector or any other appropriate vector capable of expressing the Dpc4 (Smad4)-modulating protein in that tumor cell.
  • the cancer cell may be present in a blood or bone marrow sample collected from a leukemia patient.
  • a dose of the protein expressing retrovirus vector or adenovirus vector or plasmid vector or any other appropriate vector is administered to the sample of blood or bone marrow at a dose sufficient to transduce enough cells in the sample to produce a reduction in tumor cell numbers.
  • the cell proliferation of the treated cancer cells will be slowed or terminated followed by a process similar to normal cellular differentiation or cell senescence.
  • blood or bone marrow or other tissue is treated ex vivo using an effective dose of a lipsome-encapsulated protein. Thereafter the sample may be returned to the donor or infused into another recipient.
  • the present invention provides a method for evaluating the effect of mutations on Dpc4-regulated gene expression by monitoring Dpc4 function in a variety of cell lines free of a requirement for engineered over expression of heterologous protein.
  • an improved vector pair was developed based on six tandem copies of the SBE element (p6SBE) or a non-binding mutant sequence (p ⁇ MBE) (Fig. 1). Transcriptional activation was sensitively dependent upon the SBE sequence and was responsive to the
  • TGF-b-Dpc4-SBE pathway were central to the DPC4 tumor-suppressive role, defects in this signaling system should be uniformly seen among the many types of DPC4 mutants identified in human tumors.
  • a mutant panel was tested, comprising three missense, two frameshif , and four nonsense mutations affecting the MH2 domain, and two missense mutations of the MH1 domain (Figs. 1 and 3 A), representing mutations found in human pancreatic, colorectal, and ovarian tumors " .
  • Transient transfection of wild-type DPC4 expression constructs reconstituted the SBE transactivation function in the DPC4-m ⁇ cell line MDA-MB-468 (Fig. 3 A).
  • the individual expression of nine forms of mutant Dpc4 failed to restore the pathway, while only the codon 130 and 370 mutants appeared to retain minimal wild-type function (Fig. 3A).
  • the inhibitory effects of the mutations could be caused by a failure of the mutant Dpc4 to bind DNA.
  • mutants were tested in an in vitro DNA-binding assay (Figs 3B and 3C).
  • the results established a functional grouping for DPC4 mutations: missense mutations of the MH1 domain (codon 100 arginine to threonine, codon 130 proline to serine) were deficient in SBE-binding, while all mutations of the MH2 region retained variable degrees of binding.
  • An unanticipated augmentation of the SBE-binding ability was seen for mutations at codons 515 and 516 that prevent translation of the last 37 amino acids of Dpc4 (Fig. 3A), suggesting that the C-terminal tail of Dpc4 harbors an inhibitory effect on SBE-binding. This property was obscured when more severe truncating mutations are studied (Fig. 3 A).
  • a mutation at codon 100 in the MH1 domain was sufficient to inactivate the SBE- binding, irrespective of the presence of the C-terminus, as tested by the construction of two truncated forms (343st and 515st) of the codon 100 mutant protein (Fig. 3C).
  • a potentially auto-inhibitory interaction between the MH1 domain and the C- terminus of Dpc4 could be shown for the codon 100 mutation in vitro , such an interaction is not required to explain the emergence of this mutation in tumors.
  • DNA- binding is a property of the MH1 region , and the intrinsic effect of the codon 100 mutation to inactivate this DNA-binding module is sufficient to ablate SBE-mediated gene regulation by DPC4 (Fig. 3C).
  • the codon 130 mutant appeared to produce discrepant results upon comparison of the investigations in vivo and in vitro (Figs. 3 A and 3C) indicating that the codon 130 mutant protein still retained partial SBE-binding activity.
  • the detection of this activity in vivo might be exaggerated by the use of a concatemerized SBE element in the reporter construct and/or by protein over expression.
  • This mutant was therefore further evaluated using an engineered codon 515 truncation to increase the binding sensitivity in vitro.
  • the codon 130 mutation was shown to retain residual SBE-binding (Fig. 3C).
  • the functional impairment of this mutant is likely to be more pronounced at physiologic (non- concatemerized) binding sites in the genome under the natural Dpc4 expression levels. Instructive comparisons to the well-studied transcriptional activator, p53, are possible. Similar to the inhibitory property of the last 37 amino acids of Dpc4, an analogous loss of the last 30 amino acids of the p53 gene also served to augment
  • DPC4 suffers relatively few mutations within its DNA-binding domain. It is likely that the functional inactivation by MH1 mutations is not always very effective, as a dramatically lowered binding capacity by the 130S mutation (130S-515st vs 515st, Fig 3C) only leads to a loss of half of the reporter activation (Fig. 3A). It may also be suggested that in Dpc4, there might be a limited number of residues in the MH1 subject to inactivating mutations. Also in contrast, missense mutations of the transactivation domain of p53 are essentially not observed in
  • Dpc4 MH2 domain Some functional aspects of the Dpc4 MH2 domain have been suggested by previous studies: transcriptional activation , nuclear localization , and an interaction with other Smad proteins . These are related, as nuclear localization of Dpc4 and the Drosophila homolog Medea is dependent upon signaling initiated by TGFb-related ligands and mediated by other Smad proteins 9 ' 18 ' 21 ' 29 32. To dissect among the possibilities, a means was developed to manipulate the nuclear localization of Dpc4 by engineering a chimeric construct that placed a modified murine estrogen receptor ligand- binding domain (MER) at the C-terminus of the full-length wild-type and mutant Dpc4 (Fig. 1).
  • MER murine estrogen receptor ligand- binding domain
  • Proteins fused to the MER domain are retained in the cytoplasm until induced by 4-hydroxytamoxifen (4-OHT), whereupon a redistribution of fusion proteins to the nucleus occurs 33.
  • 4-OHT-induced nuclear redistribution of Dpc4 bypassed the requirement for TGF ⁇ stimulation of wild-type Dpc4 and resurrected the ability of the three MH2 domain missense mutants (351H, 370H, and 493 H) to direct gene expression through the SBE element (Fig 4).
  • the three residues affected by the mutation lie at the interface proposed for the Dpc4-Smad interaction , suggesting that the requirement for TGFb-stimulated Dpc4 heterodimer formation might be bypassed by the forced nuclear redistribution of Dpc4.
  • the MER domain did not rescue the transactivation function of the DPC4 mutations that caused premature truncation of translation (Fig. 4), supporting the last 37 residues as critical for the actual transactivation function.
  • the p6SBE/p6MBE reporter system permitted such a survey for inherent defects in the TGFb-Dpc4-SBE pathway, and was applied to characterize a panel of cancer cells harboring alterations in DPC4 or other TGF- ⁇ pathway components.
  • Cells lacking DPC4 due to homozygous deletion and cells harboring any form of DPC4 mutation showed a defect in the TGFb- Dpc4-SBE pathway (Fig. 5A).
  • Three cell lines (Panel, Su86.86, and ZR75-30) that had a wild-type DPC4 gene showed a strong SBE-mediated response to TGF- ⁇ , indicative of an intact pathway in these cells.
  • TGF- ⁇ receptors have been shown to affect TGF ⁇ responsiveness in pancreatic and other cell lines 38 39 .
  • transi •ent transfection was used to express a constitutively active TGF ⁇ type I receptor construct 39 i •n order to bypass any potential receptor deficiency. This reconstituted the SBE response in both ZR75-1 and PL-45 but not in the DPC4-mxll lines 20.
  • DPC4-n l ⁇ cancer cells stable transfection to restore wild-type DPC4 expression was sufficient to complement the defect in TGFb-SBE responses (Fig. 5B). The level of the pathway defects could therefore be ascertained in all cell lines.
  • chimeric heterologous proteins or compounds that inco ⁇ orate a Dpc4-interaction domain and a nuclear localization signal might be useful to stimulate the expression of Dpc4-inducible genes.
  • a modular domain to redistribute Dpc4 to the nuclear compartment exhibited SBE-mediated transcriptional activation in a cell line having a TGF- ⁇ receptor defect, and was sufficient to resurrect SBE-mediated activation by C- terminal DPC4 missense mutants.
  • Substitutions and truncating mutations of the C- terminal half of DPC4 lacked the ability to regulate transcription but still retained the sequence-specific DNA-binding function. Amino acid substitutions within the N-terminal third of Dpc4 weakened or ablated DNA-binding and SBE-mediated gene regulation.
  • Smad4 Activation of Smad4 (i.e., Dpc4) was controlled by use of a fusion of Smad4 to a modular domain (a modified MER ligand-binding domain). Smad4 activation was detected by transactivation and apoptosis studies.
  • Breast cancer MDA-MB-468 cell lines were generated that stably expressed wild-type (Smad4-MER) or mutant (100T-Smad4-MER) Smad4.
  • Smad4-MER stably expressed wild-type
  • 100T-Smad4-MER 100T-Smad4-MER
  • Smad4-MER were disrupted by a single tumorigenic amino acid change (SEQ ID NO:2, Argl 00 to Thr), and thus likely to be of physiological relevance. This model permits the exploration of the phenotypic changes that occur upon the translocation of Smad4 protein to the nucleus.
  • the growth responses of two clones expressing wild-type Smad4-MER were examined. In both, 4-OHT treatment resulted in a significant decrease in cell number. Cell growth was compared under one, two or four days of 4-OHT treatment and untreated conditions. Moderate and reproducible growth suppression was present at one or two days of 4-OHT treatment.
  • the moderate and transient Gl cell cycle arrest did not fully account for the profound decrease of cell number and delayed growth suppression after 4 days of 4-OHT treatment as shown in the time course experiments (Figs. 8 and 9).
  • the nuclear translocation of Smad4 resulted in an induction of cell apoptosis ( ⁇ 5 fold by TUNEL assay, PO.05, Fig. 10, Panel A and Fig. 1 1).
  • Apoptotic induction was evident in all cell cycle phases and the sub-Gl fraction. This effect persisted after 4 days of 4-OHT exposure.
  • the apoptotic response was specific for wild-type Smad4, as this response was not observed in the MER control clone (Fig. 10, Panel B and Fig. 11).
  • nuclear translocation presents an inducible control of Smad4 (Dpc4) function and provides a method for identifying compounds which effect Smad4 nuclear translocation.
  • Dpc4 Smad4
  • endogenous TGF-b-like ligand(s) and other perhaps more indirect signals are able to activate some Smad4-related functions, hampering the distinction of Smad4-dependent effects.
  • Smad4 proteins are sequestered in the cytoplasm.
  • the present invention has identified a Gl arrest and apoptosis induction attributable to the nuclear translocation of wild-type Smad4, phenotypes that are in agreement with its tumor-suppressive role.
  • the Gl arrest was relatively moderate but reproducible.
  • Rb phosphorylation is critical for the cell entry from Gl to S phase.
  • the Smad4-mediated cell cycle arrest in MDA-MB-468 is Rb-independent as this cell line lacks the RBI gene.
  • Smad4-inducible apoptosis is of great consequence in growth control.
  • the period of the greatest growth suppression temporally was correlated with the apoptotic responses.
  • the Smad4-induced apoptosis is apparently independent of p53 and Rb proteins, as the p53 and RBI genes in the MDA-MB-468 line are inactivated.
  • the combination of apoptotic and cell cycle components observed here reflects the multiple phenotypic responses seen for ligands such as TGF-b and bone mo ⁇ hogenic proteins, whose effector pathways can be shown to be mediated by Smad4 (Dpc4).
  • the 100T-Smad4 and wild-type Smad4 protein display opposing phenotypic responses.
  • the interaction of mutant Smad4 with Smad4-binding partners may explain this observation.
  • Smad4 participates in Smad-mediated gene transactivation (in whole or in part) by interaction with other signal transduetion proteins, intact interactions of 100T-Smad4 with other binding partners including transcription factors, coupled with the inability to bind the SBE sequence, could allow 100T-Smad4 to titrate (squelch) other signal transduetion proteins or general transcription factors.
  • missense mutations in the MHl domain are infrequent.
  • the other two major categories of mutation include missense mutations in the MH2 domain which interfere with nuclear translocation, and truncation mutations in the MH2 domain which cause loss of the transactivation function.
  • the growth advantages offered by the 100T mutant in the present invention suggest that tumors containing certain mutant Smad4 may harbor specific properties that differ from SMAD4 (DPC4)-null cells.
  • Smad4 accomplishes its tumor suppressive functions as a nuclear protein.
  • Two mechanisms by which the nuclear translocation of Smad4 might effect phenotypic changes are considered.
  • 4-OHT administration may facilitate the movement of prefonned cytoplasmic hetero-complexes to allow their action at the DNA recognition sites.
  • Use of the MER domain eliminates nuclear localization as a rate-limiting step in signal transduetion by Smad hetero-complexes.
  • Smad4 may function at the transcription level independent of other Smads, controlled largely by its level of nuclear accumulation. These functions operate subsequent to and independent of heterologous interactions that bring Smad4 to the nucleus.
  • Smad4 may serve its tumor-suppressive role through participation in the signaling pathways of multiple ligands.
  • the present invention provides a means for identifying downstream targets of Smad4 (Dpc4) transcriptional activation activity. Changes in gene transcription directly attributable to Smad4 nuclear translocation are believed to be responsible for the cell cycle regulation and apoptotic effects identified here. In identifying such targets, therapeutic manipulation of multiple signaling pathways in cancer may be possible. It would be useful to identify chemical or peptide agents that would facilitate the direct nuclear translocation of individual proteins, such as Smad4.
  • the present invention provides a method for dissecting the downstream genes that mediate the phenotypic changes resulting in uncontrolled cell growth.
  • DPC4 cDNA from a human library was modified at three nucleotides within a polymerase chain reaction primer site to match a consensus sequence (ccaccATGG) for the start site of translation (M. Kozak, Mammalian Genome 7, 563-74, 1996).
  • the 1.6 kb open reading frame was subcloned into the Bam HI and Eco RI sites of pcDNA3.1 (Invitrogen), resulting in pDPC4-WT.
  • Tumor-derived mutation sequences were introduced within the DPC4 coding region of pDPC4-WT by site-directed mutagenesis (Quick-change, Stratagene).
  • SMAD2 and SMAD3 cDNAs were similarly modified with the consensus Kozak sequence and subcloned into pcDNA3.1 to form pSMAD2-WT and pSMAD3-WT.
  • pMER contains the coding sequence of the modified murine estrogen receptor ligand-binding domain 33 subcloned into the BamH I and EcoR I sites of pcDNA3.1.
  • pDPC4-WT-MER and the pDPC4-mut-MER series were constructed by the subcloning of the DPC4 and MER sequences into the Hind III and EcoR I sites of pcDNA3.1 followed by site mutagenesis. The sequences of all constructs were confirmed by restriction fragment analysis, manual complete gene sequencing (Sequenase 2.0, Amersham), and by in vitro transcription and translation (Promega).
  • SMAD4 coding sequences fused 3' to murine estrogen receptor (MER) ligand-binding domain were constructed in pcDNA3.1 (Smad4-MER or 100T-Smad4-MER, respectively; pcDNA3.1 from Invitrogen).
  • the SMAD4-null breast cancer cell line MDA-MB-468 (ATCC) was transfected with the above plasmids using FuGENE ⁇ reagent (Boehringer Mannheim), and selected with 350 mg/ml G418 (active concentrations, from Life Technologies). G418-resistant clones were screened for protein expression by immunoblot analysis.
  • the Colo357 cell line was obtained from the European Collection of Animal Cell Cultures.
  • PL-45 was obtained by in vitro culture of a pancreatic adenocarcinoma. All other cell lines are commercially available from American Type Culture Collection. The entire sequence of the DPC4 gene was previously determined for each cell line " .
  • Cell cultures at 50-70% confluence in 6-well cluster dishes were transfected with Lipofectamine (Life Technologies) according to manufacturer's instructions. In all experiments, 0.2 to 0.5 mg of the b-galactosidase expression vector pCMVb (Clontech) was cotransfected to control the transfection efficiency.
  • Reporter assays Stable cell lines were plated on 6-well cluster dishes and transfected with 0.5 mg of 6SBE-Luc which contains Smad4 consensus palindromic binding sites, 6MBE-Luc which contains the mutant form of SBE, or a TGF-b-responsive reporter, 3TP-lux. In all experiments, 0.5 mg of b-galactosidase expression vector pCMVb (Clontech) was co-transfected for normalization of transfection efficiency. Transfected cells were then treated with 100 nM 4-hydroxytamoxifen (4-OHT, Sigma), 1 ng/ml TGF-b 1 (R&D Systems) or vehicle. Reporter assays were performed as described previously.
  • Bound and unbound probe were separated on 4% polyacrylamide/0.5x TBE gels at 4°C. Competitors containing either wild-type SBE or MBE were added to the binding reactions to confirm the binding specificity. A mutant probe (the MBE) failed bind to Dpc4, and Smad2 generated in vitro was incapable of SBE binding .
  • RT-PCR Messenger RNA was isolated from monolayer cells (QuikPrep Micro, Pharmacia Biotech). Reverse transcription from 100 ng mRNA was performed by using 18-mer oligo(dT) and Superscript II (Life Technologies).
  • the PCR prime sets forward 5'-GTGGAATAGCTCCAGCTATC-3 (SEQ ID NO: 10), reverse 5'- CGGCATGGTATGAAGTACTTC-3' (SEQ ID NO:l 1)), (forward 5'-GAAATGCCACG- GTAGAAATG-3 (SEQ ID NO: 12), reverse 5 * -GGTCCATTCAGATGAAGTTC-3 (SEQ ID NO: 13)), (forward 5'-AACAGGAATGCAGCAGTGGA-3' (SEQ ID NO: 14); reverse 5'-ATGGTGCACATTCGGGTCAA-3' (SEQ ID NO: 15)), and (forward 5'- ACCCCCACTGAAAAAAGATGA-3' (SEQ ID NO.T6)); reverse 5'- GCATCTTCAAACC
  • pancreatic BxPC3 cells were transfected with pDPC4-WT or pDPC4-343st, respectively, and selected by 200 mg/ml G418 (active concentration, Life Technologies). Individual G418-resistant clones were isolated. The Z PC -expressing clones were identified by screening with RT-PCR to detect products of exons 4-5 and exons 10-1 1. Reactions lacking reverse transcriptase were used to verify the absence of amplification from DNA contamination. The pooled G418-resistant clones from BxPC3 cells transfected with pcDNA3.1 were used as the Neo control.
  • Cell counting and cell cycle analysis Cells stably expressing MER fusion proteins were plated in 6-well cluster dishes at a density of 2x10 5 cells/plate. After an overnight incubation, media were replaced with 10% FBS in the presence of 100 nM 4-OHT or vehicle. After 1, 2, and 4 days of incubation, cells were trypsinized, centrifuged and resuspended in phosphate-buffered saline. Cell numbers were determined by hemocytometer. Flow cytometry was used to determine the cell cycle distributions. Following the 4-OHT treatment, cells were trypsinized and washed with ice-cold phosphate-buffered saline. Cell suspensions were then fixed drop-wise in ice-cold 70% ethanol. Fixed cells were subsequently stained with 10 mg/ml propidium iodide containing 100 mg/ml RNase A and analyzed by flow cytometry for DNA content. Ten thousand forward scatter gated events were collected for each sample.
  • TUNEL Tdt-mediated dUTP nick end-labeling assays Stable clones were plated on 35 mm culture dishes in a density of 5x10 5 cells/dish. After an overnight incubation, cells were treated with 100 nM 4-OHT or vehicle. In some experiments, membrane-permeable caspase inhibitor I (Z-VAD-FMK) or caspase-3 inhibitor II
  • Fragmented DNA ends such as present in apoptotic cells, were labeled with dUTP-BrdU in a reaction containing terminal deoxynucleotide transferase and detected with FITC-labeled monoclonal antibody against BrdU. Cells were then stained with propidium iodide solution containing RNase A. Green FITC emissions were acquired on a log scale and red propidium iodide florescence on a linear scale. Ten thousand cells were analyzed using a Coulter Pics V flow cytometer (Coulter), and data analyzed using Multiparameter Data Acquisition and Display System-86 version 2.0 software (Coulter).
  • a window (identical for treated and untreated pairs) was selected to separate the "labeled" (inside, apoptotic) and "non-labeled” (outside) cells.
  • a subset (0.5 - 2%) of labeled cells was detectable in the absence of 4-OHT.
  • a trapezoidal window was used to reflect the higher apoptotic labeling in G2/M phase cells having a higher DNA content.
  • Total cellular protein was harvested in Laemmli buffer (without b-mercaptolethanol) and protein concentrations were determined by use of BCA reagents (Pierce). Twenty mg of total protein were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by polyclonal antiserum against the estrogen receptor Hgand binding domain (working dilution 1 :200, Santa Cruz). Immunodetection of hyper- or hypo-phosphorylated Rb protein was performed using the monoclonal antibody recognizing both forms of human Rb (clone G3-245, from PharMingen).
  • Eppert, K. et al MADR2 maps to 18q21 and encodes a TGFb-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Ce/786, 543-52 (1996).
  • Macias-Silva, M. et al. MADR2 is a substrate of the TGF-beta receptor and its phosphorylation is required for nuclear accumulation and signaling.

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Abstract

L'invention concerne des méthodes et compositions de localisation nucléaire de séquences polynucléotidiques et polypeptidiques codant un nouveau gène suppresseur de tumeurs, le Dpc4. L'invention concerne également une méthode de traitement d'un trouble de prolifération cellulaire associé à Dpc4. Dpc4 est un marqueur pouvant s'utiliser pour un diagnostic, un pronostic ou une thérapie des troubles associés à Dpc4.
PCT/US1999/018540 1998-08-14 1999-08-13 Methode de localisation nucleaire de dpc4 (smad4) WO2000009526A2 (fr)

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US8394742B2 (en) 2005-04-15 2013-03-12 The Translation Genomics Research Institute Methods, compounds and compositions with genotype selective anticancer activity
CN112823027A (zh) * 2018-07-24 2021-05-18 青春生命科学公司 将蛋白质和编码所述蛋白质的基因递送至活细胞的脂质体的应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8394742B2 (en) 2005-04-15 2013-03-12 The Translation Genomics Research Institute Methods, compounds and compositions with genotype selective anticancer activity
CN112823027A (zh) * 2018-07-24 2021-05-18 青春生命科学公司 将蛋白质和编码所述蛋白质的基因递送至活细胞的脂质体的应用
EP3826683A4 (fr) * 2018-07-24 2022-04-13 So Young Life Sciences Corporation Utilisation de liposomes permettant la transmission d'une protéine et d'un gène codant pour la protéine à une cellule vivante

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