NZ501892A

NZ501892A - Use of a reagent that modulates HIC-1 expression for treating cell proliferation

Info

Publication number: NZ501892A
Application number: NZ501892A
Authority: NZ
Inventors: Stephen B Baylin; Michele Makos Wales
Original assignee: Univ Johns Hopkins Med
Priority date: 1994-11-15
Filing date: 1995-11-15
Publication date: 2001-06-29

Abstract

The use of a reagent that modulates HIC-1 expression in the manufacture of a medicament for the treatment of cell proliferation.

Description

<div class="application article clearfix" id="description"> PATE**"* 92 NEW ZEALAND PATENTS ACT, 1953 No: Divided out.ofNZ 298554 Date: Dated 15 November 1995 COMPLETE SPECIFICATION A ° USE OF COMPOUNDS FOR TREATING CELL PROLIFERATIVE DISORDER ASSOCIATED WITH ti[C-l We, THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE of 720 Rutland Avenue, Baltimore, MD 21205, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page la) I - 8 A"' i | RECEIVED I -1o- 50 i uy This invention is a divisional of New Zealand specification no. 298554. This invention was made with government support under Grant No R01-CA43318, from the National Cancer Institute The government has certain rights in the invention. BACKGROUND OF THE INVENTION 1 Field of the Invention This invention relates generally to gene expression in normal and neoplastic cells, and specifically to a pharmaceutical use of a compound for treating cell proliferative disorder associated with HIC-1 2 Description of Related Art Advances in recombinant DNA technology have led to the discovery of normal cellular genes such as proto-oncogenes and tumor suppressor genes, which control growth, development, and differentiation Under certain circumstances, regulation of these genes is altered and they cause normal cells to assume neoplastic growth behavior There are over 40 known proto-oncogenes and tumor suppressor genes to date, which fall into various categories depending on their functional characteristics These include, (1) growth factors and growth factor receptors, (2) messengers of intracellular signal transduction pathways, for example, between the cytoplasm and the nucleus, and (3) regulatory proteins which influence gene expression and DNA replication {e.g., transcription factors) Chromosome 17p is frequently altered in human cancers, and allelic losses often coincide with mutations in the p53 gene at 17p 13 1 (Vogelstein, B , et al., Cell, 70 523, 1992) This gene is one of the most frequently altered tumor suppressor genes in human neoplasms However, in some tumor types, 17p allelic loss occurs at a high frequency in regions distal to p53 and in the absence of p53 mutations For instance, 60% of breast cancers lose 17p alleles while only 30% of these tumors contain p53 mutations (Chen, L-C , etal., Proc. Natl. Acad Sci. USA, 88 3847, 1991, Takita, K , etal.. Cancer Res, 52 3914, 1992, Deng, G, etal., Cancer Resr 54.499^1994. Corn- L ' - r. ..VI. : cr Ni i -sals 2^03 t D rr/^> r- n / p- i-h elis, RS,e/ al, Cancer Res., 54 4200, 1994) Furthermore, in one study of breast cancer, the independent loss of 17p 13 3 alleles was accompanied by increased levels of p53 mRNA. Human cancer cells typically contain somatically altered genomes, characterized by mutation, amplification, or deletion of critical genes In addition, the DNA template from human cancer cells often displays somatic changes in DNA methylation (E R Fearon, et al., Cell, 61 759, 1990, P A Jones, et al., Cancer Res., 46 461, 1986, R Holliday, Science, 238 163, 1987, A De Bustros, et al., Proc. Natl. Acad. Sci, USA, 85 5693, 1988); P A Jones, et al.. Adv. Cancer Res., 54 1, 1990, S B Baylin, et al.. Cancer Cells,._3 383, 1991; M. Makos, etal., Proc. Natl. Acad Sci., USA, 89 1929, 1992, N Ohtani-Fujita, et al, Oncogene, 8 1063, 1993) However, the precise role of abnormal DNA methylation in human tumorigenesis has not been established DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA. Several biological functions have been attributed to the methylated bases in DNA The most established biological function is the protection of the DNA from digestion by cognate restriction enzymes The restriction modification phenomenon has, so far, been observed only in bacteria Mammalian cells, however, possess a different methylase that exclusively methylates cytosine residues on the DNA that are 5' neighbors of guanine (CpG) This methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A, H, and Riggs, R D eds in DNA Methylation Biochemistry and Biological Significance, Spnnger-Verlag, New York, 1984) A CpG rich region, or "CpG island", has recently been identified at 17p 13 3, which is aberrantly hypermethylated in multiple common types of human cancers (Makos, M , et al., Proc. Natl. Acad Sci. USA, 89 1929, 1992, Makos, M , et al, Cancer Res , 53 2715, 1993, Makos, M, et al., Cancer Res 53 2719, 1993) This hypermethylation coincides with timing and frequency of 17p losses and p53 mutations in brain, colon, and renal cancers Silenced gene transcription associated with hypermethylation of the normally unmethylated promoter region CpG islands has been implicated as an alternative mechanism to mutations of coding regions for inactivation of tumor suppressor genes (Baylin, SB, et al, Cancer Cells, 3 383, 1991, Jones, P A and Buckley, J D , Adv. Cancer Res ,54 1-23, 1990) This change has now been associated with the loss of expression of VHL, a renal cancer tumor suppressor gene on 3p (J G Herman, et al, Proc. Natl. Acad Sci. USA, 91 9700-9704, 1994), the estrogen receptor gene on 6q (Ottaviano, Y L , etal., Cancer Res, 54 2552, 1994) and the H19 gene on 1 lp (Steenman, M.J C , et al, Nature Genetics, 7 433, 1994) For several human tumor types, a second tumor suppressor gene may reside distal to, and be interactive with, the p53 gene at chromosome 17pl3 1 There is a need to identify tumor suppressor genes in order to develop the appropriate methodologies for increasing or decreasing their expression in cells where aberrant expression is observed Through characterization of a 17pl3 3 CpG island which is aberrantly hypermethylated in multiple common human tumor types, the present invention provides such a gene HIC-1 (hypermethylated in cancer) is a novel zinc finger transcription factor gene which is ubiquitously expressed in normal tissues, but under-expressed in tumor cells (e.g, breast, lung, colon, fibroblasts) where it is hypermethylated A p53 binding site is located in the 5' flanking region of HIC-1 Over-expression of a wild-type p53 gene in colon cancer cells containing only a mutant p53 allele, results in 20-fold activation of HIC-1 expression The present invention shows that many human cancers exhibit decreased HIC-1 expression relative to their tissues of origin The limitation and failings of the prior art to provide meaningful markers which correlate with the presence of cell proliferative disorders, such as cancer, has created a need for markers which can be used diagnostically, prognostically, and therapeutically over the course of such disorders The present invention goes some way towards fulfilling such a need or at least provides the public with a useful choice -4- 50 1892 SUMMARY OF THE INVENTION According to the present invention there is provided a use of a therapeutically effective amount of reagent which modulates HIC-1 expression in the manufacture of a medicament for treating a cell proliferative disorder associated with HIC-1. For example, since HIC-1 associated disorders typically involve hypermethylation of HIC-1 polynucleotide sequence, a polynucleotide sequence which contains a non-methylatable nucleotide analog is utilized for treatment of a subject. According to a second aspect, the invention provides an expression vector comprising a nucleotide sequence encoding HIC-1, in operable linkage with a promoter in the manufacture of a medicament for use in a method of gene therapy, whereby said medicament is introduced into cells of a host subject The parent invention NZ 298554 is based on the seminal discovery of a novel tumor suppressor gene, HIC-1 (hypermethylated in cancer), which is aberrantly hypermethylated in multiple common human tumor types, and provides a HIC-1 polypeptide as well as a polynucleotide sequence encoding the polypeptide and antibodies which bind to the polypeptide. In one embodiment, the invention of parent NZ 298554 provides a diagnostic method for detecting a cell proliferative disorder associated with HIC-1 in a tissue of a subject, comprising contacting a target cellular component containing HIC-1 with a reagent which detects HIC-1. Such cellular components include nucleic acid and protein. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1A is a diagram showing a map of an 110 kb region of cosmid C-13A which contains a 50 kb human DNA insert harboring the region of chromosome 17p 13 3 previously shown to have hypermethylation in multiple human tumor types (Makos, M , et al., Proc. Natl. Acad. Sci. USA, 89 1929, 1992, Makos, M, et al., Cancer Res , 53 2715, 1993, Makos, M., et al., Cancer Res 53 2719, 1993) The position of the YNZ22 probe, EcoRI (E) restriction site and the location of a series of cosmid subclones which were prepared to span the area are shown FIGURE IB is a schematic for the HIC-1 gene which was found to be encompassed within the region shown m FIGURE 1A and for which the ammo acid sequence is shown in FIGURE 2B Shown are- potential p53 binding site; TATAA = the TATA box sequence 40 bp upstream from the transcription start site, 5' UTR = the 1st untranslated exon, ATG = the most 5' translation start site, ZIN (zinc finger N-terminus) = the 478bp exon encompassing the highly conserved region (FIGURE 2A) of the Zin domain subfamily of zinc finger transcription factors, rectangle with shaded bars represents the 2015 bp last exon of HIC-1 and each shaded bar represents one of the 5 zinc fingers (FIGURE 2B) clustered m this 3' region of the gene, TAG = translation stop site in the HIC-1 gene; AATAAA = polyadenylation signal site found 835 bp from the translation stop site FIGURE 1C and SEQ ED NO 1 and 2 show the nucleotide and deduced ammo acid sequence of HIC-1 FIGURE 2A and SEQ ED NO 3 show the amino acid sequences of HIC-1 The HIC-1 amino acid sequence is compared with the conserved N-termmus region of the other members of the Zm domain zinc finger family In the parentheses, the numbers indicate the position of the conserved region relative to the translation start site of each gene The darkest shading shows position of amino acids which are identical for at least five of the 9 proteins and the lighter shading shows position of conservative ammo acid differences between the family members D = drosophila, M = murine, H = human The bracket of amino acids at the bottom represents an area in HIC-1 not found at this position m the other family members FIGURE 2B shows the entire coding region of the HIC-1 gene The deduced amino acid sequence for the two coding exons of HIC-1, as defined by the sequence analyses and expression strategies outlined in the text, are shown The 5 zinc fingers in the 3' half of the protein are shown by the shaded boxes FIGURE 3 shows a Northern analyses of HIC-1 gene expression S = spleen, The = thymus, P= prostate, Te = testis, O = ovary, SI = small intestine, B = peripheral blood cells The band above the 4 4 kb marker co-hybridizes with ribosomal RNA The ~1 1 kb band has not yet been identified but could be an alternate splice product since it was not detected with probes from the zinc finger or 3' untranslated regions of HIC-1 FIGURE 4A shows RNAse protection assays of HIC-1 gene expression in a variety of normal and neoplastic human tissues In all panels, the top asterisk marks the position of the undigested 360bp HIC-1 gene RNA probe which was derived from the region containing the zinc fingers in cosmid subclone 600 (FIGURE 1A) The protected HIC-1 fragment (300bp) is labeled HIC-1 FIGURE 4A compares expression in 10 ug of total RNA from 2 established culture lines of normal human fibroblasts (WI-38 and IMR-90) to the HT 1080 culture line of fibrosarcoma cells (Fibro-C), from 3 different samples of normal colon (Colon - N) to the colon carcinoma cell line, CaC02 (Colon-C), and from a sample of normal lung (Lung-N) to the established line of human small cell lung carcinoma, NCI-H209 (Lung-C) 501892 -7- FIGURE 4B shows the RNAse protection assay for 10 ug of RNA from 6 different established culture lines of breast carcinoma (lane 1 MDA231, lane 2 HS58T, lane 3 MDA468, lane 4 T47D, lane 5 MCF7; lane 6 MDA453), each of which has extensive methylation of Not I sites of the HIC-1 CpG island 5 FIGURE 4C shows the RNAse protection assay for 10 ug of RNA from normal fetal brain (B) compared to a series of non-cultured brain tumors (1 anaplastic astrocytoma (AA) and 8 more advanced glioblastomas (lanes 1-8) FIGURE 5 shows an RNAse protection assay, as detailed in FIGURE 4, after infection of an adenoviral vector containing either the [3-galactosidase gene or the 10 wild type human p53 gene into the SW480 line of human colon cancer cells (Uninfected, normal, control human fibroblasts (F), uninfected SW480 cells (U), SW480 cells infected with the p-galactosidase gene (GAL), and SW480 cells infected with the p53 gene (p53)). Positions of the undigested HIC-1 and GAPDH probes and of the HIC-1 and GAPDH transcripts are marked exactly as in FIGURE 4 DETAILED DESCRIPTION OF THE INVENTION -| 5 The present invention provides a use of a therapeutically effective amount of reagent which modulates HIC-1 expression m the manufacture of a medicament for treating a cell proliferative disorder associated with HIC-1. The novel tumor suppressor gene, HIC-1 (hypermethylated in cancer) which is used in the present invention is disclosed in parent NZ 298554. HIC-1 is located on chromosome 17pl3.3, distal to the tumor suppressor gene, p53, at 17pl3 1, within a CpG island which is abnormally 20 methylated in many different types of tumors This abnormally methylated CpG island completely encompasses the coding region of HIC-1 gene The present invention may make use of the substantially pure HIC-1 polypeptide including the amino acid sequence shown in FIGURE 2B and SEQ ID NO 3 HIC-1 polypeptide is characterized as having a distinct amino acid homology to a highly conserved N-terminal motif, termed the Zin (Zinc finger 25 N-terminal) domain, which is present in each member of subset of zinc finger transcription factors In addition, it also has five Kruppel type C^s,-His2 ?inc fingers^ characteristic of the 3' region of those same proteins I • i- : - !' , ■ , v / S _ *• • - ML'3 21K The term "substantially pure" as used herein refers to HIC-1 polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated One skilled in the art can purify HIC-1 using standard techniques for protein purification The substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel The purity of the HIC-1 polypeptide can also be determined by ammo-terminal amino acid sequence analysis The invention may also make use of a functional peptide, HIC-1, and functional fragments thereof As used herein, the term "functional polypeptide" refers to a polypeptide which possesses a biological function or activity which is identified through a defined functional assay and which is associated with a particular biologic, morphologic, or phenotypic alteration in the cell Functional fragments of the HIC-1 polypeptide, include fragments of HIC-1 which retain the activity of e.g, tumor suppressor activity, of HIC-1 Smaller peptides containing the biological activity of HIC-1 are included in the invention The biological function, for example, can vary from a polypeptide fragment as small as an epitope to which an antibody molecule can bind to a large polypeptide which is capable of participating in the characteristic induction or programming of phenotypic changes within a cell A "functional polynucleotide" denotes a polynucleotide which encodes a functional polypeptide as described herein Minor modifications of the HIC-1 primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the HIC-1 polypeptide described herein Such 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 tumor suppressor activity of HIC-1 is present Further, deletion of one or more ammo acids can also result in a modification of the structure of the resultant molecule without significantly altering its activity This can lead to the development of a smaller active molecule which would have broader utility For example, it is possible to remove ammo or carboxy terminal amino acids which may not be required for HIC-1 activity The HIC-1 polypeptide disclosed also includes conservative variations of the polypeptide sequence The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of argimne for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide The invention may also make use of an isolated polynucleotide sequence including a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ED NO 3 The polynucleotide sequence also includes the 5' and 3' untranslated sequences and includes regulatory sequences, for example The term "isolated" as used herein includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode HIC-1 It is understood that all polynucleotides encoding all or a portion of HIC-1 are also included herein, as long as they encode a polypeptide with HIC-1 activity Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides For example, HIC-1 polynucleotide may be subjected to site-directed mutagenesis The polynucleotide sequence for HIC-1 also includes antisense sequences. The disclosed polynucleotides include sequences that are degenerate as a result of the genetic code There are 20 natural amino acids, most of which are specified by more than one codon Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of HIC-1 polypeptide encoded by the nucleotide sequence is functionally unchanged In addition, a polynucleotide consisting essentially of a polynucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO 3 and having at least one epitope for an antibody immunoreactive with HIC-1 polypeptide is also disclosed. The polynucleotide encoding HIC-1 includes the nucleotide sequence in FIGURE 1C (SEQ ID NO 1 and 2), as well as nucleic acid sequences complementary to that sequence A complementary sequence may include an antisense nucleotide When the sequence is RNA the deoxynucleotides A, G, C, and T of FIGURE 1C (SEQ ED NO 1 and 2) are replaced by ribonucleotides A G, C, and U, respectively Also included are fragments of the above-descnbed nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA that encodes the protein of FIGURE 2B (SEQ ED NO 3) under physiological conditions and under moderately stringent conditions Specifically disclosed herein is a DNA sequence for H3C-1 which schematically is illustrated in FIGURES 1A and IB (see also, FIGURE 1C and SEQ ED NO 2) The transcribed exon encompasses 5 zinc fingers and extends 359 bp from the last zinc finger to the stop site The transcription proceeds 239 bp past the stop site, in an apparent 3' untranslated region (UTR) There is also a polyadenylation signal, AATAAA at position 835 bp from the stop site In addition, after the Zin domain and before the zinc finger exons, there is a consensus splice donor and an acceptor site separated by an intron region The complete coding region of HIC-1 is encompassed by two exons within the CpG rich 3 0 kb region between Not I sites N3 and N7 The disclosed DNA sequences can be obtained by several methods. For example, 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 and 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features Prefereably the disclosed HIC-1 polynucleotide 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 Oligonucleotide 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 This includes a heterogeneous mixture of denatured double-stranded DNA For such screening, 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 In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al, Nucl Acid Res., 9 879, 1981) The development of specific DNA sequences encoding HIC-1 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. Of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures, the isolation of genomic DNA isolates is the least common This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of mtrons The synthesis of DNA sequences is frequently the method of choice when the entire sequence of ammo acid residues of the desired polypeptide product is known When the entire sequence of ammo 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 -12- synthesis of cDNA sequences Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrymg cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of gene expression When used in combination with polymerase chain reaction technology, even rare expression products can be cloned In those cases where significant portions of the ammo acid sequence of the polypeptide are known, 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, etal., Nucl. Acid Res., H 2325, 1983) A cDNA expression library, such as lambda gtl 1, can be screened indirectly for HIC-1 peptides having at least one epitope, using antibodies specific for HIC-1 Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of HIC-1 cDNA DNA sequences encoding HIC-1 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 In the present invention, the HIC-1 polynucleotide sequences may be inserted into a recombinant expression vector The term "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 HIC-1 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 -13- 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, metallothionem I, or polyhedrin promoters) Polynucleotide sequences encoding HIC-1 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. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the HIC-1 coding sequence and appropriate transcriptional/translational control signals These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic techniques See, for example, the techniques described in Maniatis, et al, 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N Y A variety of host-expression vector systems may be utilized to express the HIC-1 coding sequence These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA plasmid DNA or cosmid DNA expression vectors containing the H3C-1 coding sequence, yeast transformed with recombinant yeast expression vectors containing the HIC-1 coding sequence, plant cell systems infected with recombinant virus expression vectors {e.g, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors {eg, Ti plasmid) containing the HIC-1 coding sequence, insect cell systems infected with recombinant virus expression vectors {eg, baculovirus) containing the HIC-1 coding sequence, or animal cell -14- systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccinia virus) containing the HIC-1 coding sequence, or transformed animal cell systems engineered for stable expression Since HIC-1 has not been confirmed to contain carbohydrates, both bacterial expression systems as well as those that provide for translational and post-translational modifications may be used, e g, mammalian, insect, yeast or plant expression systems Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc may be used in the expression vector (see e.g., Bitter, et al., Methods in Enzymology 153 516-544, 1987) For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage y, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g, metallothionein promoter) or from mammalian viruses (e.g, the retrovirus long terminal repeat, the adenovirus late promoter; the vaccinia virus 7 5K promoter) may be used Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the inserted HIC-1 coding sequence In addition, the endogenous HIC-1 promoter may also be used to provide transcription machinery of HIC-1 In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the expressed For example, when large quantities of HIC-1 are to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable Those which are engineered to contain a cleavage site to aid in recovering are preferred Such vectors include but are not limited to the E coli expression vector pUR278 (Ruther, etal., EMBO J. 2 1791, 1983), m which the HIC-1 coding sequence may be ligated into the vector m frame with the lac Z coding region so that a hybrid -lac Z protein is produced, pIN vectors (Inouye & Inouye, Nucleic Acids Res., 13 3101-3109, 1985, Van Heeke & Schuster, J Biol Chem 264 5503-5509, 1989), glutathione-S-transferase (GST) and the like -15- In yeast, a number of vectors containing constitutive or inducible promoters may be used For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed Ausubel, et al., Greene Publish Assoc & Wiley Interscience, Ch 13, Grant, et al, 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds Wu & Grossman, 31987, Acad Press, N Y, Vol 153, pp 516-544, Glover, 1986, DNA Cloning, Vol II, IRL Press, Wash, D C , Ch. 3, and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds Berger & Kimmel, Acad Press, NY, Vol 152, pp 673-684, and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds Strathera, etal, Cold Spring Harbor Press, Vols I and II A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch 3, R. Rothstem In DNA Cloning Vol 11, A Practical Approach, Ed DM Glover, 1986, IRL Press, Wash, D C ). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome In cases where plant expression vectors are used, the expression of the HIC-1 coding sequence may be driven by any of a number of promoters For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Bnsson, et al, Nature 310-511-514, 1984), or the coat protein promoter to TMV (Takamatsu, et al, EMBO J 6 307-311, 1987) may be used, alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi, et al, EMBO J 3 1671-1680, 1984, Broglie, et al., Science 224 838-843, 1984), or heat shock promoters, e.g, soybean hspl7 5-E or hspl7 3-B (Gurley, et al., Mol. Cell. Biol. 6 559-565, 1986) may be used These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc For reviews of such techniques see, for example, Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463, and Gnerson & Corey, 1988, Plant Molecular Biology, 2d Ed , Blackie, London, Ch 7-9 An alternative expression system which could be used to express is an insect system In one such system, Autographa califormca nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes The virus grows in Spodoptera frugiperda cells The HIC-1 coding sequence may be cloned into non-essential regions (for example the polyhednn gene) of the virus and placed under control of an AcNPV promoter (for example the polyhednn promoter) Successful insertion of the HIC-1 coding sequence will result in mactivation of the polyhednn gene and production of non-occluded recombinant virus (i.e , virus lacking the protemaceous coat coded for by the polyhedrin gene) These recombinant viruses are then used to infect Spodopterafrugiperda cells in which the inserted gene is expressed (e.g., see Smith, etal, 1983, J Viol. 46 584. U S Smith, Patent No 4,215,051) Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and advantageously, secretion of the gene product may be used as host cells for the expression of HIC-1 Mammalian cell lines may be preferable Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, -293, and WI38 Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the HIC-1 coding sequence may be ligated to an adenovirus transcription/-translation control complex, e.g, the late promoter and tripartite leader sequence This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non-essential region of the viral genome (e.g, region El or E3) will result m a recombinant virus that is viable and capable of expressing the protein in infected hosts (e.g, see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 813655-3659, 1984) Alternatively, the vaccinia virus 7 5K promoter may be used (e.g, see, Mackett, etal., 1982, Proc. Natl. Acad Sci. USA 79 7415-7419, Mackett, etal, J Virol. 49 857-864, 1984, Panicali, etal., Proc. Natl. Acad. Sci. USA 79 4927-4931, 1982) Of particular interest are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al., Mol. Cell. Biol. 1 486, 1981) Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell Transcnption of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression These vectors can be used for stable expression by including a selectable marker m the plasmid, such as, for example, the neo gene Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the HIC-1 gene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA 8_L 6349-6353, 1984) High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters For long-term, high-yield production of recombinant proteins, stable expression is preferred Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the HIC-1 cDNA controlled by appropriate expression control elements {e.g, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc ), and a selectable marker The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines For example, following the introduction of foreign DNA engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al. Cell, H.223, 1977), hypoxanthme-guamne phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad Sci. USA, 48 2026, 1962), and adenine phosphoribosyltransferase (Lowy, etal., Cell, 22 817, 1980) genes can be employed m tk", hgprt or ap'rt cells respectively Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., Natl Acad. Sci USA, 77 3567, 1980, O'Hare, et al., Proc. Natl. Acad Sci. USA , 78 1527, 1981), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci USA, 78 2072, 1981, neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J Mol Biol, 150 1, 1981), and hygro, which confers resistance to hygromycin (Santerre, et al, Gene, 30 147, 1984) genes Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize -18- indole in place of tryptophan, hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad Sci. USA, 85 8047, 1988), and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L, 1987, In Current Communications in Molecular Biology, Cold Spnng Harbor Laboratory, ed ) 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 Where the host is prokaryotic, such as E. coh, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art Alternatively, MgCl2 or RbCl can be used Transformation can also be performed after forming a protoplast of the host cell if desired When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased m liposomes, or virus vectors may be used Eukaryotic cells can also be cotransformed with DNA sequences encoding the HIC-1 of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes 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 Spnng Harbor Laboratory, Gluzman, ed , 1982) Isolation and punfication of microbial or host cell expressed polypeptide, or fragments thereof, provided by the invention, may be earned out by conventional means including preparative chromatography and affinity and immunological separations involving monoclonal or polyclonal antibodies -19- The invention of parent NZ 298554 includes antibodies lmmunoreactive with HIC-1 polypeptide (SEQ ID NO 3) or lmmunoreactive fragments thereof Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art (Kohler, et al., Nature, 256 495, 1975) The term antibody as used in this invention is meant to include intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding an epitopic determinant on HIC-1 The invention of parent NZ 298554 also provides a method for detecting a cell proliferative disorder associated with HIC-1 in a subject, comprising contacting a target cellular component suspected of having a HIC-1 associated disorder, with a reagent which reacts with or binds to HIC-1 and detecting HIC-1 The target cell component can be nucleic acid, such as DNA or RNA or it can be protein When the component is nucleic acid, the reagent is typically a nucleic acid probe or PCR primer When the cell component is protein, the reagent is typically an antibody probe The target cell component may be detected directly in situ or it may be isolated from other cell components by common methods known to those of skill in the art before contacting with a probe (See for example, Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y, 1989, Current Protocols in Molecular Biology, 1994, Ed Ausubel, et al, Greene Publ Assoc & Wiley Interscience) Detection methods include Southern and Northern blot analyses, RNase protection, immunoassays and other detection assays that are known to those of skill in the art The probes can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a biolummescent compound, a chemilummescent compound, a metal chelator, or an enzyme Those of ordinary skill m the art will know of other suitable labels for binding to the probes or will be able to ascertain such, using routine experimentation -20- Since the applicants have shown that a decreased level of HIC-1 transcription is often the result of hypermethylation of the HIC-1 gene, it is often desirable to directly determine whether the HIC-1 gene is hypermethylated In particular, the cytosine rich areas terms "CpG islands" which lie in the 5' regulatory regions of genes are normally unmethylated The term "hypermethylation" includes any methylation of cytosine which is normally unmethylated in the HIC-1 gene sequence can be detected by restriction endonuclease treatment of HIC-1 polynucleotide (gene) and Southern blot analysis for example. Therefore, in a disclosed method, when the cellular component detected is DNA, restriction endonuclease analysis is preferable to detect hypermethylation of the HIC-1 gene Any restriction endonuclease that includes CG as part of its recognition site and that is inhibited when the C is methylated, can be utilized Methylation sensitive restriction endonucleases such as BssHll, Mspl, Notl or Hpall, used alone or in combination are examples of such endonucleases Other methylation sensitive restriction endonucleases will be known to those of skill m the art In addition, PCR can be utilized to detect the methylation status of the HIC-1 gene Oligonucleotide primers based on any coding sequence region in the HIC-1 sequence are useful for amplyifying DNA by PCR An antibody or nucleic acid probe specific for HIC-1 may be used to detect the presence of HIC-1 polypeptide (using antibody) or polynucleotide (using nucleic acid probe) in biological fluids or tissues Oligonucleotide primers based on any coding sequence region in the HIC-1 sequence are useful for amplifying DNA for example by PCR Any specimen containing a detectable amount of HIC-1 polynucleotide or HIC-1 polypeptide antigen can be used Nucleic acid can also be analyzed by RNA in situ methods which are known to those of skill in the art. A preferred sample of this method is tissue of heart, renal, brain, colon, breast, urogenital, uterine, hematopoietic, prostate, thymus, lung, testis, and ovarian Preferably the subject is human. Various disorders which are detectable by the disclosed method include astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, colon cancer, -21- lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, endometrial cancer and neuroblastoma Monoclonal antibodies used in the disclosed method are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and noncompetitive immunoassays in either a direct or indirect format Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometnc) assay Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation The term "immunometric assay" or "sandwich immunoassay", includes simultaneous sandwich, forward sandwich and reverse sandwich immunoassays These terms are well understood by those skilled in the art Those of skill will also appreciate that antibodies according to the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future These are intended to be included within the scope of the present invention Monoclonal antibodies can be bound to many different earners and used to detect the presence of HIC-1 Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite The nature of the carrier can be 25 either soluble or insoluble for purposes of the invention Those skilled in the art will know of other suitable earners for binding monoclonal antibodies, or will be able to ascertain such using routine expenmentation 5 10 15 20 -22- In performing the assays it may be desirable to include certain "blockers" in the incubation medium (usually added with the labeled soluble antibody) The "blockers" are added to assure that non-specific proteins, proteases, or anti-heterophilic immunoglobulins to anti-HIC-1 immunoglobulins present m the experimental sample do not 5 cross-link or destroy the antibodies on the solid phase support, or the radiolabeled indicator antibody, to yield false positive or false negative results The selection of "blockers" therefore may add substantially to the specificity of the assays described herein. It has been found that a number of nonrelevant (i e , nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g., IgGl, IgG2a, IgM, etc) can be used as "blockers" The concentration of the "blockers" (normally 1-100 p.g/fj.1) may be important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in the specimen In using a monoclonal antibody for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective The term "diagnostically effective" means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the H3C-1 antigen for which the monoclonal antibodies are specific The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having HIC-1 is detectable compared to the background Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio As a rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis 25 will vary depending on such factors as age, sex, and extent of disease of the individual The dosage of monoclonal antibody can vary from about 0 001 mg/m2 to about 500 mg/m2, preferably 0 1 mg/m2 to about 200 mg/m , most preferably about 0 1 mg/m2 to about 10 mg/A Such dosages may vary, for example, depending on 15 20 -23- whether multiple injections are given, tumor burden, and other factors known to those of skill in the art For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope The radioisotope chosen must have a type of 5 decay which is detectable for a given type of instrument Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized Ideally, a radioisotope used for in vivo imaging will lack a particle 10 emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to 15 immunoglobulins are the Afunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are mIn, 97Ru, 67Ga, 68Ga, 72As, ® 89Zr, and 201T1. 20 A monoclonal antibody useful in the method of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR) In general, any conventional method for visualizing diagnostic imaging can be utilized Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic 25 isotopes for MRI Elements which are particularly useful in such techniques include I57Gd, 55Mn, 162Dy, 52Cr, and 56Fe -24- ^ w; i o y 2 The present invention provides a use of a therapeutically effective amount of reagent which modulates HIC-1 expression in the manufacture of a medicament for treating a cell proliferative disorder associated with HIC-1. In brain, breast and renal cancer cells, for example, the HIC-1 5 nucleotide sequence is under-expressed as compared to expression in a normal cell, therefore, it is possible to design appropriate therapeutic or diagnostic techniques directed to this sequence Thus, where a cell-proliferative disorder is associated with the expression of HIC-1 associated with malignancy, nucleic acid sequences that modulate HIC-1 expression at the transcriptional or translational level can be used 10 In cases when a cell proliferative disorder or abnormal cell phenotype is associated with the under expression of HIC-1, for example, nucleic acid sequences encoding HIC-1 (sense) could be administered to the subject with the disorder The term "cell-proliferative disorder" denotes malignant as well as non-malignant cell populations which often appear to differ from the surrounding tissue both 15 morphologically and genotypically Such disorders may be associated, for example, with absence of expression of HIC-1. Essentially, any disorder which is etiologically linked to expression of HIC-1 could be considered susceptible to treatment with a reagent of the invention which modulates HIC-1 expression The term "modulate" envisions the suppression of methylation of HIC-1 20 polynucleotide when HIC-1 is under-expressed When a cell proliferative disorder is associated with HIC-1 expression, such methylation suppressive reagents as 5-azacytadine can be introduced to a cell Alternatively, when a cell proliferative disorder is associated with unaer-expression of HIC-1 polypeptide, a sense polynucleotide sequence (the DNA coding strand) encoding HIC-1 polypeptide, or 5' 25 regulatory nucleotide sequences (i e , promoter) of HIC-1 in operable linkage with HIC-1 polynucleotide can be introduced into the cell Demethylases known in the art could also be used to remove methylation The invention further provides a use of an expression vector comprising a nucleotide sequence encoding HIC-1, in operable linkage with a promoter in the manufacture of a medicament for use in a method of gene therapy, whereby said medicament is introduced into cells of a host subject Such therapy would achieve lts^herapeutjic v > - j l 3 r- » , r- j \ / j- nj "y* It -25- effect by introduction of the appropriate HIC-1 polynucleotide which contains a HIC-1 structural gene (sense), into cells of subjects having the proliferative disorder Delivery of sense HIC-1 polynucleotide constructs can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system The polynucleotide sequences used in the method of the invention may be the native, unmethylated sequence or, alternatively, may be a sequence in which a nonmethylatable analog is substituted within the sequence Preferably, the analog is a nonmethylatable analog of cytidine, such as 5-azacytadine Other analogs will be known to those of skill in the art Alternatively, such nonmethylatable analogs could be administered to a subject as drug therapy, alone or simultaneously with a sense structural gene for HIC-1 or sense promoter for HIC-1 operably linked to HIC-1 structural gene In another embodiment, a HIC-1 structural gene is operably linked to a tissue specific heterologous promoter and used for gene therapy For example, a HIC-1 gene can be ligated to prostate specific antigen (PSA) - prostate specific promoter for expression of HIC-1 in prostate tissue Other tissue specific promoters will be known to those of skill in the art Alternatively, the promoter for another tumor suppressor gene can be linked to the HIC-1 structural gene and used for gene therapy Various 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 Preferably, the retroviral vector is a derivative of a munne or avian retrovirus Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to Moloney munne leukemia virus (MoMuLV), Harvey munne sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV) Most preferably, a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV), thereby providing a broader host range than murine vectors, for example -26- A number of additional 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 HIC-1 sense or antisense polynucleotide Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles This assistance can be provided, for example, by using 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 Y2, PA317 and PA12, for example These cell lines produce empty virions, since no genome is packaged If 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 Another targeted deliveiy system for HIC-1 polynucleotide is a colloidal dispersion system 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 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 Biochem. Sci, -27- 6 77, 1981) In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells 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., Biotechmques, 6 682, 1988) The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol Other phospholipids or other lipids may also be used The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations Examples of lipids useful m liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated Illustrative phospholipids 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, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand 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 m a variety of ways In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer Various linking groups can be used for joining the lipid chains to the targeting ligand. In general, 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 ligand may be any compound of interest which will bind to another compound, such as a receptor In general, surface membrane proteins which bind to specific effector molecules are referred to as receptors In the present invention, antibodies are preferred receptors Antibodies can be used to target liposomes to specific cell-surface ligands For example, certain antigens expressed specifically on tumor cells, referred to as tumor-associated antigens (TAAs), may be exploited for the purpose of targeting HIC-1 antibody-containing liposomes directly to the malignant tumor Since the HIC-1 gene product may be indiscriminate with respect to cell type m its action, a targeted delivery system offers a significant improvement over randomly injecting non-specific liposomes Preferably, the target tissue is human brain, colon, breast, lung, and renal origin A number of procedures can be used to covalently attach either polyclonal or -29- monoclonal antibodies to a liposome bilayer Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab')2, as long as they bind efficiently to an antigenic epitope on the target cells Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors For use in the diagnostic research and therapeutic applications suggested above, kits are also disclosed. Such a kit may comprise a carrier means being compartmentalized to receive m close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method For example, one of the container means may comprise a probe which is or can be detectably labelled Such probe may be an antibody or nucleotide specific for a target protein or a target nucleic acid, respectively, wherein the target is indicative, or correlates with, the presence of HIC-1 of the invention Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotm-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radionucleotide label Also disclosed is a method for identifying a tumor suppressor gene by detecting abnormal nucleic acid methylation, in particular, detecting CpG island hypermethylation m the regions of frequent allelic loss The present applicant has shown that aberrant methylation of normally unmethylated CpG islands can function as a "mutation" to silence tumor suppressor gene transcription during tumor progression The occurrence of the 17p 13 3 hypermethylation appears to correlate with both the timing and incidence of these allelic losses in the progression of brain, colon, and renal cancers It is shown by the present applicant that this CpG island harbors a tumor suppressor HIC-1 gene which is silenced by abnormal methylation In other words, identification of such CpG islands has constituted an important -30- strategy for isolation of the new tumor suppressor HIC-1 gene Therefore, the finding of this abnormality in chromosome areas which frequently undergo the tumor associated allelic losses that broadly define candidate tumor suppressor regions could facilitate the localization of the responsible genes The common methods used for detecting abnormal nucleic acid methylation are well known in the art and those skilled in the art should be able to use one of the methods accordingly for the purpose of practicing the present invention The following Examples are intended to illustrate, but not to limit the invention While such Examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. EXAMPLES HIC-1 expression is ubiquitous in normal adult tissues However, in cultured tumor cells and in primary cancers which exhibit hypermethylation of the associated CpG island, HIC-1 expression is reduced or absent For example, the expression of HIC-1 is absent in tumors with CpG island hypermethylation, including lung, colon, breast and brain tumors This expression pattern is consistent with a tumor suppressor gene function for HIC-1 EXAMPLE 1 MATERIALS AND METHODS 1. Subcloning of cosmid DNA Subclones of cosmid C13A DNA (FIGURE 1A) were prepared by isolation of multiple restriction fragments on agarose gels and ligation of these into pBluescnpt plasmid (Stratagene) 2. DNA sequencing Single stranded DNA was first isolated by growing plasmid DNA in 2xYT broth with 75ug/ml ampicillm and in the presence of 107-10s pfii/ml of VCSM13 (Stratagene) (helper phage) for 2 hrs After isolation, the DNA was sequenced using the GIBCO BRL cycle sequencing kit Generally, 22 base pair primers were end labeled with y-32P and cycle conditions were 95°C for 1 cycle followed by 20 cycles of 95°C for 10 sec and 65°C for 10 sec Reaction products were analyzed on 10% acrylamide/8 M urea gels 3. Southern and Northern hybridizations Isolation procedures for DNA and poly A+ RNA agarose gel running conditions, a-32P labelling of probes, filter hybridization and wash conditions are as previously described (Baylin, S B , et al, Cancer Cells, 3 383-390, 1991, Jones, P A, et al., Cancer Res., 54 1-23, 1990; Herman, J G, et al., Proc. Nat'l Acad. Sci., in press, 1994, Ottaviano, Y L , et al., Cancer Res., 54 2552-2555, 1994, Issa, J-P , et al, Nature Genetics, in press; Steenman, M.J C , etal., Nature Genetics, 7 433-439, 1994, and Gish, W, etal. Nature Genetics, 3 266-272, 1993) Radioautograms were either exposed at -70°C for various times or in a phosphoimager casette, followed by exposure and analysis in the phosphoimager Image Quant program (Molecular Dynamics) Preparation of single strand, a-32P-labeled RNA probes for use in some Northern hybridizations was accomplished by in vitro transcription, using T3 or T7 polymerase, of DNA inserts in the various cosmid sublcones shown in FIGURE 1 A. 4. RNAse protection assays Preparation of a-32P-labeled RNA probes from the various cosmid subclones (FIGURE 1 A), liquid hybridization to RNA samples, and post-hybridization digestion by RNAse were all performed with the Ambion MAXIscnpt and RPAII kits according to the manufacturer's specifications In general, 8x104 cpm of probe was hybridized to 10 |ig of total RNA for 12-15 h at 45°C Products of RNAse digestion were analyzed on a 6% acrylamide/8 M urea gel Lengths of hybndization probes were determined by positions of various restnction cuts of the plasmid insert DNA For assessment of RNA loading, a 250 bp GAPDH probe was prepared by Hinc II restriction and co-hybridized with RNA m all reactions 5. Exon trapping -32- Exon trapping was performed with subclone 26 (FIGURE 1 A) using the GIBCO BRL Exon Trapping System, as per manufacturer's protocol 6. Cell cultures and tissue specimens Normal human fibroblast lines WI-38 and IMR-90 and colon cancer line, CaC02, were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD) The NCI-H209 line of human small cell lung carcinoma has been previously described (Carney, D N, etal, Recent Results Cancer Res. ,99 157-166, 1985) All established breast cancer lines were utilized, as detailed in FIGURE 5, in a recent study (Herman, J G, et al, Proc. Nat'l. Acad. Sci., 919700-9704, 1994) and were kindly provided by Dr Nancy Davidson A cell fusion system of tumor progression consisting of normal donor fibroblast line GM229 and the HT1080 line of fibrosarcoma cells, plus their fusion products, SFTH 300 and SFTH 300 TR1, were a gift from Dr B Weismann All samples of fresh, non-cultured, normal and neoplastic human tissues were those obtained as described (Herman, J G, et al, supra-, Ottaviano, Y L, etal, supra; Issa, J-P , et al, supra, Steenman, M.J C , et al, supra, and Gish, W , et al, supra) EXAMPLE 2 IDENTIFICATION OF NEW TIJMOR SUPPRESSOR GENE To characterize the region encompassing the aberrantly methylated CpG island, a series of subclones were prepared (FIGURE 1A) from the 17p cosmid C-13A (Ledbetter, D H., etal, Proc. Natl. Acad. Sci. USA, 86 5136, 1989, El-Deiry, W S , et al, Nature Genetics, \ 45-49, 1992, Kern, SE,e/ al, Science, 252 1708, 1991, Funk, W D, et al, Mol. & Cell. Biol., 12 2866, 1992) previously shown to contain the cluster of methylation sensitive Not I sites hypermethylated in tumors Using these as probes for "zoo blots", three regions (FIGURE 1A plasmids CI, CII, and 400) were found which hybridized, under stringent conditions, to restriction fragments in bovine and munne DNA Traditional positional cloning approaches were impeded by high non-specific hybndization of these probes to human DNA and cDNA libranes, probably due to the high GC content of the area Therefore, most of the 11 kb region (FIGURE 1 A) was sequenced and analyzed by the Grail computer program (Gish, W, et al, D J , Nature Genetics, 3 266, 1993) FIGURE 1A is a diagram showing a map of an 11 0 kb region of cosmid C-13A which contains a 50 kb human DNA insert harboring the region of chromosome 17p 13 3 previously shown to have hypermethylation in multiple human tumor types (Makos, M., et al, Proc. Natl. Acad. Sci. USA, 89 1929, 1992, Makos, M., etal, Cancer Res, 53 2715, 1993, Makos, M , et al, Cancer Res 53 2719, 1993) The position of the YNZ22 probe, EcoRI (E) restriction site and the location of a series of cosmid subclones which were prepared to span the area are shown FIGURE IB is a schematic for the HIC-1 gene which was found to be encompassed within the region shown in FIGURE 1A and for which the amino acid sequence is shown in FIGURE 2B Shown are potential p53 binding site, TATAA = the TATA box sequence 40 bp upstream from the transcription start site, 5' UTR = the 1st untranslated exon, ATG = the most 5' translation start site, ZIN (zinc finger N-terminus) = the 478bp exon encompassing the highly conserved region (FIGURE 2A) of the Zin domain subfamily of zinc finger transcription factors, rectangle with shaded bars represents the 2015 bp last exon of HIC-1 and each shaded bar represents one of the 5 zinc fingers (FIGURE 2B) clustered in this 3' region of the gene, TAG = translation stop site in the HIC-1 gene, AATAAA = polyadenylation signal site found 835 bp from the translation stop site FIGURE 1C shows the nucleotide and deduced amino acid sequence of HIC-1 Two independent regions of excellent coding potential were revealed between the N3 to N7 Not I restriction sites (FIGURE 1 A) Blast program (Altschul, S F , et al., J Mol. Biol., 215 403. 1990) analysis revealed distinct amino acid homologies (FIGURES IB and 2A), within one of the independent regions, to a highly conserved N-terminal motif, termed the Zin (zinc finger N-terminal) domain, which is present in each member of a recently defined subset of zinc finger transcription factors (Harrison and Travers, EMBO J 9 207, 1990, di Bello, etal, Genetics, 129 385, 1991, Numoto, etal, Nucleic Acids Res 21 3767, 1993, Chardin, etal., Nucleic Acids Res. 19 1431, 1991) In addition to the Zm domain, five Kruppel type Cys^-His^ zmc fingers (Ruppert, J M, etal, Mol. & Cell. Biol.,8 3104-3113,1988) characteristic of the 3' region of these same proteins, were also identified (FIGURES IB and 2B) This novel gene was named HIC-1 (hypermethylated in cancer) EXAMPLE 3 CHARACTERIZATION OF HTC-t A combination of RNAse protection strategies, exon trapping studies, and Northern blot analyses, were utilized to characterize expression of HIC-1 and to define the genomic structure of the gene (FIGURES IB and 1C, SEQ ID NO 1 and 2) The start of transcription was identified within 40 bp downstream from a TATA box sequence (FIGURE IB) which precedes an untranslated first exon The putative ATG site and the Zin domain are located in a 476 bp second exon and are m a similar position to those of the 8 other Zin domain proteins (FIGURE 2A) The 5 zinc fingers (FIGURES IB and 2B) reside in a 2015 bp final exon, containing a translation stop site 835 bp upstream from the polyadenylation signal, AATAAA The HIC-1 gene (FIGURES 1C and 2B ), structured similarly to the other Zin domain proteins, is encompassed by three exons within the CpG rich 3 0 kb region between Not I sites N3 and N7 (FIGURE 1) FIGURE 2A and SEQ ID NO.2 show the amino acid sequences of HIC-1 The HIC-1 amino acid sequence is compared with the conserved N-terminus region of the other members of the Zm domain zinc finger family In the parentheses, the numbers indicate the position of the conserved region relative to the translation start site of each gene The darkest shading shows position of amino acids which are identical for at least five of the 9 proteins and the lighter shading shows position of conservative amino acid differences between the family members D = drosophila, M = murine, H = human The bracket of amino acids at the bottom represents an area in HIC-1 not found at this position in the other family members FIGURE 2B and SEQ ID NO 3 show the entire coding region of the HIC-1 gene The deduced amino acid sequence for the two coding exons of HIC-1, as defined by the sequence analyses and expression strategies outlined in the text, are shown The 5 zinc fingers m the 3' half of the protein are shown by the shaded boxes -35- EXAMPLE 4 ANALYSIS OF HIC-1 GENE EXPRESSION HIC-1 was found to be ubiquitously expressed gene By Northern analysis of poly A+ RNA from multiple normal tissues, probes from the HIC-1 Zin domain, zinc finger regions, and 3' untranslated regions inclusive of the polyadenylation site, all identified the same predominant 3 0 kb transcript FIGURE 3 shows a Northern analyses of HIC-1 gene expression S = spleen, The = thymus, P= prostate, Te = testis, 0 = ovary; SI = small intestine, B = peripheral blood cells The band above the 4 4 kb marker co-hybridizes with ribosomal RNA The -1 1 kb band has not yet been identified but could be an alternate splice product since it was not detected with probes from the zinc finger or 3' untranslated regions of HIC-1. FIGURE 4 A shows RNAse protection assays of HIC-1 gene expression in a variety of normal and neoplastic human tissues In all panels, the top asterisk marks the position of the undigested 360bp HIC-1 gene RNA probe which was derived from the region containing the zinc fingers m cosmid subclone 600 (FIGURE 1A) The protected HIC-1 fragment (300bp) is labeled HIC-1 FIGURE 4A compares expression in 10 ug of total RNA from 2 established culture lines of normal human fibroblasts (WI-38 and IMR-90) to the HT 1080 culture line of fibrosarcoma cells (Fibro-C), from 3 different samples of normal colon (Colon - N) to the colon carcinoma cell line, CaC02 (Colon-C), and from a sample of normal lung (Lung-N) to the established line of human small cell lung carcinoma, NCI-H209 (Lung-C) FIGURE 4B shows the RNAse protection assay for 10 ug of RNA from 6 different established culture lines of breast carcinoma (lane 1 MDA231, lane 2 HS58T, lane 3 MDA468, lane 4 T47D, lane 5 MCF7, lane 6 MDA453), each of which has extensive 25 methylation of Not I sites of the HIC-1 CpG island FIGURE 4C shows the RNAse protection assay for 10 ug of RNA from normal fetal brain (B) compared to a series of non-cultured brain tumors (1 anaplastic astrocytoma (AA) and 8 more advanced glioblastomas (lanes 1-8) 5 10 15 20 The 3 0 kb transcript was found in all adult tissues tested with especially high levels in lung, colon, prostate, thymus, testis, and ovary (FIGURE 3) With the Zin domain probe, a 1 1 kb transcript was also detected in some tissues which may represent an alternatively spliced product (FIGURE 3) RNase protection assays (RPAZ Kit-Ambion), using a probe from plasmid 600 (FIGURE 1A), validated the ubiquitous expression of HIC-1, protecting transcripts of predicted size in cultured fibroblasts (FIGURE 4A) and non-cultured colon mucosa (FIGURE 4A), lung (FIGURE 4A), and brain (FIGURE 4C) By RNAse protection assays, HIC-1 expression was found to be absent or decreased in neoplastic cells which have aberrant HIC-1 CpG island methylation Little or no expression (FIGURE 4A) was detected in cultured cancer cell lines of colon, lung, and fibroblast, all previously shown to be fully methylated at Not I sites 3 through 7 The same finding was true for 6 cultured breast cancers (FIGURE 4B), all of which exhibited hypermethylation of Not I sites 3 through 7 Furthermore, in primary colon tumors, HIC-1 expression was 2 to 17-fold decreased in a non-cultured human colon polyp and 3 primary colon tumors, as compared to the corresponding normal colon Finally, the absence of HIC-1 expression in primary, non-cultured brain tumors was found in tumors that exhibited aberrant hypermethylation of the CpG island An anaplastic astrocytoma which exhibited a full methylation pattern of the HIC-1 CpG island, did not express this gene (FIGURE 4C), as compared to normal brain In 4 glioblastomas, m which both DNA and RNA were available, two expressed HIC-1 either weakly (FIGURE 4C, lane 1) or not at all (FIGURE 4C, lane 4) and had predominantly hypermethylated alleles, while two with unmethylated alleles expressed the gene at levels equal to adjacent normal brain (FIGURE 4C, lanes 2 and 3) Four additional glioblastomas for which RNA was available were also studied One expressed HIC-1 weakly (FIGURE 4C, lane 5), one had no expression (FIGURE 4C, lane 6), and two tumors expressed this gene (FIGURE 4C, lanes 7-8) -37- In addition, hypermethylation of HIC-1 was analyzed in several primary tumors and cultured cell lines by DNA analysis as follows Southern analyses of DNA from control and 24 hour infected cells which was digested with EcoRI (12U/ug DNA) plus Not I (20U/ug), were probed with a-32P-labeled YNZ22 (FIGURE 1A) exactly as detailed in previous studies (Makos, etal., supra, 1992, 1993) Filters were imaged in the Phosphoimager (Molecular Dynamics) The results shown in Table 1 indicate that HIC-1 is found to be hypermethylated in a variety of tumors and cell lines from various origins including brain, colon, renal, hematopoietic, and prostate cancers and tumors TABLE 1 HYPERMETHYLATION OF HIC-1 IN TTJMQRS AND CELL LINES PRIMARY TUMORS BRAIN TTJMQRS CULTURED CELL LINES Low Grade Astrocytomas Anaplastic Astrocytomas Glioblastoma Multiforme Medulloblastoma COLON CANCSRS Polyps Carcinomas LONG CANCSRS I 7 METH % 7 100 4 80 6 75 4 80 6 100 1 METH % 90 Glials 2 2 100 Carcinoma 6 7 85 Carcinomas Carcinoma IS 12 75 -38- TABLE 1 (CON'T) RENAL CANCSRS Early Stage 8 4 50 Late Stage 3 2 67 Late Stage 21 16 5 LETJKEMIAS Lymphomas 3 1 33 Lymphomas 8 5 CML/Blast 8 7 87 AML 13 10 80 ALL 10 8 80 10 I METH % I METH BREAST CANCERS Cancer 24 15 62 Cancers 6 6 PROSTATE CANCERS Cancer 17 17 100 Cancer 5 4 15 ENDOMETRIAL CANCER Cancer 6 4 67 NEUROBLASTOMAS early/late stage 12 2 IS Cancers 4 4 (amount of 20 methylation LOW) EXAMPLE 5 INTERACTION OF P53 WITH HIC-1 EXPRESSION Consistent with the hypothesis that a suppressor gene exists at 17pl3 3 which may interact with p53, the present invention identifies a potential p53 binding site 4 kb 5' 25 to the TATA box in the HIC-1 gene (FIGURE IB) Therefore, the p53 response of the HIC-1 gene was tested by using a colon cancer cell line (SW480) in which the p53 responsive gene, WAF-1, had been shown previously to be induced by expression of wild type p53 (El-Deiry, et al., Cell, 75 817-825, 1993) This cell line contains one 17p chromosome, a mutant p53 allele, and a fully methylated HIC-1 CpG island 30 Furthermore, the cell line SW480 is severely growth arrested by exogenously expressing the wild type p53 gene (Baker, S J , et al., Science, 249 912-915, 1990) expressing the wild type p53 gene (Baker, S J, et al., Science, 249 912-915, 1990) FIGURE 5 shows an RNAse protection assay, as detailed m FIGURE 4, after infection of an adenoviral vector containing either the P-galactosidase gene or the wild type human p53 gene into the SW480 line of human colon cancer cells (Uninfected, normal, control human fibroblasts (F), uninfected SW480 cells (U), SW480 cells infected with the P-galactosidase gene (GAL), and SW480 cells infected with the p53 gene (p53)) Positions of the undigested HIC-1 and GAPDH probes and of the HIC-1 and GAPDH transcripts are marked exactly as in FIGURE 4 HIC-1 is expressed at only low levels in this cells line (Fig 5 A - U) When the wild type p53 gene is exogenously expressed in the SW480 cells, the level of HIC-1 expression is upregulated 20 fold (Fig 5 - p53), as compared to control cells (U & GAL) These results suggest that the tumor suppressor gene p53 activates HIC-1 expression, either directly or indirectly However, since a p53 binding sites has been identified 4 Okb upstream from the transcription start site (see enclosed map), it suggests a direct interaction between p53 and HIC-1 We are working to validate this type of interaction SUMMARY OF EXAMPT.FS HIC-1 plays a significant role in normal and neoplastic cells At least four other genes have thus far been identified as potential downstream targets of p53, including WAF1 (El-Deiry, W S , et al, supra.) MDM2 (Chen, CY, etal., Proc. Natl. Acad. Sci. USA, 91 2684-2688, 1994), GADD45 (Kastan, MB , etal., Cell, 21 587-597, 1992) and BAX (Miyashita, T, etal., Oncogene, 9 1799-1805, 1994) HIC-1 probably functions as a transcription factor, as inferred by its structure and the characteristics of the other members of the Zin domain family Two drosophila members, tram-track and broad complex, are transcriptional repressors which help regulate segmental development (Harrison and Travers, EMBO J 9 207, 1990, di Bello, et al., Genetics, 129 385, 1991) A third drosophila protein, GAGA appears to function by dynamically blocking the formation of nucleosomal structures which would impede transcriptional activation of promoter regions (Tsukiyama, T , et al., Nature, 367 525-532, 1994) The munne Zm domain gene, MZF5, has tn-vitro transcriptional repressor reactivity for c-myc and thymidine kinase promoters (Numoto, et al, Nucleic Acids Res , 2J. 3767, 1993) Finally, two of the 4 other human Zin domain proteins were found as components of translocations in human neoplasms (Chardin, et al, Nucleic Acids Res., 19 1431, 1991, Hromas, etal, J. Biol. Chem., 266 14183, 1991, Chen, et al ,EMBO J., 12 1161, 1993) Second, it is necessary to determine the precise interaction between p53 and the HIC-1 promoter In summary, the present invention identifies a new gene at 17pl3 3, HIC-1, for which the expression pattern, structural motifs, chromosomal location, and p53 responsiveness are suggestive of an important function in tumorgenesis Identification of the precise p53 pathway in which HIC-1 is involved should clarify the role of this gene in normal and neoplastic cells Finally, the results suggest that in tumor DNA, identification of hypermethylated CpG islands associated with regions of allelic loss could facilitate the localization and cloning of candidate tumor suppressor genes as well as function as markers for recurrent abnormal growth or cells which may be resistant to particular therapeutic regimens The foregoing is meant to illustrate, but not to limit, the scope of the invention Indeed, those of ordmaiy skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation -41- SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The Johns Hopkins University School of Medicine 5 (11) TITLE OF INVENTION: NOVEL TUMOR SUPPRESSOR GENE, HIC-1 (111) NUMBER OF SEQUENCES: 3 (IV) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fish & Richardson, P.C. 10 (B) STREET: 4225 Executive Square, Suite 1400 (C) CITY: La Jolla (D) STATE: California (E) COUNTRY: USA (F) ZIP: 92037 15 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 20 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT/US95/ (B) FILING DATE: 15-NOV-1995 (C) CLASSIFICATION: (Vlll) ATTORNEY/AGENT INFORMATION: 25 (A) NAME: Haile, Ph.D., Lisa A. (B) REGISTRATION NUMBER: 38,347 (C) REFERENCE/DOCKET NUMBER: 07265/039W01 (IX) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (619) 678-5070 30 (B) TELEFAX: (619) 678-5099 (2) INFORMATION FOR SEQ ID NO:1: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4616 base pairs (B) TYPE: nucleic acid 35 (C) STRANDEDNESS: single (D) TOPOLOGY: linear -42- (ii) MOLECULE TYPE. DNA (genomic) (vi1) IMMEDIATE SOURCE (B) CLONE HIC-1 polynucleotide (ix) FEATURE. (A) NAME/KEY. CDS (B) LOCATION. 1 461S (xi) SEQUENCE DESCRIPTION. SEQ ID NO:l CCCGGCCCGC CGGGACCGCA GGTAACGGGC CGCGGGGCCC CGCGGGCCAG GAGGGGAACG 60 GGGTCGGGCG GGCGAGCAGC GGGCAGGGGA GCTCAGGGCT CGGCTCCGGG CTCTGCCGCC 120 10 GGATTTGGGG GCCGCGAGGA AGAGCTGCGA GCCGAGGGCC TGGGGCCGGC GCACTCCTCC 18 0 CGCCCTGTCT GCAGTTGGAA AACTTTTCCC CAAGTTTGGG GCGGCGGAGT TCCGGGGGAG 24 0 AAGGGGCCGG GGGAGCCGCG GAGGGAGGCG CCGGGCCCGC GCGTGTAGGG CCCAGGCCGA 3 00 GGCCGGGACG CGGGTGGGGC GCAGGCCCGG GTCAGGGCCG CAGCCGGCTG TGCGCCGTGC 3 60 CCGCCCGGGG CGCTGCCCCC TCCCTCCCCT GGGAGCTGCG TGGCTCCCCC CTCCCCCCCA 420 15 CCTGCTTCCT GCCTCAGCCT CCTGCCCCGA TATAACGCCC TCCCCGCGCC GGGCCCGGCC 430 TTCGCGCTCT GCCCGCCACG GCAGCCGCTG CCTCCGCTCC CCGCGCGGCC GCCGCCCGGG 54 0 CCCCGACCGA GGGTTGACAG CCCCCGGCCA GGGCGGCGCC AGGGCGGGCA CCGCGCTCCC 600 CTCCTCCGTA TCACTTCCCC CAACTGGGGC AACTTCTCCC GAGGCGGGAG GCGCTGGTTC 660 CTCGGCTCCC TTTCTCCCTA CTTGGGTAAA GTTCTCCGCC CTGAATGACT TTTCCTGAAG 720 20 CGGACATTTT ACTTAAATCG GGTAACTGTC TCCAAAAGGG TCACTGCGCC TGAACAGTTT 78 0 TCTTCTCGGA AGCCCCAGCA CCCAGCCAGG TGCCCTGGGG CGTGCAGGCC GCCCTGGCCT 84 0 CCCCTCCACC GGCGGCCGCT CACCTCCTGC TCCTTCTCCT GGTCCGGGCG GGCCGGCCTG 90 0 GGCTCCCACT CCAGAGGGCA GCTGGTCCTT CGCCGGTGCC CAGGCCGCAG GGCTGATGCC 96 0 CCCGCTCAGC TGAGGGAAGG GGAAGTGGAG GGGAGAAGTG CCGGGCTGGG GCCAGGCGGC 102 0 25 CAGGGCGCCG CACGGCTCTC ACCCGGCCGG TGTGTGTCCC CGCAGGAGAG TGTGCTGGGC 10 8 0 AGACGATGCT GGACACGATG GAGGCGCCCG ACAACCAGCG CACCAAGGGC TTCTTGTGCG TCCGCGCGCA CAAGAACGTG CTGGCGGCCA ATGACAACCT GCTCAACCTG GACCATGACA 5 TGGACTTCAT CTACACCGGC CGCCTGGCTG TGGCCCCGGG GGCTGAGCCG AGCCTGGGCG TCCCCGACCT CGTGGCGCTG TGCAAGAAAC TGCGGGGCGG CGGCGGCGGC GGCGGCGGCT TGCGGGCCGC CACGCCGTCA TCCAGGCCTG 10 GCCTGCCGCG GAGCCGCCCT CGGGCCCAGA GTACGCGTCG GGACCCGGCC CGGCCGCCGC TCTTTGTGGC CTGGACCTGT CCAAGAAGAG GCTGGCTGAG CGCGAGCTGC CCCCGCGCCC CTACAAGGAG CCGCCTCTCG CCCTGCCGTC 15 GGAGGCCGCA CCGCCTTCCG ACCCATTTCG CCCCGGCCGC CCCAACGGGC CTAGTCTCCT GGGTAGCTAT GGCGACGAGC TGGGCCGGGA GCGTGGTGGG GACGCGGCCG TCTCGCCCGG GCGCTACCCT GGCAGCCTGG ACGGGCCCGG 20 CAGCAGCGAG GAGACCGGTA GCAGCGAGGA GCTACCCATG CCCGCACCTG GCCTATGGCG TGTGCATTCC GTGCGGCAAG GGCTTCCCCA CTCACGTGGA GGAGGAGGAA GCGCTGTACG GGGCCGCCGG CCTAGGGCCC CCTTTTGGAG -43- GCCACTCCAG GCAGCTGCTG CTGCAGCTCA 114 0 ACGTGATCAT CGTGGTGCAG AACGCCCTCT 120 0 GCAGCGCCTA CCTCAAGTCC CTGGTGGTGC 1260 TGGTGAGCCC GGCCGTGTTC CGCCTGGTGC 13 20 ACGGCGCAGA GGCGGCTGCG GCCGCGGCCG 13 80 CCGTGCTGGC CGCCGCCAGC TACCTGCAGA 144 0 GCCTCAAGCG CCACGGCAAG TACTGCCACC 1500 ACGCGCCCTA TGGTCGGCCG GGCCGGGGCC 1560 CTACCCGTCC CCAGTCGGGC CTCCGCCGCC 1520 GGCCGCGGTC AACACGCACT GCGCCGAGCT 1680 ACTCTGTGCC TCGGAGCGCC GCTGCTCCCC 174 0 CCCGCCGGGC TCCGCGGCGC CAGAGCGGCC 1800 GGACAGCCCT CCCAGCGCCG GCCCCGCCGC 1860 GCTGCCGCCG CTGCCCTTCC AGAAGCTGGA 1920 CGGCGGCAGC GGCAGCCCGG GACCCGAGCC 1980 CTATCGCTGG ATGAAGCACG AGCCGGGCCT 204 0 GCGCGGCTCC CCCAGCGAGC GCTGCGAAGA 210 0 GGGGCCCCCG CTCGGCCTGG CGCCGCCGCC 216 0 CGCGGGCGGC GACGGCGACG ACTACAAGAG 222 0 CCCCAGCACC GCCTGGCGGC CACCTCGAGG 22 8 0 AGCCCGAGAG CTTCGGTGAC AACCTGTACG 23 4 0 GCTCTGAGCA GCTGAACGCG CACGTGGAGG 24 0 0 GCAGGGCCGA GGCGGCCGAA GTGGCCGCTG 24SO GCGGCGGGGA CAAGGTCGCC GGGGCTCCGG 252 0 GTGGCCTGGG AGAGCTGCTG CGGCCCTACC ACCCGGCCAC GCTGCGGCAG CACGAGAAGA CCATCTGCGG GAAGAAGTTC ACGCAGCGTG TGGGCCTCAA GCCCTTCGCG TGCGACGCGT 5 TCACCCGGAC GCACATGCGC ATCCACCCTC GCGGCGGCAA GTTCGCACAG CAACGCAACC GGGGCGCGGC GGCGCGGCCG GGGCGCTGGC CCCCGACGGC AAGGGCAAGC TCGACTTCCC GCCGAGCAGC TGAGCCTGAA GCAGCAGGAC 10 ACCACGCACT TCCTGCACGA CCCCAAGGTG TTCACGGCCG AGCTGGGCCT CAGCCCCGAC CACCTGGCGG CCGGGCCCGA CGGCGGACCA CTCGCCAGCC CGCTCTGTCG CTGCTGCGCG GGCGGCGCGC AGGGCCCACT GTGCCCGGGA 15 ACCTCTCGGC GGCCTCACCT GGCCTCACTG AACCCCGGGA CGGGGTGGGA TGGGGTAAGG CAAAGGAGAC CCCAGGCCCC TCCCGCCTCT TCCGCGCTGC TCTTAGAGGG GGAGGGGTGT GGCCCTTGCG ACCACACCCA TTCTCACTGT 20 AGAGTTGGGG AGTGGGGAGG GGACTGAGCC CCACCCCGGG ACTGATAATG TGAAGTTCCT CAACCCTTCC TTCCTCAGTC ACCAAGGGCG TACCACCAGG TCTCCCACTC CCGCGGTGCC TATTTATTGC ATGCGCCCCG GCGGCCCCCC -44- GCTGCGGCTC GTGCGACAAG AGCTACAAGG 2580 CGCACTGGCT GACCCGGCCC TACCCATGCA 264 0 GGACCATGAC GCGCCACATG CGCAGCCACC 270 0 GCGGCATGCG GTTCACGCGC CAGTACCGCC 2 76 0 GCGGCGAGAA GCCCTACGAG TGCCAGGTGT 2820 TCATCAGCCA CATGAAGATG CACGCCGTGG 2880 GGGCTTGGGG GGGCTCCCCG GCGTCCCCGG 294 0 CGAGGGCGTC TTTGCTGTGG CTCGCTCACG 3 0 00 AAGGCGGCCG CGACCGAGCT GCTGGCGCAG 3060 GCGCTGGAGA GCCTCTACCC GCTGGCCAAG 3120 AAGGCGGCCG AGGTGCTGAG CCAGGGCGCT 3180 TCGACCGTTT CTCTCCCACC TAGAGCGCCC 3 24 0 GCCCTGGCCC GCACCCCAGG GAGCGGCGGG 3 3 00 CAACCGCAGC GTCGCCACAG TGGCGGCTCC 3 3 60 CTTCGTGCCT TAGCTCGGGG GTCGGGGGAG 3420 GAAATTTATA TTTTTGATAT CAGCTTTGAC 34 80 TCCTGTGGTT CGTCGGCCCC CTCCCCCGGC 3 54 0 CACTGTCGGG GCACTCCTAG CCCTACCTCC 3 60 0 GAATCTCCCC GCTGGGTCGG AGCGTCGGGC 3 66 0 GGCCGGAGGC CCCCGCACCC CCGCTCCCAC 3 72 0 CATTTTGCAC AAGTGGCACT AGCCCAGGGC 3 78 0 GGGAGTTCTG GAGTCGGAAG GCGAAGAGCC 3 84 0 CTCCCTTCCC TTCCCTGCGG CCCCGGACCA 3 900 ATCCCGAGCC CAGGCTGGGC TGGGCTGGAA 3 96 0 -45- CGCGGTCTCT TTAGCTCCCT CCTCTTCGTT TGTATATTTC CTACCTTGTA CACAGCTCTT 4 020 CCAGAGCCGC TTCCATTTTC TATACTCGAA CCAAACAGCA ATAAAGCAGT AACCAAGGAC 4080 CCCGACCCCG CTGCTCTCTT CTGCCCCTGC ACAAGGACCT GGATGCTGCG CCCGCTGGGT 414 0 GGAGGAGCCA GAAAGGGCCA CCCTCACACA GGTGCAGAGG CTTGGACCTG CCTCCCTCCC 42 0 0 5 CAGTCCCAGA AACAGATCAG CAAGAGGTCA GGTATGTTTC ATAACTAAAA ATTTATTAAG 42SO GAAACAAAAC CAGTGCTGCA AACGGGACAG AAAGGAGAGC TGGGTCTCCC TCCCGACCAC 43 2 0 CCAGTCATCG GCCTTCCAGC TGGGGAGAGA ATCTTAAAGG AGAGGCCGGG GACCCTGTAC 43 80 TCCAAAGAGC CCAGTCTTCT GAGACTCTAG GGGACTCCTA CCCCCAAACT ACTGGCCTTG 444 0 GCTCCCCTAC ACGGTACCCC ATCGCTTCTG GCATAGTCCT GGGCCTCAGG GAGGGCAGAG 4 500 10 CTGCGCACCC ATCCTCCAGG CAGGCTGTGC AGTCAGGCCA TGGGCTCTGG GGTATCCCCC 4 560 ACTGGTCCCA TTAAGATTTG CCCCTGGCTC CACCGAAAAC CCCGTCTTCC CCTAAG 4 616 (2) INFORMATION FOR SEQ ID NO.2: (l) SEQUENCE CHARACTERISTICS (A) LENGTH 4112 base pairs 15 (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY, linear (ii) MOLECULE TYPE: DNA (genomic) (vn) IMMEDIATE SOURCE: 20 (B) CLONE. HIC-1 coding polynucleotide (ix) FEATURE (A) NAME/KEY. CDS (B) LOCATION 1086 .2726 (xi) SEQUENCE DESCRIPTION SEQ ID NO.2 25 CCCGGCCCGC CGGGACCGCA GGTAACGGGC CGCGGGGCCC CGCGGGCCAG GAGGGGAACG 60 GGGTCGGGCG GGCGAGCAGC GGGCAGGGGA GCTCAGGGCT CGGCTCCGGG CTCTGCCGCC 120 GGATTTGGGG GCCGCGAGGA AGAGCTGCGA GCCGAGGGCC TGGGGCCGGC GCACTCCTCC 18 0 CGCCCTGTCT GCAGTTGGAA AACTTTTCCC CAAGTTTGGG GCGGCGGAGT TCCGGGGGAG 24 0 -46- AAGGGGCCGG GGGAGCCGCG GAGGGAGGCG CCGGGCCCGC GCGTGTAGGG CCCAGGCCGA 300 GGCCGGGACG CGGGTGGGGC GCAGGCCCGG GTCAGGGCCG CAGCCGGCTG TGCGCCGTGC 360 CCGCCCGGGG CGCTGCCCCC TCCCTCCCCT GGGAGCTGCG TGGCTCCCCC CTCCCCCCCA 4 20 CCTGCTTCCT GCCTCAGCCT CCTGCCCCGA TATAACGCCC TCCCCGCGCC GGGCCCGGCC 4 80 5 TTCGCGCTCT GCCCGCCACG GCAGCCGCTG CCTCCGCTCC CCGCGCGGCC GCCGCCCGGG 54 0 CCCCGACCGA GGGTTGACAG CCCCCGGCCA GGGCGGCGCC AGGGCGGGCA CCGCGCTCCC 600 CTCCTCCGTA TCACTTCCCC CAACTGGGGC AACTTCTCCC GAGGCGGGAG GCGCTGGTTC 66 0 CTCGGCTCCC TTTCTCCCTA CTTGGGTAAA GTTCTCCGCC CTGAATGACT TTTCCTGAAG 72 0 CGGACATTTT ACTTAAATCG GGTAACTGTC TCCAAAAGGG TCACTGCGCC TGAACAGTTT 780 10 TCTTCTCGGA AGCCCCAGCA CCCAGCCAGG TGCCCTGGGG CGTGCAGGCC GCCCTGGCCT 84 0 CCCCTCCACC GGCGGCCGCT CACCTCCTGC TCCTTCTCCT GGTCCGGGCG GGCCGGCCTG 900 GGCTCCCACT CCAGAGGGCA GCTGGTCCTT CGCCGGTGCC CAGGCCGCAG GGCTGATGCC 960 CCCGCTCAGC TGAGGGAAGG GGAAGTGGAG GGGAGAAGTG CCGGGCTGGG GCCAGGCGGC 1020 CAGGGCGCCG CACGGCTCTC ACCCGGCCGG TGTGTGTCCC CGCAGGAGAG TGTGCTGGGC 1080 15 AGACG ATG CTG GAC ACG ATG GAG GCG CCC GGC CAC TCC AGG CAG CTG 112 7 Met Leu Asp Thr Met Glu Ala Pro Gly His Ser Arg Gin Leu 15 10 20 CTG CTG CAG CTC AAC AAC CAG CGC ACC AAG GGC TTC TTG TGC GAC GTG Leu Leu Gin Leu Asn Asn Gin Arg Thr Lys Gly Phe Leu Cys Asp Val 15 20 25 30 1175 ATC ATC GTG GTG CAG AAC GCC CTC TTC CGC GCG CAC AAG AAC GTG CTG 1223 lie lie Val Val Gin Asn Ala Leu Phe Arg Ala His Lys Asn Val Leu 35 40 45 GCG GCC AGC AGC GCC TAC CTC AAG TCC CTG GTG GTG CAT GAC AAC CTG 1271 25 Ala Ala Ser Ser Ala Tyr Leu Lys Ser Leu Val Val His Asp Asn Leu 50 55 60 CTC AAC CTG GAC CAT GAC ATG GTG AGC CCG GCC GTG TTC CGC CTG GTG Leu Asn Leu Asp His Asp Met Val Ser Pro Ala Val Phe Arg Leu Val 65 70 75 1319 -47- CTG GAC TTC ATC TAC ACC GGC CGC CTG GCT GAC GGC GCA GAG GCG GCT 13 67 Leu Asp Phe lie Tyr Thr Gly Arg Leu Ala Asp Gly Ala Glu Ala Ala 80 85 90 GCG GCC GCG GCC GTG GCC CCG GGG GCT GAG CCG AGC CTG GGC GCC GTG 1415 5 Ala Ala Ala Ala Val Ala Pro Gly Ala Glu Pro Ser Leu Gly Ala Val 95 100 105 110 CTG GCC GCC GCC AGC TAC CTG CAG ATC CCC GAC CTC GTG GCG CTG TGC 14 63 Leu Ala Ala Ala Ser Tyr Leu Gin lie Pro Asp Leu Val Ala Leu Cys 115 120 125 10 AAG AAA CGC CTC AAG CGC CAC GGC AAG TAC TGC CAC CTG CGG GGC GGC 1511 Lys Lys Arg Leu Lys Arg His Gly Lys Tyr Cys His Leu Arg Gly Gly 130 135 140 GGC GGC GGC GGC GGC GGC TAC GCG CCC TAT GCT ATG GCG ACG AGC TGG 1559 Gly Gly Gly Gly Gly Gly Tyr Ala Pro Tyr Ala Met Ala Thr Ser Trp 15 145 150 155 GCC GGG AGC GCG GCT CCC CCA GCG AGC GCT GCG AAG AGC GTG GTG GGG 1607 Ala Gly Ser Ala Ala Pro Pro Ala Ser Ala Ala Lys Ser Val Val Gly 160 165 170 ACG CGG CCG TCT CGC CCG GGG GGC CCC CGC TCG GCC TGG CGC CGC CGC 1655 20 Thr Arg Pro Ser Arg Pro Gly Gly Pro Arg Ser Ala Trp Arg Arg Arg 175 180 185 190 CGC GCT ACC CTG GCA GCC TGG ACG GGC CCG GCG CGG GCG GCG ACG GCG 1703 Arg Ala Thr Leu Ala Ala Trp Thr Gly Pro Ala Arg Ala Ala Thr Ala 195 200 205 25 ACG ACT ACA AGA GCA GCA GCG AGG AGA CCG GTA GCA GCG AGG ACC CCA 1751 Thr Thr Thr Arg Ala Ala Ala Arg Arg Pro Val Ala Ala Arg Thr Pro 210 215 220 GCA CCG CCT GGC GGC CAC CTC GAG GGC TAC CCA TGC CCG CAC CTG GCC 1799 Ala Pro Pro Gly Gly His Leu Glu Gly Tyr Pro Cys Pro His Leu Ala 30 225 230 235 TAT GGC GAG CCC GAG AGC TTC GGT GAC AAC CTG TAC GTG TGC ATT CCG 184 7 Tyr Gly Glu Pro Glu Ser Phe Gly Asp Asn Leu Tyr Val Cys lie Pro 240 245 250 TGC GGC AAG GGC TTC CCC AGC TCT GAG CAG CTG AAC GCG CAC GTG GAG 13 95 35 Cys Gly Lys Gly Phe Pro Ser Ser Glu Gin Leu Asn Ala His Val Glu 255 260 265 270 -48- GCT CAC GTG GAG GAG GAG GAA GCG CTG TAC GGC AGG GCC GAG GCG GCC 1943 Ala His Val Glu Glu Glu Glu Ala Leu Tyr Gly Arg Ala Glu Ala Ala 275 280 285 GAA GTG GCC GCT GGG GCC GCC GGC CTA GGG CCC CCT TTT GGA GGC GGC 19 91 5 Glu Val Ala Ala Gly Ala Ala Gly Leu Gly Pro Pro Phe Gly Gly Gly 290 295 300 GGG GAC AAG GTC GCC GGG GCT CCG GGT GGC CTG GGA GAG CTG CTG CGG 203 9 Gly Asp Lys Val Ala Gly Ala Pro Gly Gly Leu Gly Glu Leu Leu Arg 305 310 315 10 CCC TAC CGC TGC GGC TCG TGC GAC AAG AGC TAC AAG GAC CCG GCC ACG 20 87 Pro Tyr Arg Cys Gly Ser Cys Asp Lys Ser Tyr Lys Asp Pro Ala Thr 320 325 330 CTG CGG CAG CAC GAG AAG ACG CAC TGG CTG ACC CGG CCC TAC CCA TGC 213 5 Leu Arg Gin His Glu Lys Thr His Trp Leu Thr Arg Pro Tyr Pro Cys 1 5 335 340 345 350 ACC ATC TGC GGG AAG AAG TTC ACG CAG CGT GGG ACC ATG ACG CGC CAC 2183 Thr lie Cys Gly Lys Lys Phe Thr Gin Arg Gly Thr Met Thr Arg His 355 360 365 ATG CGC AGC CAC CTG GGC CTC AAG CCC TTC GCG TGC GAC GCG TGC GGC 2231 20 Met Arg Ser His Leu Gly Leu Lys Pro Phe Ala Cys Asp Ala Cys Gly 370 375 380 ATG CGG TTC ACG CGC CAG TAC CGC CTC ACC CGG ACG CAC ATG CGC ATC 2279 Met Arg Phe Thr Arg Gin Tyr Arg Leu Thr Arg Thr His Met Arg lie 385 390 395 25 CAC CCT CGC GGC GAG AAG CCC TAC GAG TGC CAG GTG TGC GGC GGC AAG 2327 His Pro Arg Gly Glu Lys Pro Tyr Glu Cys Gin Val Cys Gly Gly Lys 400 405 410 TTC GCA CAG CAA CGC AAC CTC ATC AGC CAC ATG AAG ATG CAC GCC GTG 23 75 Phe Ala Gin Gin Arg Asn Leu lie Ser His Met Lys Met His Ala Val 30 415 420 425 430 GGG GGC GCG GCG GCG CGG CCG GGG CGC TGG CGG GCT TGG GGG GGC TCC 2423 Gly Gly Ala Ala Ala Arg Pro Gly Arg Trp Arg Ala Trp Gly Gly Ser 435 440 445 35 CCG GCG TCC CCG GCC CCG ACG GCA AGG GCA AGC TCG ACT TCC CCG AGG Pro Ala Ser Pro Ala Pro Thr Ala Arg Ala Ser Ser Thr Ser Pro Arg 450 455 460 2471 -49- GCO TCT TTG CTG TGG CTC GCT CAC GGC CGA GCA GCT GAG CCT GAA GCA 2519 Ala Ser Leu Leu Trp Leu Ala His Gly Arg Ala Ala Glu Pro Glu Ala 465 470 475 GCA GGA CAA GGC GGC CGC GAC CGA GCT GCT GGC GCA GAC CAC GCA CTT 2 567 5 Ala Gly Gin Gly Gly Arg Asp Arg Ala Ala Gly Ala Asp His Ala Leu 480 485 490 CCT GCA CGA CCC CAA GGT GGC GCT GGA GAG CCT CTA CCC GCT GGC CAA 2615 Pro Ala Arg Pro Gin Gly Gly Ala Gly Glu Pro Leu Pro Ala Gly Gin 495 500 505 510 10 GTT CAC GGC CGA GCT GGG CCT CAG CCC CGA CAA GGC GGC CGA GGT GCT 2663 Val His Gly Arg Ala Gly Pro Gin Pro Arg Gin Gly Gly Arg Gly Ala 515 520 525 GAG CCA GGG CGC TCA CCT GGC GGC CGG GCC CGA CGG CGG ACC ATC GAC 2711 Glu Pro Gly Arg Ser Pro Gly Gly Arg Ala Arg Arg Arg Thr lie Asp 15 530 535 540 CGT TTC TCT CCC ACC TAGAGCGCCC CTCGCCAGCC CGCTCTGTCG CTGCTGCGCG 2766 Arg Phe Ser Pro Thr 545 GCCCTGGCCC GCACCCCAGG GAGCGGCGGG GGCGGCGCGC AGGGCCCACT GTGCCCGGGA 2826 20 CAACCGCAGC GTCGCCACAG TGGCGGCTCC ACCTCTCGGC GGCCTCACCT GGCCTCACTG 28 86 CTTCGTGCCT TAGCTCGGGG GTCGGGGGAG AACCCCGGGA CGGGGTGGGA TGGGGTAAGG 294 6 GAAATTTATA TTTTTGATAT CAGCTTTGAC CAAAGGAGAC CCCAGGCCCC TCCCGCCTCT 3 006 TCCTGTGGTT CGTCGGCCCC CTCCCCCGGC TCCGCGCTGC TCTTAGAGGG GGAGGGGTGT 3 066 CACTGTCGGG GCACTCCTAG CCCTACCTCC GGCCCTTGCG ACCACACCCA TTCTCACTGT 3126 25 GAATCTCCCC GCTGGGTCGG AGCGTCGGGC AGAGTTGGGG AGTGGGGAGG GGACTGAGCC 3186 GGCCGGAGGC CCCCGCACCC CCGCTCCCAC CCACCCCGGG ACTGATAATG TGAAGTTCCT 3246 CATTTTGCAC AAGTGGCACT AGCCCAGGGC CAACCCTTCC TTCCTCAGTC ACCAAGGGCG 33 06 GGGAGTTCTG GAGTCGGAAG GCGAAGAGCC TACCACCAGG TCTCCCACTC CCGCGGTGCC 33 66 CTCCCTTCCC TTCCCTGCGG CCCCGGACCA TATTTATTGC ATGCGCCCCG GCGGCCCCCC 3 4 25 30 ATCCCGAGCC CAGGCTGGGC TGGGCTGGAA CGCGGTCTCT TTAGCTCCCT CCTCTTCGTT 3486 -50- TGTATATTTC CTACCTTGTA CACAGCTCTT CCAGAGCCGC TTCCATTTTC TATACTCGAA 354 6 CCAAACAGCA ATAAAGCAGT AACCAAGGAC CCCGACCCCG CTGCTCTCTT CTGCCCCTGC 3 606 ACAAGGACCT GGATGCTGCG CCCGCTGGGT GGAGGAGCCA GAAAGGGCCA CCCTCACACA 3 666 GGTGCAGAGG CTTGGACCTG CCTCCCTCCC CAGTCCCAGA AACAGATCAG CAAGAGGTCA 3726 5 GGTATGTTTC ATAACTAAAA ATTTATTAAG GAAACAAAAC CAGTGCTGCA AACGGGACAG 3 786 AAAGGAGAGC TGGGTCTCCC TCCCGACCAC CCAGTCATCG GCCTTCCAGC TGGGGAGAGA 3 846 ATCTTAAAGG AGAGGCCGGG GACCCTGTAC TCCAAAGAGC CCAGTCTTCT GAGACTCTAG 3 906 GGGACTCCTA CCCCCAAACT ACTGGCCTTG GCTCCCCTAC ACGGTACCCC ATCGCTTCTG 3 966 GCATAGTCCT GGGCCTCAGG GAGGGCAGAG CTGCGCACCC ATCCTCCAGG CAGGCTGTGC 4 026 10 AGTCAGGCCA TGGGCTCTGG GGTATCCCCC ACTGGTCCCA TTAAGATTTG CCCCTGGCTC 4 086 CACCGAAAAC CCCGTCTTCC CCTAAG 4112 (2) INFORMATION FOR SEQ ID NO-3 (i) SEQUENCE CHARACTERISTICS (A) LENGTH- 54 7 amino acids (B) TYPE ammo acid (D) TOPOLOGY, linear (li) MOLECULE TYPE protein (xi) SEQUENCE DESCRIPTION. SEQ ID NO 3 Met Leu Asp Thr Met Glu Ala Pro Gly His Ser Arg Gin Leu Leu Leu Gin Leu Asn Asn Gin Arg Thr Lys Gly Phe Leu Cys Asp Val lie lie Val Val Gin Asn Ala Leu Phe Arg Ala His Lys Asn Val Leu Ala Ala Leu Asp His Asp Met Val Ser Pro Ala Val Phe Arg Leu Val Leu Asp -51- Phe lie Tyr Thr Gly Arg Leu Ala Asp Gly Ala Glu Ala Ala Ala Ala 85 90 95 Ala Ala Val Ala Pro Gly Ala Glu Pro Ser Leu Gly Ala Val Leu Ala 100 105 HO Ala Ala Ser Tyr Leu Gin lie Pro Asp Leu Val Ala Leu Cys Lys Lys 115 120 125 Arg Leu Lys Arg His Gly Lys Tyr Cys His Leu Arg Gly Gly Gly Gly 130 135 140 Gly Gly Gly Gly Tyr Ala Pro Tyr Ala Met Ala Thr Ser Trp Ala Gly 145 150 155 160 Ser Ala Ala Pro Pro Ala Ser Ala Ala Lys Ser Val Val Gly Thr Arg 165 170 175 Pro Ser Arg Pro Gly Gly Pro Arg 180 Thr Leu Ala Ala Trp Thr Gly Pro 195 200 Thr Arg Ala Ala Ala Arg Arg Pro 210 215 Pro Gly Gly His Leu Glu Gly Tyr 225 230 Glu Pro Glu Ser Phe Gly Asp Asn 245 Lys Gly Phe Pro Ser Ser Glu Gin 260 Val Glu Glu Glu Glu Ala Leu Tyr 275 280 Ser Ala Trp Arg Arg Arg Arg Ala 185 190 Ala Arg Ala Ala Thr Ala Thr Thr 205 Val Ala Ala Arg Thr Pro Ala Pro 220 Pro Cys Pro His Leu Ala Tyr Gly 235 240 Leu Tyr Val Cys lie Pro Cys Gly 250 255 Leu Asn Ala His Val Glu Ala His 265 270 Gly Arg Ala Glu Ala Ala Glu Val 285 Ala Ala Gly Ala Ala Gly Leu Gly Pro Pro Phe Gly Gly Gly Gly Asp 290 295 300 Lys Val Ala Gly Ala Pro Gly Gly Leu Gly Glu Leu Leu Arg Pro Tyr 305 310 315 320 Arg Cys Gly Ser Cys Asp Lys Ser Tyr Lys Asp Pro Ala Thr Leu Arg 325 330 335 -52- Gln His Glu Lys Thr His Trp Leu Thr Arg Pro Tyr Pro Cys Thr lie 340 345 350 Cys Gly Lys Lys Phe Thr Gin Arg Gly Thr Met Thr Arg His Met Arg 355 360 365 Ser His Leu Gly Leu Lys Pro Phe Ala Cys Asp Ala Cys Gly Met Arg 370 375 380 Phe Thr Arg Gin Tyr Arg Leu Thr Arg Thr His Met Arg lie His Pro 385 390 395 400 Arg Gly Glu Lys Pro Tyr Glu Cys Gin Val Cys Gly Gly Lys Phe Ala 405 410 415 Gin Gin Arg Asn Leu lie Ser His Met Lys Met His Ala Val Gly Gly 420 425 430 Ala Ala Ala Arg Pro Gly Arg Trp Arg Ala Trp Gly Gly Ser Pro Ala 435 440 445 Ser Pro Ala Pro Thr Ala Arg Ala Ser Ser Thr Ser Pro Arg Ala Ser 450 455 460 Leu Leu Trp Leu Ala His Gly Arg Ala Ala Glu Pro Glu Ala Ala Gly 465 470 475 480 Gin Gly Gly Arg Asp Arg Ala Ala Gly Ala Asp His Ala Leu Pro Ala 485 490 495 Arg Pro Gin Gly Gly Ala Gly Glu Pro Leu Pro Ala Gly Gin Val His 500 505 510 Gly Arg Ala Gly Pro Gin Pro Arg Gin Gly Gly Arg Gly Ala Glu Pro 515 520 525 Gly Arg Ser Pro Gly Gly Arg Ala Arg Arg Arg Thr lie Asp Arg Phe 530 535 540 Ser Pro Thr 545 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> -53- WHAT WE CLAIM IS:

1. A use of a therapeutically effective amount of reagent which modulates HIC-1 expression in the manufacture of a medicament for treating a cell proliferative disorder associated with HIC-1.

2. A use of claim 1, wherein the reagent is a polynucleotide sequence comprising a HIC-1 sense polynucleotide sequence.

3. A use of claim 2, wherein the reagent further includes a polynucleotide sequence which encodes a promoter in operable linkage to the HIC-1 polynucleotide sequence.

4 A use of claim 2, wherein the polynucleotide sequence is in an expression vector.

5 A use 0f claim 1, wherein the disorder is associated with a tissue selected from the group consisting of brain, urogenital, lung, colon, renal, hematopoietic, breast, thymus, testis, ovarian, and uterine

6. A use of claim 5, wherein the disorder is selected from the group consisting of low grade astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma, colon cancer, lung cancer, renal cancer, leukemia, breast cancer, prostate cancer, endometrial cancer and neuroblastoma

7. A use of claim 1, wherein the HIC-1 associated cellular proliferative disorder is associated wiin hypermethylation of HIC-1 nucleotide sequence

8. A use of an expression vector comprising a nucleotide sequence encoding HIC-1, in operable linkage with a promoter in the manufacture of a medicament for use in a method of gene therapy, whereby said medicament is introduced into cells of a host subject -54- "N A use of claim 8, wherein the expression vector is introduced into the subject's cells ex vivo and the cells are then reintroduced into the subject A use of claim 8, wherein the expression vector is an RNA virus A use of claim 10, wherein the RNA virus is a retrovirus A use of claim 8, wherein the subject is a human A use of claim 8, wherein the disorder is associated with hyper methylation of HIC-1 polynucleotide A use as claimed in claim 1 or 8 substantially as herein described or exemplified with reference to the accompanying drawings </div>