Polynucleotide and polypeptide sequences encoding a novel tumor suppressor, HIC-1, are provided. Also included is a method for detecting a cell proliferative disorder associated, with HIC-1. HIC-1 is a marker which can be used diagnostically, prognostically and therapeutically over the course of such disorders.

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 novel tumor suppressor gene, 
HIC-1, and its gene product. 
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 17p13.1 (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., et al., Proc. Natl. Acad. Sci. USA, 88:3847, 1991; 
Takita, K., et al., Cancer Res., 52:3914, 1992; Deng, G., et al., Cancer 
Res., 54:499, 1994; Comelis, R. S., et al., Cancer Res., 54:4200, 1994). 
Furthermore, in one study of breast cancer, the independent loss of 
17p13.3 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, et al., 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, 
Springer-Verlag, New York, 1984). 
A CpG rich region, or "CpG island", has recently been identified at 
17p13.3, 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, S.B., 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., et al., Cancer Res., 
54:2552, 1994) and the H19 gene on 11p (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 17p13.1. 
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 
17p13.3 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 underexpressed 
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. Overexpression 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 fulfills such a need. 
SUMMARY OF THE INVENTION 
The present invention 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. The invention 
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 present invention 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. 
In another embodiment, the present invention provides a method of treating 
a cell proliferative disorder associated with HIC-1, comprising 
administering to a subject with the disorder, a therapeutically effective 
amount of reagent which modulates HIC-1 expression. 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. 
Further, the invention provides a method of gene therapy comprising 
introducing into cells of a host subject, an expression vector comprising 
a nucleotide sequence encoding HIC-1, in operable linkage with a promoter.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a novel tumor suppressor gene, HIC-1 
(hypermethylated in cancer). HIC-1 is located on chromosome 17p13.3, 
distal to the tumor suppressor gene, p53, at 17p13.1, within a CpG island 
which is abnormally methylated in many different types of tumors. This 
abnormally methylated CpG island completely encompasses the coding region 
of HIC-1 gene. 
In a first embodiment, the present invention provides a substantially pure 
HIC-1 polypeptide consisting essentially of the amino acid sequence shown 
in FIG. 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 N-terminal) domain, which is present in each 
member of subset of zinc finger transcription factors. In addition, it 
also has five Kruppel type Cys.sub.2 -His.sub.2 zinc fingers 
characteristic of the 3' region of those same proteins. 
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 amino-terminal amino acid sequence analysis. 
The invention includes a functional polypeptide, 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 amino 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 amino or 
carboxy terminal amino acids which may not be required for HIC-1 activity. 
The HIC-1 polypeptide of the invention 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 arginine 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 also provides an isolated polynucleotide sequence consisting 
essentially of a polynucleotide sequence encoding a polypeptide having the 
amino acid sequence of SEQ ID NO:3. The polynucleotide sequence of the 
invention 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 polynucleotides of the 
invention 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, the invention also includes 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. 
The polynucleotide encoding HIC-1 includes the nucleotide sequence in FIG. 
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 FIG. 1C (SEQ ID NO: 1 and 2) are replaced by ribonucleotides A, G, C, 
and U, respectively. Also included in the invention are fragments of the 
above-described 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 FIG. 2B (SEQ ID NO: 3) under 
physiological conditions and under moderately stringent conditions. 
Specifically disclosed herein is a DNA sequence for HIC-1 which 
schematically is illustrated in FIGS. 1A and 1B (see also, FIG. 1C and SEQ 
ID 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 N.sub.3 and N.sub.7. 
DNA sequences of the invention 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. 
Preferably the HIC-1 polynucleotide of the invention is derived from a 
mammalian organism, and most preferably from human. Screening procedures 
which rely on nucleic acid hybridization make it possible to isolate any 
gene sequence from any organism, provided the appropriate probe is 
available. 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 
introns. 
The synthesis of DNA sequences is frequently the method of choice when the 
entire sequence of amino acid residues of the desired polypeptide product 
is known. When the entire sequence of amino acid residues of the desired 
polypeptide is not known, the direct synthesis of DNA sequences is not 
possible and the method of choice is the synthesis of cDNA sequences. 
Among the standard procedures for isolating cDNA sequences of interest is 
the formation of plasmid- or phage-carrying cDNA libraries which are 
derived from reverse transcription of mRNA which is abundant in donor 
cells that have a high level of 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 amino 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, et al., 
Nucl. Acid Res., 11:2325, 1983). 
A cDNA expression library, such as lambda gt11, 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 the transformed cells. Vectors suitable for use in 
the present invention include, but are not limited to the T7-based 
expression vector for expression in bacteria (Rosenberg, et al., Gene 
56:125, 1987), the pMSXND expression vector for expression in mammalian 
cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) and 
baculovirus-derived vectors for expression in insect cells. The DNA 
segment can be present in the vector operably linked to regulatory 
elements, for example, a promoter (e.g., T7, metallothionein I, or 
polyhedrin promoters). 
Polynucleotide sequences encoding 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 HIC-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 
(e.g., Ti plasmid) containing the HIC-1 coding sequence; insect cell 
systems infected with recombinant virus expression vectors (e.g., 
baculovirus) containing the HIC-1 coding sequence; or animal cell 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 .gamma., 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, et al., 
EMBO J. 2:1791, 1983), in which the HIC-1 coding sequence may be ligated 
into the vector in 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. 
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, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast 
Saccharomyces, 1982, Eds. Strathern, et aL, 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. 
Rothstein 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 
(Brisson, 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 of RUBISCO 
(Coruzzi, et al., EMBOJ. 3:1671-1680,1984; Broglie, et al., Science 
224:838-843, 1984); or heat shock promoters, e.g., soybean hsp17.5-E or 
hsp17.3-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 Grierson & 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 californica 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 polyhedrin gene) 
of the virus and placed under control of an AcNPV promoter (for example 
the polyhedrin promoter). Successful insertion of the HIC-1 coding 
sequence will result in inactivation of the polyhedrin gene and production 
of non-occluded recombinant virus (i.e., virus lacking the proteinaceous 
coat coded for by the polyhedrin gene). These recombinant viruses are then 
used to infect Spodoptera frugiperda cells in which the inserted gene is 
expressed. (e.g., see Smith, et al., 1983, J. Viol. 46:584; Smith U.S. 
Pat. 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 W138. 
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 E1 or 
E3) will result in 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, 81:3655-3659, 1984). Alternatively, the vaccinia 
virus 7.5K promoter may be used (e.g., see, Mackett, et al., 1982, Proc. 
Natl. Acad. Sci. USA 79:7415-7419; Mackett, et al., J. Virol. 49:857-864, 
1984; Panicali, et al., 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. 
Transcription 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 in 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 81:6349-6353, 
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, 11:223, 1977), hypoxanthine-guanine 
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. 
USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al., 
Cell, 22: 817, 1980) genes can be employed in tk.sup.-, hgprt.sup.- or 
aprt.sup.- 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 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 
(omithine decarboxylase) which confers resistance to the ornithine 
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue 
L., 1987, In: Current Communications in Molecular Biology, Cold Spring 
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. coli, competent cells which are 
capable of DNA uptake can be prepared from cells harvested after 
exponential growth phase and subsequently treated by the CaCl.sub.2 method 
using procedures well known in the art. Alternatively, MgCl.sub.2 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 in 
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 
Spring Harbor Laboratory, Gluzman, ed., 1982). 
Isolation and purification of microbial or host cell expressed polypeptide, 
or fragments thereof, provided by the invention, may be carried out by 
conventional means including preparative chromatography and affinity and 
immunological separations involving monoclonal or polyclonal antibodies. 
The invention includes antibodies immunoreactive with HIC-1 polypeptide 
(SEQ ID NO:3) or immunoreactive 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').sub.2, which are capable of binding an 
epitopic determinant on HIC-1. 
The invention 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 bioluminescent compound, a chemiluminescent 
compound, a metal chelator, or an enzyme. Those of ordinary skill in the 
art will know of other suitable labels for binding to the probes or will 
be able to ascertain such, using routine experimentation. 
Since the present invention shows 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 method of 
the invention, 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 BssHII, 
MspI, NotI or HpaII, used alone or in combination are examples of such 
endonucleases. Other methylation sensitive restriction endonucleases will 
be known to those of skill in 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. 
For purposes of the invention, 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 invention 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 method of the invention 
include astrocytoma, anaplastic astrocytoma, glioblastoma, 
medulloblastoma, colon cancer, lung cancer, renal cancer, leukemia, breast 
cancer, prostate cancer, endometrial cancer and neuroblastoma. 
Monoclonal antibodies used in the method of the invention 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 non-competitive 
immunoassays in either a direct or indirect format. Examples of such 
immunoassays are the radioimmunoassay (RIA) and the sandwich 
(immunometric) 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 carriers 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 either soluble or insoluble for purposes 
of the invention. Those skilled in the art will know of other suitable 
carriers for binding monoclonal antibodies, or will be able to ascertain 
such using routine experimentation. 
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 in the experimental sample do not 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 in the present invention. 
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., IgG1, IgG2a, IgM, etc.) can be used as "blockers". The 
concentration of the "blockers" (normally 1-100 .mu.g/.mu.l) 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 HIC-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 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/m.sup.2 to about 500 mg/m.sup.2, preferably 0.1 mg/m.sup.2 
to about 200 mg/m.sup.2, most preferably about 0.1 mg/m.sup.2 to about 10 
mg/m.sup.2. Such dosages may vary, for example, depending on 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 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 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 immunoglobulins are the bifunctional 
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 .sup.111 In, .sup.97 Ru, .sup.67 Ga, .sup.68 Ga, 
.sup.72 As, .sup.89 Zr, and .sup.201 T1. 
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 isotopes for MRI. Elements which are 
particularly useful in such techniques include .sup.157 Gd, .sup.55 Mn, 
.sup.162 Dy, .sup.52 Cr, and .sup.56 Fe. 
The present invention also provides a method for treating a subject with a 
cell proliferative disorder associated with of HIC-1 comprising 
administering to a subject with the disorder a therapeutically effective 
amount of reagent which modulates HIC-1 expression. In brain, breast and 
renal cancer cells, for example, the HIC-1 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. 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 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 
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 under-expression of HIC-1 
polypeptide, a sense polynucleotide sequence (the DNA coding strand) 
encoding HIC-1 polypeptide, or 5' 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 present invention also provides gene therapy for the treatment of cell 
proliferative disorders which are mediated by HIC-1. Such therapy would 
achieve its therapeutic 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 murine or avian retrovirus. Examples of retroviral vectors 
in which a single foreign gene can be inserted include, but are not 
limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma 
virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus 
(RSV). 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. 
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 .PSI.2, 17 and 
2, 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 delivery 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., 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., Biotechniques, 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 in 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 in 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 in 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 
monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes 
can include monoclonal or polyclonal antibodies or fragments thereof such 
as Fab, or F(ab').sub.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 provided by the invention. Such a kit may comprise a 
carrier means being compartmentalized to receive in 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 
biotin-binding protein, such as avidin or streptavidin, bound to a 
reporter molecule, such as an enzymatic, florescent, or radionucleotide 
label. 
The invention also provides a method for identifying a tumor suppressor 
gene by detecting abnormal nucleic acid methylation, in particular, 
detecting CpG island hypermethylation in the regions of frequent allelic 
loss. The present invention 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 17p13.3 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 invention 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 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 (FIG. 1A) were prepared by isolation of 
multiple restriction fragments on agarose gels and ligation of these into 
pBluescript plasmid (Stratagene). 
2. DNA sequencing 
Single stranded DNA was first isolated by growing plasmid DNA in 2xYT broth 
with 75 ug/ml ampicillin and in the presence of 10.sup.7 -10.sup.8 pfu/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 .gamma.-.sup.32 P and cycle conditions 
were 95.degree. C. for 1 cycle followed by 20 cycles of 95.degree. C. for 
10 sec. and 65.degree. C. for 10 sec. Reaction products were analyzed on 
10% acrylamide/8M urea gels. 
3. Southern and Northern hybridizations 
Isolation procedures for DNA and poly A+ RNA, agarose gel running 
conditions, .alpha.-.sup.32 P 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., et al., Nature 
Genetics, 7:433-439, 1994; and Gish, W., et al., Nature Genetics, 
3:266-272, 1993). Radioautograms were either exposed at -70.degree. 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, .alpha.-.sup.32 P-labeled RNA probes for use 
in some Northern hybridizations was accomplished by in vitro 
transcription, using T.sub.3 or T.sub.7 polymerase, of DNA inserts in the 
various cosmid sublcones shown in FIG. 1A. 
4. RNAse protection assays 
Preparation of .alpha.-.sup.32 P-labeled RNA probes from the various cosmid 
subclones (FIG. 1A), liquid hybridization to RNA samples, and 
post-hybridization digestion by RNAse were all performed with the Ambion 
MAXIscript and RPAII kits according to the manufacturer's specifications. 
In general, 8.times.10.sup.4 cpm of probe was hybridized to 10 .mu.g of 
total RNA for 12-15 h at 45.degree. C. Products of RNAse digestion were 
analyzed on a 6% acrylamide/8M urea gel. Lengths of hybridization probes 
were determined by positions of various restriction 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 in all 
reactions. 
5. Exon trapping 
Exon trapping was performed with subclone 26 (FIG. 1A) 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, 
CaCO.sub.2, 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., et al., Recent 
Results Cancer Res., 99:157-166, 1985). All established breast cancer 
lines were utilized, as detailed in FIG. 5, in a recent study (Herman, J. 
G., et al., Proc. Nat'l. Acad. Sci., 91:9700-9704, 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 TRI, 
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., et al., 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 TUMOR SUPPRESSOR GENE 
To characterize the region encompassing the aberrantly methylated CpG 
island, a series of subclones were prepared (FIG. 1A) from the 17p cosmid 
C-13A (Ledbetter, D. H., et al., Proc. Natl. Acad. Sci. USA, 86:5136, 
1989; El-Deiry, W. S., et al., Nature Genetics, 1:45-49, 1992; Kern, S. 
E., et 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 (FIG. 1A: plasmids CI, CII, and 
400) were found which hybridized, under stringent conditions, to 
restriction fragments in bovine and murine DNA. Traditional positional 
cloning approaches were impeded by high non-specific hybridization of 
these probes to human DNA and cDNA libraries, probably due to the high GC 
content of the area. Therefore, most of the 11 kb region (FIG. 1A) was 
sequenced and analyzed by the Grail computer program (Gish, W., et al., D. 
J., Nature Genetics, 3:266, 1993). 
FIG. 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 
17p13.3 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. 
FIG. 1B is a schematic for the HIC-1 gene which was found to be encompassed 
within the region shown in FIG. 1A and for which the amino acid sequence 
is shown in FIG. 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 
(FIG. 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 (FIG. 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. FIG. 1C shows the nucleotide and deduced amino acid 
sequence of HIC-1. 
Two independent regions of excellent coding potential were revealed between 
the N.sub.3 to N.sub.7 Not I restriction sites (FIG. 1A). Blast program 
(Altschul, S. F., et al., J Mol. Biol., 215:403, 1990) analysis revealed 
distinct amino acid homologies (FIGS. 1B 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, et al., Genetics, 129:385, 1991; 
Numoto, et al., Nucleic Acids Res. 21:3767, 1993; Chardin, et al., Nucleic 
Acids Res. 19:1431, 1991). In addition to the Zin domain, five Kruppel 
type Cys.sub.2 -His.sub.2 zinc fingers (Ruppert, J. M., et al., Mol & 
Cell. Biol, 8:3104-3113, 1988) characteristic of the 3' region of these 
same proteins, were also identified (FIGS. 1B and 2B). This novel gene was 
named HIC-1 (hypermethylated in cancer). 
EXAMPLE 3 
CHARACTERIZATION OF HIC-1 
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 (FIGS. 1B and 1C; SEQ ID 
NO:1 and 2). The start of transcription was identified within 40 bp 
downstream from a TATA box sequence (FIG. 1B) which precedes an 
untranslated first exon. The putative ATG site and the Zin domain are 
located in a 476 bp second exon and are in a similar position to those of 
the 8 other Zin domain proteins (FIG. 2A). The 5 zinc fingers (FIGS. 1B 
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 
(FIGS. 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 N.sub.3 and N.sub.7 (FIG. 1). 
FIG. 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 Zin 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. 
FIG. 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 in the 3' half of the protein are 
shown by the shaded boxes. 
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. FIG. 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 .about.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. 
FIG. 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 
(FIG. 1A). The protected HIC-1 fragment (300bp) is labeled HIC-1. FIG. 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, CaCO.sub.2 (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). 
FIG. 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. 
FIG. 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). 
The 3.0 kb transcript was found in all adult tissues tested with especially 
high levels in lung, colon, prostate, thymus, testis, and ovary (FIG. 3). 
With the Zin domain probe, a 1.1 kb transcript was also detected in some 
tissues which may represent an alternatively spliced product (FIG. 3). 
RNase protection assays (RPAZ Kit-Ambion), using a probe from plasmid 600 
(FIG. 1A), validated the ubiquitous expression of HIC-1, protecting 
transcripts of predicted size in cultured fibroblasts (FIG. 4A) and 
non-cultured colon mucosa (FIG. 4A), lung (FIG. 4A), and brain (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 4C), as compared to normal brain. 
In 4 glioblastomas, in which both DNA and RNA were available, two 
expressed HIC-1 either weakly (FIG. 4C, lane 1) or not at all (FIG. 4C, 
lane 4) and had predominantly hypermethylated alleles, while two with 
unmethylated alleles expressed the gene at levels equal to adjacent normal 
brain (FIG. 4C, lanes 2 and 3). 
Four additional glioblastomas for which RNA was available were also 
studied. One expressed HIC-1 weakly (FIG. 4C, lane 5), one had no 
expression (FIG. 4C, lane 6), and two tumors expressed this gene (FIG. 4C, 
lanes 7-8). 
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 
.alpha.-.sup.32 P-labeled YNZ22 (FIG. 1A) exactly as detailed in previous 
studies (Makos, et al., 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 TUMORS AND CELL LINES 
PRIMARY TUMORS CULTURED CELL LINES 
# METH % # METH % 
______________________________________ 
BRAIN 
TUMORS 
Low Grade 
7 7 100 
Astrocytomas 
Anaplastic 
5 4 80 
Astrocytomas 
Glioblastoma 
8 6 75 Glials 2 2 100 
Multiforme 
Medullo- 5 4 80 
blastoma 
COLON 
CANCERS 
Polyps 6 6 100 
Carcinomas 
8 7 90 Carcinoma 
6 7 85 
LUNG 
CANCERS 
Carcinomas 
5 0 0 Carcinoma 
16 12 75 
RENAL 
CANCERS 
Early Stage 
8 4 50 
Late Stage 
3 2 67 Late Stage 
21 16 80 
LEUKEMIAS 
Lymphomas 
3 1 33 Lymphomas 
8 5 60 
CML/Blast 
8 7 67 
AML 13 10 80 
ALL 10 8 80 
BREAST 
CANCERS 
Cancer 24 15 62 Cancers 6 6 100 
PROSTATE 
CANCERS 
Cancer 17 17 100 Cancer 5 4 80 
ENDO- 
METRIAL 
CANCER 
Cancer 6 4 67 
NEURO- 
BLASTOMAS 
early/late stage 
12 2 16 Cancers 4 4 100 
(amount of 
methylation 
LOW) 
______________________________________ 
EXAMPLE 5 
INTERACTION OF P53 WITH HIC-1 EXPRESSION 
Consistent with the hypothesis that a suppressor gene exists at 17p13.3 
which may interact with p53, the present invention identifies a potential 
p53 binding site 4 kb 5' to the TATA box in the HIC-1 gene (FIG. 1B). 
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 (E1-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. 
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). 
FIG. 5 shows an RNAse protection assay, as detailed in FIG. 4, after 
infection of an adenoviral vector containing either the 
.beta.-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 
.beta.-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 FIG. 4. 
HIC-1 is expressed at only low levels in this cells line (FIG. 5A-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 4.0 kb 
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 EXAMPLES 
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 WAFI (E1-Deiry, W. S., et al., supra.) MDM2 
(Chen, C. Y., et al., Proc. Natl. Acad. Sci. USA, 91:2684-2688,1994), 
GADD45 (Kastan, M. B., et al., Cell, 71:587-597, 1992) and BAX (Miyashita, 
T., et al., 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 J9:207, 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 
murine Zin domain gene, MZF5, has in-vitro transcriptional repressor 
activity for c-myc and thymidine kinase promoters (Numoto, et al., Nucleic 
Acids Res., 21:3767, 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, etal.,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 17p13.3, 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 ordinary skill in the art can readily envision 
and produce further embodiments, based on the teachings herein, without 
undue experimentation. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 14 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4616 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: HIC-1 polynucleotide 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..4616 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCCGGCCCGCCGGGACCGCAGGTAACGGGCCGCGGGGCCCCGCGGGCCAGGAGGGGAACG60 
GGGTCGGGCGGGCGAGCAGCGGGCAGGGGAGCTCAGGGCTCGGCTCCGGGCTCTGCCGCC120 
GGATTTGGGGGCCGCGAGGAAGAGCTGCGAGCCGAGGGCCTGGGGCCGGCGCACTCCTCC180 
CGCCCTGTCTGCAGTTGGAAAACTTTTCCCCAAGTTTGGGGCGGCGGAGTTCCGGGGGAG240 
AAGGGGCCGGGGGAGCCGCGGAGGGAGGCGCCGGGCCCGCGCGTGTAGGGCCCAGGCCGA300 
GGCCGGGACGCGGGTGGGGCGCAGGCCCGGGTCAGGGCCGCAGCCGGCTGTGCGCCGTGC360 
CCGCCCGGGGCGCTGCCCCCTCCCTCCCCTGGGAGCTGCGTGGCTCCCCCCTCCCCCCCA420 
CCTGCTTCCTGCCTCAGCCTCCTGCCCCGATATAACGCCCTCCCCGCGCCGGGCCCGGCC480 
TTCGCGCTCTGCCCGCCACGGCAGCCGCTGCCTCCGCTCCCCGCGCGGCCGCCGCCCGGG540 
CCCCGACCGAGGGTTGACAGCCCCCGGCCAGGGCGGCGCCAGGGCGGGCACCGCGCTCCC600 
CTCCTCCGTATCACTTCCCCCAACTGGGGCAACTTCTCCCGAGGCGGGAGGCGCTGGTTC660 
CTCGGCTCCCTTTCTCCCTACTTGGGTAAAGTTCTCCGCCCTGAATGACTTTTCCTGAAG720 
CGGACATTTTACTTAAATCGGGTAACTGTCTCCAAAAGGGTCACTGCGCCTGAACAGTTT780 
TCTTCTCGGAAGCCCCAGCACCCAGCCAGGTGCCCTGGGGCGTGCAGGCCGCCCTGGCCT840 
CCCCTCCACCGGCGGCCGCTCACCTCCTGCTCCTTCTCCTGGTCCGGGCGGGCCGGCCTG900 
GGCTCCCACTCCAGAGGGCAGCTGGTCCTTCGCCGGTGCCCAGGCCGCAGGGCTGATGCC960 
CCCGCTCAGCTGAGGGAAGGGGAAGTGGAGGGGAGAAGTGCCGGGCTGGGGCCAGGCGGC1020 
CAGGGCGCCGCACGGCTCTCACCCGGCCGGTGTGTGTCCCCGCAGGAGAGTGTGCTGGGC1080 
AGACGATGCTGGACACGATGGAGGCGCCCGGCCACTCCAGGCAGCTGCTGCTGCAGCTCA1140 
ACAACCAGCGCACCAAGGGCTTCTTGTGCGACGTGATCATCGTGGTGCAGAACGCCCTCT1200 
TCCGCGCGCACAAGAACGTGCTGGCGGCCAGCAGCGCCTACCTCAAGTCCCTGGTGGTGC1260 
ATGACAACCTGCTCAACCTGGACCATGACATGGTGAGCCCGGCCGTGTTCCGCCTGGTGC1320 
TGGACTTCATCTACACCGGCCGCCTGGCTGACGGCGCAGAGGCGGCTGCGGCCGCGGCCG1380 
TGGCCCCGGGGGCTGAGCCGAGCCTGGGCGCCGTGCTGGCCGCCGCCAGCTACCTGCAGA1440 
TCCCCGACCTCGTGGCGCTGTGCAAGAAACGCCTCAAGCGCCACGGCAAGTACTGCCACC1500 
TGCGGGGCGGCGGCGGCGGCGGCGGCGGCTACGCGCCCTATGGTCGGCCGGGCCGGGGCC1560 
TGCGGGCCGCCACGCCGTCATCCAGGCCTGCTACCCGTCCCCAGTCGGGCCTCCGCCGCC1620 
GCCTGCCGCGGAGCCGCCCTCGGGCCCAGAGGCCGCGGTCAACACGCACTGCGCCGAGCT1680 
GTACGCGTCGGGACCCGGCCCGGCCGCCGCACTCTGTGCCTCGGAGCGCCGCTGCTCCCC1740 
TCTTTGTGGCCTGGACCTGTCCAAGAAGAGCCCGCCGGGCTCCGCGGCGCCAGAGCGGCC1800 
GCTGGCTGAGCGCGAGCTGCCCCCGCGCCCGGACAGCCCTCCCAGCGCCGGCCCCGCCGC1860 
CTACAAGGAGCCGCCTCTCGCCCTGCCGTCGCTGCCGCCGCTGCCCTTCCAGAAGCTGGA1920 
GGAGGCCGCACCGCCTTCCGACCCATTTCGCGGCGGCAGCGGCAGCCCGGGACCCGAGCC1980 
CCCCGGCCGCCCCAACGGGCCTAGTCTCCTCTATCGCTGGATGAAGCACGAGCCGGGCCT2040 
GGGTAGCTATGGCGACGAGCTGGGCCGGGAGCGCGGCTCCCCCAGCGAGCGCTGCGAAGA2100 
GCGTGGTGGGGACGCGGCCGTCTCGCCCGGGGGGCCCCCGCTCGGCCTGGCGCCGCCGCC2160 
GCGCTACCCTGGCAGCCTGGACGGGCCCGGCGCGGGCGGCGACGGCGACGACTACAAGAG2220 
CAGCAGCGAGGAGACCGGTAGCAGCGAGGACCCCAGCACCGCCTGGCGGCCACCTCGAGG2280 
GCTACCCATGCCCGCACCTGGCCTATGGCGAGCCCGAGAGCTTCGGTGACAACCTGTACG2340 
TGTGCATTCCGTGCGGCAAGGGCTTCCCCAGCTCTGAGCAGCTGAACGCGCACGTGGAGG2400 
CTCACGTGGAGGAGGAGGAAGCGCTGTACGGCAGGGCCGAGGCGGCCGAAGTGGCCGCTG2460 
GGGCCGCCGGCCTAGGGCCCCCTTTTGGAGGCGGCGGGGACAAGGTCGCCGGGGCTCCGG2520 
GTGGCCTGGGAGAGCTGCTGCGGCCCTACCGCTGCGGCTCGTGCGACAAGAGCTACAAGG2580 
ACCCGGCCACGCTGCGGCAGCACGAGAAGACGCACTGGCTGACCCGGCCCTACCCATGCA2640 
CCATCTGCGGGAAGAAGTTCACGCAGCGTGGGACCATGACGCGCCACATGCGCAGCCACC2700 
TGGGCCTCAAGCCCTTCGCGTGCGACGCGTGCGGCATGCGGTTCACGCGCCAGTACCGCC2760 
TCACCCGGACGCACATGCGCATCCACCCTCGCGGCGAGAAGCCCTACGAGTGCCAGGTGT2820 
GCGGCGGCAAGTTCGCACAGCAACGCAACCTCATCAGCCACATGAAGATGCACGCCGTGG2880 
GGGGCGCGGCGGCGCGGCCGGGGCGCTGGCGGGCTTGGGGGGGCTCCCCGGCGTCCCCGG2940 
CCCCGACGGCAAGGGCAAGCTCGACTTCCCCGAGGGCGTCTTTGCTGTGGCTCGCTCACG3000 
GCCGAGCAGCTGAGCCTGAAGCAGCAGGACAAGGCGGCCGCGACCGAGCTGCTGGCGCAG3060 
ACCACGCACTTCCTGCACGACCCCAAGGTGGCGCTGGAGAGCCTCTACCCGCTGGCCAAG3120 
TTCACGGCCGAGCTGGGCCTCAGCCCCGACAAGGCGGCCGAGGTGCTGAGCCAGGGCGCT3180 
CACCTGGCGGCCGGGCCCGACGGCGGACCATCGACCGTTTCTCTCCCACCTAGAGCGCCC3240 
CTCGCCAGCCCGCTCTGTCGCTGCTGCGCGGCCCTGGCCCGCACCCCAGGGAGCGGCGGG3300 
GGCGGCGCGCAGGGCCCACTGTGCCCGGGACAACCGCAGCGTCGCCACAGTGGCGGCTCC3360 
ACCTCTCGGCGGCCTCACCTGGCCTCACTGCTTCGTGCCTTAGCTCGGGGGTCGGGGGAG3420 
AACCCCGGGACGGGGTGGGATGGGGTAAGGGAAATTTATATTTTTGATATCAGCTTTGAC3480 
CAAAGGAGACCCCAGGCCCCTCCCGCCTCTTCCTGTGGTTCGTCGGCCCCCTCCCCCGGC3540 
TCCGCGCTGCTCTTAGAGGGGGAGGGGTGTCACTGTCGGGGCACTCCTAGCCCTACCTCC3600 
GGCCCTTGCGACCACACCCATTCTCACTGTGAATCTCCCCGCTGGGTCGGAGCGTCGGGC3660 
AGAGTTGGGGAGTGGGGAGGGGACTGAGCCGGCCGGAGGCCCCCGCACCCCCGCTCCCAC3720 
CCACCCCGGGACTGATAATGTGAAGTTCCTCATTTTGCACAAGTGGCACTAGCCCAGGGC3780 
CAACCCTTCCTTCCTCAGTCACCAAGGGCGGGGAGTTCTGGAGTCGGAAGGCGAAGAGCC3840 
TACCACCAGGTCTCCCACTCCCGCGGTGCCCTCCCTTCCCTTCCCTGCGGCCCCGGACCA3900 
TATTTATTGCATGCGCCCCGGCGGCCCCCCATCCCGAGCCCAGGCTGGGCTGGGCTGGAA3960 
CGCGGTCTCTTTAGCTCCCTCCTCTTCGTTTGTATATTTCCTACCTTGTACACAGCTCTT4020 
CCAGAGCCGCTTCCATTTTCTATACTCGAACCAAACAGCAATAAAGCAGTAACCAAGGAC4080 
CCCGACCCCGCTGCTCTCTTCTGCCCCTGCACAAGGACCTGGATGCTGCGCCCGCTGGGT4140 
GGAGGAGCCAGAAAGGGCCACCCTCACACAGGTGCAGAGGCTTGGACCTGCCTCCCTCCC4200 
CAGTCCCAGAAACAGATCAGCAAGAGGTCAGGTATGTTTCATAACTAAAAATTTATTAAG4260 
GAAACAAAACCAGTGCTGCAAACGGGACAGAAAGGAGAGCTGGGTCTCCCTCCCGACCAC4320 
CCAGTCATCGGCCTTCCAGCTGGGGAGAGAATCTTAAAGGAGAGGCCGGGGACCCTGTAC4380 
TCCAAAGAGCCCAGTCTTCTGAGACTCTAGGGGACTCCTACCCCCAAACTACTGGCCTTG4440 
GCTCCCCTACACGGTACCCCATCGCTTCTGGCATAGTCCTGGGCCTCAGGGAGGGCAGAG4500 
CTGCGCACCCATCCTCCAGGCAGGCTGTGCAGTCAGGCCATGGGCTCTGGGGTATCCCCC4560 
ACTGGTCCCATTAAGATTTGCCCCTGGCTCCACCGAAAACCCCGTCTTCCCCTAAG4616 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4112 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vii) IMMEDIATE SOURCE: 
(B) CLONE: HIC-1 coding polynucleotide 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1086..2726 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CCCGGCCCGCCGGGACCGCAGGTAACGGGCCGCGGGGCCCCGCGGGCCAGGAGGGGAACG60 
GGGTCGGGCGGGCGAGCAGCGGGCAGGGGAGCTCAGGGCTCGGCTCCGGGCTCTGCCGCC120 
GGATTTGGGGGCCGCGAGGAAGAGCTGCGAGCCGAGGGCCTGGGGCCGGCGCACTCCTCC180 
CGCCCTGTCTGCAGTTGGAAAACTTTTCCCCAAGTTTGGGGCGGCGGAGTTCCGGGGGAG240 
AAGGGGCCGGGGGAGCCGCGGAGGGAGGCGCCGGGCCCGCGCGTGTAGGGCCCAGGCCGA300 
GGCCGGGACGCGGGTGGGGCGCAGGCCCGGGTCAGGGCCGCAGCCGGCTGTGCGCCGTGC360 
CCGCCCGGGGCGCTGCCCCCTCCCTCCCCTGGGAGCTGCGTGGCTCCCCCCTCCCCCCCA420 
CCTGCTTCCTGCCTCAGCCTCCTGCCCCGATATAACGCCCTCCCCGCGCCGGGCCCGGCC480 
TTCGCGCTCTGCCCGCCACGGCAGCCGCTGCCTCCGCTCCCCGCGCGGCCGCCGCCCGGG540 
CCCCGACCGAGGGTTGACAGCCCCCGGCCAGGGCGGCGCCAGGGCGGGCACCGCGCTCCC600 
CTCCTCCGTATCACTTCCCCCAACTGGGGCAACTTCTCCCGAGGCGGGAGGCGCTGGTTC660 
CTCGGCTCCCTTTCTCCCTACTTGGGTAAAGTTCTCCGCCCTGAATGACTTTTCCTGAAG720 
CGGACATTTTACTTAAATCGGGTAACTGTCTCCAAAAGGGTCACTGCGCCTGAACAGTTT780 
TCTTCTCGGAAGCCCCAGCACCCAGCCAGGTGCCCTGGGGCGTGCAGGCCGCCCTGGCCT840 
CCCCTCCACCGGCGGCCGCTCACCTCCTGCTCCTTCTCCTGGTCCGGGCGGGCCGGCCTG900 
GGCTCCCACTCCAGAGGGCAGCTGGTCCTTCGCCGGTGCCCAGGCCGCAGGGCTGATGCC960 
CCCGCTCAGCTGAGGGAAGGGGAAGTGGAGGGGAGAAGTGCCGGGCTGGGGCCAGGCGGC1020 
CAGGGCGCCGCACGGCTCTCACCCGGCCGGTGTGTGTCCCCGCAGGAGAGTGTGCTGGGC1080 
AGACGATGCTGGACACGATGGAGGCGCCCGGCCACTCCAGGCAGCTG1127 
MetLeuAspThrMetGluAlaProGlyHisSerArgGlnLeu 
1510 
CTGCTGCAGCTCAACAACCAGCGCACCAAGGGCTTCTTGTGCGACGTG1175 
LeuLeuGlnLeuAsnAsnGlnArgThrLysGlyPheLeuCysAspVal 
15202530 
ATCATCGTGGTGCAGAACGCCCTCTTCCGCGCGCACAAGAACGTGCTG1223 
IleIleValValGlnAsnAlaLeuPheArgAlaHisLysAsnValLeu 
354045 
GCGGCCAGCAGCGCCTACCTCAAGTCCCTGGTGGTGCATGACAACCTG1271 
AlaAlaSerSerAlaTyrLeuLysSerLeuValValHisAspAsnLeu 
505560 
CTCAACCTGGACCATGACATGGTGAGCCCGGCCGTGTTCCGCCTGGTG1319 
LeuAsnLeuAspHisAspMetValSerProAlaValPheArgLeuVal 
657075 
CTGGACTTCATCTACACCGGCCGCCTGGCTGACGGCGCAGAGGCGGCT1367 
LeuAspPheIleTyrThrGlyArgLeuAlaAspGlyAlaGluAlaAla 
808590 
GCGGCCGCGGCCGTGGCCCCGGGGGCTGAGCCGAGCCTGGGCGCCGTG1415 
AlaAlaAlaAlaValAlaProGlyAlaGluProSerLeuGlyAlaVal 
95100105110 
CTGGCCGCCGCCAGCTACCTGCAGATCCCCGACCTCGTGGCGCTGTGC1463 
LeuAlaAlaAlaSerTyrLeuGlnIleProAspLeuValAlaLeuCys 
115120125 
AAGAAACGCCTCAAGCGCCACGGCAAGTACTGCCACCTGCGGGGCGGC1511 
LysLysArgLeuLysArgHisGlyLysTyrCysHisLeuArgGlyGly 
130135140 
GGCGGCGGCGGCGGCGGCTACGCGCCCTATGCTATGGCGACGAGCTGG1559 
GlyGlyGlyGlyGlyGlyTyrAlaProTyrAlaMetAlaThrSerTrp 
145150155 
GCCGGGAGCGCGGCTCCCCCAGCGAGCGCTGCGAAGAGCGTGGTGGGG1607 
AlaGlySerAlaAlaProProAlaSerAlaAlaLysSerValValGly 
160165170 
ACGCGGCCGTCTCGCCCGGGGGGCCCCCGCTCGGCCTGGCGCCGCCGC1655 
ThrArgProSerArgProGlyGlyProArgSerAlaTrpArgArgArg 
175180185190 
CGCGCTACCCTGGCAGCCTGGACGGGCCCGGCGCGGGCGGCGACGGCG1703 
ArgAlaThrLeuAlaAlaTrpThrGlyProAlaArgAlaAlaThrAla 
195200205 
ACGACTACAAGAGCAGCAGCGAGGAGACCGGTAGCAGCGAGGACCCCA1751 
ThrThrThrArgAlaAlaAlaArgArgProValAlaAlaArgThrPro 
210215220 
GCACCGCCTGGCGGCCACCTCGAGGGCTACCCATGCCCGCACCTGGCC1799 
AlaProProGlyGlyHisLeuGluGlyTyrProCysProHisLeuAla 
225230235 
TATGGCGAGCCCGAGAGCTTCGGTGACAACCTGTACGTGTGCATTCCG1847 
TyrGlyGluProGluSerPheGlyAspAsnLeuTyrValCysIlePro 
240245250 
TGCGGCAAGGGCTTCCCCAGCTCTGAGCAGCTGAACGCGCACGTGGAG1895 
CysGlyLysGlyPheProSerSerGluGlnLeuAsnAlaHisValGlu 
255260265270 
GCTCACGTGGAGGAGGAGGAAGCGCTGTACGGCAGGGCCGAGGCGGCC1943 
AlaHisValGluGluGluGluAlaLeuTyrGlyArgAlaGluAlaAla 
275280285 
GAAGTGGCCGCTGGGGCCGCCGGCCTAGGGCCCCCTTTTGGAGGCGGC1991 
GluValAlaAlaGlyAlaAlaGlyLeuGlyProProPheGlyGlyGly 
290295300 
GGGGACAAGGTCGCCGGGGCTCCGGGTGGCCTGGGAGAGCTGCTGCGG2039 
GlyAspLysValAlaGlyAlaProGlyGlyLeuGlyGluLeuLeuArg 
305310315 
CCCTACCGCTGCGGCTCGTGCGACAAGAGCTACAAGGACCCGGCCACG2087 
ProTyrArgCysGlySerCysAspLysSerTyrLysAspProAlaThr 
320325330 
CTGCGGCAGCACGAGAAGACGCACTGGCTGACCCGGCCCTACCCATGC2135 
LeuArgGlnHisGluLysThrHisTrpLeuThrArgProTyrProCys 
335340345350 
ACCATCTGCGGGAAGAAGTTCACGCAGCGTGGGACCATGACGCGCCAC2183 
ThrIleCysGlyLysLysPheThrGlnArgGlyThrMetThrArgHis 
355360365 
ATGCGCAGCCACCTGGGCCTCAAGCCCTTCGCGTGCGACGCGTGCGGC2231 
MetArgSerHisLeuGlyLeuLysProPheAlaCysAspAlaCysGly 
370375380 
ATGCGGTTCACGCGCCAGTACCGCCTCACCCGGACGCACATGCGCATC2279 
MetArgPheThrArgGlnTyrArgLeuThrArgThrHisMetArgIle 
385390395 
CACCCTCGCGGCGAGAAGCCCTACGAGTGCCAGGTGTGCGGCGGCAAG2327 
HisProArgGlyGluLysProTyrGluCysGlnValCysGlyGlyLys 
400405410 
TTCGCACAGCAACGCAACCTCATCAGCCACATGAAGATGCACGCCGTG2375 
PheAlaGlnGlnArgAsnLeuIleSerHisMetLysMetHisAlaVal 
415420425430 
GGGGGCGCGGCGGCGCGGCCGGGGCGCTGGCGGGCTTGGGGGGGCTCC2423 
GlyGlyAlaAlaAlaArgProGlyArgTrpArgAlaTrpGlyGlySer 
435440445 
CCGGCGTCCCCGGCCCCGACGGCAAGGGCAAGCTCGACTTCCCCGAGG2471 
ProAlaSerProAlaProThrAlaArgAlaSerSerThrSerProArg 
450455460 
GCGTCTTTGCTGTGGCTCGCTCACGGCCGAGCAGCTGAGCCTGAAGCA2519 
AlaSerLeuLeuTrpLeuAlaHisGlyArgAlaAlaGluProGluAla 
465470475 
GCAGGACAAGGCGGCCGCGACCGAGCTGCTGGCGCAGACCACGCACTT2567 
AlaGlyGlnGlyGlyArgAspArgAlaAlaGlyAlaAspHisAlaLeu 
480485490 
CCTGCACGACCCCAAGGTGGCGCTGGAGAGCCTCTACCCGCTGGCCAA2615 
ProAlaArgProGlnGlyGlyAlaGlyGluProLeuProAlaGlyGln 
495500505510 
GTTCACGGCCGAGCTGGGCCTCAGCCCCGACAAGGCGGCCGAGGTGCT2663 
ValHisGlyArgAlaGlyProGlnProArgGlnGlyGlyArgGlyAla 
515520525 
GAGCCAGGGCGCTCACCTGGCGGCCGGGCCCGACGGCGGACCATCGAC2711 
GluProGlyArgSerProGlyGlyArgAlaArgArgArgThrIleAsp 
530535540 
CGTTTCTCTCCCACCTAGAGCGCCCCTCGCCAGCCCGCTCTGTCGCTGCTGCGCG2766 
ArgPheSerProThr 
545 
GCCCTGGCCCGCACCCCAGGGAGCGGCGGGGGCGGCGCGCAGGGCCCACTGTGCCCGGGA2826 
CAACCGCAGCGTCGCCACAGTGGCGGCTCCACCTCTCGGCGGCCTCACCTGGCCTCACTG2886 
CTTCGTGCCTTAGCTCGGGGGTCGGGGGAGAACCCCGGGACGGGGTGGGATGGGGTAAGG2946 
GAAATTTATATTTTTGATATCAGCTTTGACCAAAGGAGACCCCAGGCCCCTCCCGCCTCT3006 
TCCTGTGGTTCGTCGGCCCCCTCCCCCGGCTCCGCGCTGCTCTTAGAGGGGGAGGGGTGT3066 
CACTGTCGGGGCACTCCTAGCCCTACCTCCGGCCCTTGCGACCACACCCATTCTCACTGT3126 
GAATCTCCCCGCTGGGTCGGAGCGTCGGGCAGAGTTGGGGAGTGGGGAGGGGACTGAGCC3186 
GGCCGGAGGCCCCCGCACCCCCGCTCCCACCCACCCCGGGACTGATAATGTGAAGTTCCT3246 
CATTTTGCACAAGTGGCACTAGCCCAGGGCCAACCCTTCCTTCCTCAGTCACCAAGGGCG3306 
GGGAGTTCTGGAGTCGGAAGGCGAAGAGCCTACCACCAGGTCTCCCACTCCCGCGGTGCC3366 
CTCCCTTCCCTTCCCTGCGGCCCCGGACCATATTTATTGCATGCGCCCCGGCGGCCCCCC3426 
ATCCCGAGCCCAGGCTGGGCTGGGCTGGAACGCGGTCTCTTTAGCTCCCTCCTCTTCGTT3486 
TGTATATTTCCTACCTTGTACACAGCTCTTCCAGAGCCGCTTCCATTTTCTATACTCGAA3546 
CCAAACAGCAATAAAGCAGTAACCAAGGACCCCGACCCCGCTGCTCTCTTCTGCCCCTGC3606 
ACAAGGACCTGGATGCTGCGCCCGCTGGGTGGAGGAGCCAGAAAGGGCCACCCTCACACA3666 
GGTGCAGAGGCTTGGACCTGCCTCCCTCCCCAGTCCCAGAAACAGATCAGCAAGAGGTCA3726 
GGTATGTTTCATAACTAAAAATTTATTAAGGAAACAAAACCAGTGCTGCAAACGGGACAG3786 
AAAGGAGAGCTGGGTCTCCCTCCCGACCACCCAGTCATCGGCCTTCCAGCTGGGGAGAGA3846 
ATCTTAAAGGAGAGGCCGGGGACCCTGTACTCCAAAGAGCCCAGTCTTCTGAGACTCTAG3906 
GGGACTCCTACCCCCAAACTACTGGCCTTGGCTCCCCTACACGGTACCCCATCGCTTCTG3966 
GCATAGTCCTGGGCCTCAGGGAGGGCAGAGCTGCGCACCCATCCTCCAGGCAGGCTGTGC4026 
AGTCAGGCCATGGGCTCTGGGGTATCCCCCACTGGTCCCATTAAGATTTGCCCCTGGCTC4086 
CACCGAAAACCCCGTCTTCCCCTAAG4112 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 547 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetLeuAspThrMetGluAlaProGlyHisSerArgGlnLeuLeuLeu 
151015 
GlnLeuAsnAsnGlnArgThrLysGlyPheLeuCysAspValIleIle 
202530 
ValValGlnAsnAlaLeuPheArgAlaHisLysAsnValLeuAlaAla 
354045 
SerSerAlaTyrLeuLysSerLeuValValHisAspAsnLeuLeuAsn 
505560 
LeuAspHisAspMetValSerProAlaValPheArgLeuValLeuAsp 
65707580 
PheIleTyrThrGlyArgLeuAlaAspGlyAlaGluAlaAlaAlaAla 
859095 
AlaAlaValAlaProGlyAlaGluProSerLeuGlyAlaValLeuAla 
100105110 
AlaAlaSerTyrLeuGlnIleProAspLeuValAlaLeuCysLysLys 
115120125 
ArgLeuLysArgHisGlyLysTyrCysHisLeuArgGlyGlyGlyGly 
130135140 
GlyGlyGlyGlyTyrAlaProTyrAlaMetAlaThrSerTrpAlaGly 
145150155160 
SerAlaAlaProProAlaSerAlaAlaLysSerValValGlyThrArg 
165170175 
ProSerArgProGlyGlyProArgSerAlaTrpArgArgArgArgAla 
180185190 
ThrLeuAlaAlaTrpThrGlyProAlaArgAlaAlaThrAlaThrThr 
195200205 
ThrArgAlaAlaAlaArgArgProValAlaAlaArgThrProAlaPro 
210215220 
ProGlyGlyHisLeuGluGlyTyrProCysProHisLeuAlaTyrGly 
225230235240 
GluProGluSerPheGlyAspAsnLeuTyrValCysIleProCysGly 
245250255 
LysGlyPheProSerSerGluGlnLeuAsnAlaHisValGluAlaHis 
260265270 
ValGluGluGluGluAlaLeuTyrGlyArgAlaGluAlaAlaGluVal 
275280285 
AlaAlaGlyAlaAlaGlyLeuGlyProProPheGlyGlyGlyGlyAsp 
290295300 
LysValAlaGlyAlaProGlyGlyLeuGlyGluLeuLeuArgProTyr 
305310315320 
ArgCysGlySerCysAspLysSerTyrLysAspProAlaThrLeuArg 
325330335 
GlnHisGluLysThrHisTrpLeuThrArgProTyrProCysThrIle 
340345350 
CysGlyLysLysPheThrGlnArgGlyThrMetThrArgHisMetArg 
355360365 
SerHisLeuGlyLeuLysProPheAlaCysAspAlaCysGlyMetArg 
370375380 
PheThrArgGlnTyrArgLeuThrArgThrHisMetArgIleHisPro 
385390395400 
ArgGlyGluLysProTyrGluCysGlnValCysGlyGlyLysPheAla 
405410415 
GlnGlnArgAsnLeuIleSerHisMetLysMetHisAlaValGlyGly 
420425430 
AlaAlaAlaArgProGlyArgTrpArgAlaTrpGlyGlySerProAla 
435440445 
SerProAlaProThrAlaArgAlaSerSerThrSerProArgAlaSer 
450455460 
LeuLeuTrpLeuAlaHisGlyArgAlaAlaGluProGluAlaAlaGly 
465470475480 
GlnGlyGlyArgAspArgAlaAlaGlyAlaAspHisAlaLeuProAla 
485490495 
ArgProGlnGlyGlyAlaGlyGluProLeuProAlaGlyGlnValHis 
500505510 
GlyArgAlaGlyProGlnProArgGlnGlyGlyArgGlyAlaGluPro 
515520525 
GlyArgSerProGlyGlyArgAlaArgArgArgThrIleAspArgPhe 
530535540 
SerProThr 
545 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGTCTTGTGCAGAGGCATGGTC22 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyAspTyrGlyThrSerLeuValSerAlaIleGlnLeuLeuArgCys 
151015 
HisGlyAspLeuValAspCysThrLeuAlaAlaGlyGlyArgSerPhe 
202530 
ProAlaHisLysIleValLeuCysAlaAlaSerProPheLeuLeuAsp 
354045 
LeuLeuLysAsnThrProCysLysHisProValValMetLeuAlaGly 
505560 
ValAsnAlaAsnAspLeuGluAlaLeuLeuGluPheValTyrArgGly 
65707580 
GluValSerValAspHisAlaGlnLeuProSerLeuLeuGlnAlaAla 
859095 
GlnCysLeuAsnIleGlnGlyLeu 
100 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AsnAsnHisGlnSerAsnLeuLeuSerValPheAspGlnLeuLeuHis 
151015 
AlaGluThrPheValAspValThrLeuAlaValGluGlyGlnHisLeu 
202530 
LysAlaHisLysMetValLeuSerAlaCysSerProTyrPheAsnThr 
354045 
LeuPheValSerHisProGluLysHisProIleValIleLeuLysAsp 
505560 
ValProTyrSerAspMetLysSerLeuLeuAspPheMetTyrArgGly 
65707580 
GluValSerValAspGlnGluArgLeuThrAlaPheLeuArgValAla 
859095 
GluSerLeuArgIleLysGlyLeu 
100 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AsnAsnTyrGlnSerSerIleThrSerAlaPheGluAsnLeuArgAsp 
151015 
AspGluAlaPheValAspValThrLeuAlaCysGluGlyArgSerIle 
202530 
LysAlaHisArgValValLeuSerAlaCysSerProTyrPheArgGlu 
354045 
LeuLeuLysSerThrProCysLysHisProValIleLeuLeuGlnAsp 
505560 
ValAsnPheMetAspLeuHisAlaLeuValGluPheIleTyrHisGly 
65707580 
GluValAsnValHisGlnLysSerLeuGlnSerPheLeuLysThrAla 
859095 
GluValLeuArgValSerGlyLeu 
100 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 105 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AspAspHisLysThrLeuPheLeuLysThrLeuAsnGluGlnArgLeu 
151015 
GluGlyGluPheCysAspIleAlaIleValValGluAspValLysPhe 
202530 
ArgAlaHisArgCysValLeuAlaAlaCysSerThrTyrPheLysLys 
354045 
LeuPheLysLysLeuGluValAspSerSerSerValIleGluIleAsp 
505560 
PheLeuArgSerAspIlePheGluGluValLeuAsnTyrMetTyrThr 
65707580 
AlaLysIleSerValLysLysGluAspValAsnLeuMetMetSerSer 
859095 
GlyGlnIleLeuGlyIleArgPheLeu 
100105 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 104 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AlaSerHisSerLeuValLeuLeuGlnGlnLeuAsnMetGlnArgGlu 
151015 
PheGlyPheLeuCysAspCysThrValAlaIleGlyAspValTyrPhe 
202530 
LysAlaHisArgAlaValLeuAlaAlaPheSerAsnTyrPheLysMet 
354045 
IlePheIleHisGlnThrSerGluCysIleLysIleGlnProThrAsp 
505560 
IleGlnProAspIlePheSerTyrLeuLeuHisIleMetTyrThrGly 
65707580 
LysGlyProLysGlnIleValAspHisSerArgLeuGluGluGlyIle 
859095 
ArgPheLeuHisAlaAspTyrLeu 
100 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 106 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
ThrArgHisAlaSerAspValLeuLeuAsnLeuAsnArgLeuArgSer 
151015 
ArgAspIleLeuThrAspValValIleValValSerArgGluGlnPhe 
202530 
ArgAlaHisLysThrValLeuMetAlaCysSerGlyLeuPheTyrSer 
354045 
IlePheThrAspGlnLeuLysCysAsnLeuSerValIleAsnLeuAsp 
505560 
ProGluIleAsnProGluGlyPheCysIleLeuLeuAspPheMetTyr 
65707580 
ThrSerArgLeuAsnLeuArgGluGlyAsnIleMetAlaValMetAla 
859095 
ThrAlaMetTyrLeuGlnMetGluHisVal 
100105 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
ProSerHisProThrGlyLeuLeuCysLysAlaAsnGlnMetArgLeu 
151015 
AlaGlyThrLeuCysAspValValIleMetValAspSerGlnGluPhe 
202530 
HisAlaHisArgThrValLeuAlaCysThrSerMetPheGluIleLeu 
354045 
PheArgHisArgAsnSerGlnHisTyrThrLeuAspPheLeuSerPro 
505560 
LysThrPheGlnGlnIleLeuGluTyrAlaTyrThrAlaThrLeuGln 
65707580 
AlaLysAlaGluAspLeuAspAspLeuLeuTyrAlaAlaGluIleLeu 
859095 
GluIleGluTyrLeu 
100 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 102 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
ValGlnHisSerValArgValLeuGlnGluLeuAsnLysGlnArgGlu 
151015 
LysGlyGlnTyrCysAspAlaThrLeuAspValGlyGlyLeuValPhe 
202530 
LysAlaHisTrpSerValLeuAlaCysCysSerHisPhePheGlnSer 
354045 
LeuTyrGlyAspGlySerGlyGlySerValValLeuProAlaGlyPhe 
505560 
AlaGluIlePheGlyLeuLeuLeuAspPhePheTyrThrGlyHisLeu 
65707580 
AlaLeuThrSerGlyAsnArgAspGlnValLeuLeuAlaAlaArgGlu 
859095 
LeuArgValProGluAla 
100 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
ProGlyHisSerArgGlnLeuLeuLeuGlnLeuAsnAsnGlnArgThr 
151015 
LysGlyPheLeuCysAspValIleIleValValGlnAsnAlaLeuPhe 
202530 
ArgAlaHisLysAsnValLeuAlaAlaSerSerAlaTyrLeuLysSer 
354045 
LeuValValHisAspAsnLeuLeuAsnLeuAspHisAspMetValSer 
505560 
ProAlaValPheArgLeuValLeuAspPheIleTyrThrGlyArgLeu 
65707580 
AlaAlaGluProSerLeuGlyAlaValLeuAlaAlaAlaSerTyrLeu 
859095 
GlnIleProAspLeu 
100 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AspGlyAlaGluAlaAlaAlaAlaAlaAlaValAlaProGly 
1510 
__________________________________________________________________________