U.S. patent application number 10/663189 was filed with the patent office on 2005-02-03 for methods of diagnosing and treating hepatic cell proliferative disorders.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Bakker, Jila, Lin, Xiaohui, Nelson, William G., Tchou, Julia C..
Application Number | 20050026158 10/663189 |
Document ID | / |
Family ID | 31980885 |
Filed Date | 2005-02-03 |
United States Patent
Application |
20050026158 |
Kind Code |
A1 |
Nelson, William G. ; et
al. |
February 3, 2005 |
Methods of diagnosing and treating hepatic cell proliferative
disorders
Abstract
Provided are methods and compositions useful for the diagnosis,
prognosis and treatment of hepatic cellular proliferative
disorders. The methods include the modulation or analysis of
hypemethylated glutathione-S-transferase nuleic acid sequence in
hepatic samples and biological fluids.
Inventors: |
Nelson, William G.; (Towson,
MD) ; Lin, Xiaohui; (Baltimore, MD) ; Tchou,
Julia C.; (Baltimore, MD) ; Bakker, Jila;
(Baltimore, MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
31980885 |
Appl. No.: |
10/663189 |
Filed: |
September 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10663189 |
Sep 15, 2003 |
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09687246 |
Oct 12, 2000 |
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6709818 |
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60159168 |
Oct 13, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2523/125 20130101; C12Q 2600/154 20130101; G01N 33/573
20130101; C12Q 2521/331 20130101; G01N 2333/91171 20130101; C12Q
2600/106 20130101; G01N 33/576 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Goverment Interests
[0002] The U.S. Government may have certain rights in this
invention pursuant to Grant No.: CA58236 and CA70196 by the
National Institute of Health (NIH).
Claims
1-75. (canceled).
76. A kit useful for the detection of a methylated CpG-containing
nucleic acid in a GSTP1 promoter comprising carrier means
containing one or more containers comprising a first container
containing a reagent which modifies unmethylated cytosine and a
second container containing primers for amplification of the
CpG-containing nucleic acid, wherein the primers distinguish
between modified methylated and nonmethylated nucleic acid.
77. The kit of claim 76, wherein the modifying regent is
bisulfite.
78. The kit of claim 76, wherein said reagent modifies cytosine to
uracil.
79. The kit of claim 76, wherein the primer hybridizes with a
target polynucleotide sequence having the sequence from about -539
to -239 upstream from GSTP1 transcription start site.
80. The kit of claim 79, wherein the primers are SEQ ID Nos: 1, 2,
7, 8, 9, 10, 11, 12, or 13 or any combination thereof.
81. The kit of claim 76, further comprising nucleic acid
amplification buffer.
82. Isolated oligonucleotide primer(s) for detection of a
methylated CpG-containing nucleic acid wherein the primer
hybridizes with a target polynucleotide sequence having the
sequence in the region from about -539 to -239 upstream from GSTP1
transcription start site.
83. The primers of claim 81, wherein the primers are SEQ ID Nos: 1,
2, 7, 8, 9, 10, 11, 12, or 13 or any combination thereof.
84-85. (canceled).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e)(1) to U.S. Provisional Application No. 60/159,168,
filed Oct. 13, 1999, the disclosure of which is incorporated herein
by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the diagnosis of
cancer and specifically to identification of a hypermethylated
glutathione-S-transferase (GSTP1) gene as a diagnostic indicator of
hepatic cell proliferative disorders.
BACKGROUND
[0004] Hepatocellular carcinoma (HCC) constitutes one of the most
common life-threatening cancers in world. Most HCC cases arise in
the setting of chronic hepatitis virus infection. Dietary
carcinogens, such as alflatoxin B1, likely also contribute to
hepatic carcinogenesis. Glutathione S-transferases (GSTs) may help
defend normal hepatocytes against a variety of potentially
promutagenic stresses, including reactive oxygen species associated
with chronic hepatic inflammation, and reactive electrophilic
compounds associated with the hepatic metabolism of dietary
carcinogens. Therapeutic elevation of the expression of GSTs and
other carcinogen detoxification enzymes has been demonstrated to
attenuate hepatic carcinogenesis in animal models. Oltipraz, an
inducer of carcinogen detoxification enzyme expression in
hepatocytes, is currently under development as a chemoprotective
agent for human HCC.
[0005] In higher order eukaryotes DNA is methylated only at
cytosines located 5' to guanosine in the CpG dinucleotide. This
modification has important regulatory effects on gene expression,
especially when involving CpG rich areas, known as "CpG islands,"
located in the promoter regions of many genes. While almost all
gene-associated islands are protected from methylation on autosomal
chromosomes, extensive methylation of CpG islands has been
associated with transcriptional inactivation of selected imprinted
genes and genes on the inactive X-chromosome of females. Abberant
methylation of normally unmethylated CpG islands has been described
as a frequent event in immortalized and transformed cells, and has
been associated with transcriptional inactivation of defined tumor
suppressor genes in human cancers.
[0006] Somatic "CpG island" DNA hypermethylation changes have been
frequently detected in human cancer cell genomes. Several tumor
suppressor genes, such as Rb, VHL, and p16, have been reported to
be inactivated by "CpG island" DNA hypermethylation in different
human cancer types. For HCC, changes in DNA methylation at a number
of gene loci have been found to frequently accompany
carcinogenesis. In one study, somatic "CpG island" hypermethylation
affecting E-cadherin was detected in the majority (67%) of human
HCC specimens and in many (46%) liver tissues showing chronic
hepatitis or cirrhosis. In another study, abnormal DNA methylation
changes at several loci along chromosome 16, a chromosome
frequently exhibiting allelic losses in HCC, were also detected in
HCC DNA and DNA from liver tissues with chronic hepatitis or
cirrhosis.
[0007] 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. 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.
[0008] 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. 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. This change has now been associated with the loss
of expression of VHL, a renal cancer tumor suppressor gene on 3p,
the estrogen receptor gene on 6q and the H19 gene on 11p.
[0009] In eukaryotic cells, methylation of cytosine residues that
are immediately 5' to a guanosine, occurs predominantly in CG poor
regions. In contrast, discrete regions of CG dinucleotides called
CpG islands remain unmethylated in normal cells, except during
X-chromosome inactivation and parental specific imprinting where
methylation of 5' regulatory regions can lead to transcriptional
repression. De novo methylation of the Rb gene has been
demonstrated in a small fraction of retinoblastomas, and recently,
a more detailed analysis of the VHL gene showed aberrant
methylation in a subset of sporadic renal cell carcinomas.
Expression of a tumor suppressor gene can also be abolished by de
novo DNA methylation of a normally unmethylated 5.degree. CpG
island.
[0010] Identification of the earliest genetic changes in
tumorigenesis is a major focus in molecular cancer research.
Diagnostic approaches based on identification of these changes are
likely to allow implementation of early detection strategies and
novel therapeutic approaches targeting these early changes might
lead to more effective cancer treatment.
SUMMARY OF THE INVENTION
[0011] The present invention provides for the first time that
ability to detect and treat hepatic cell proliferative disorders by
detecting a methylated CpG-containing
glutathione-S-transferase.
[0012] In one embodiment, the invention provides a method for
detecting a hepatic cell proliferative disorder by detecting a
methylated CpG-containing glutathione-S-transferase (GST) nucleic
acid in a hepatic specimen or biological fluid wherein a methylated
GST nucleic acid is indicative a hepatic cell proliferative
disorder. The method of detecting may include contacting a nucleic
acid-containing hepatic specimen or biological fluid with an agent
that modifies unmethylated cytosine, amplifying the CpG-containing
nucleic acid in the specimen by means of CpG-specific
oligonucleotide primers, wherein the oligonucleotide primers
distinguish between modified methylated and nonmethylated nucleic
acid, and detecting the methylated nucleic acid based on the
presence or absence of amplification products produced in during
amplification. Alternatively, the detection may be performed by
contacting a target nucleic acid in the hepatic specimen or
biological fluid with a reagent which detects methylation of the
promoter region of the GST when the target nucleic acid is DNA, or
wherein the reagent detects the level of GST RNA when the target
nucleic acid is RNA, and detecting the GST target nucleic acid,
wherein hypermethylation of the promoter of GST DNA, or decreased
levels of GST RNA, as compared with the level of GST RNA in a
normal cell, is indicative of a GST-associated cell proliferative
disorder in hepatic tissue. The GST can be a .pi. family GST (e.g.,
GSTP1).
[0013] In another embodiment, the invention provides a method for
detecting a hepatic cell proliferative disorder associated with a
glutathione-S-transferase (GST) in a subject by contacting a target
nucleic acid in a sample of hepatic tissue or biological fluid from
the subject with a reagent which detects the GST, wherein the
reagent detects methylation of the promoter region of the GST when
the target nucleic acid is DNA, and wherein the reagent detects the
level of GST RNA when the target nucleic acid is RNA, and detecting
the GST target nucleic acid, wherein hypermethylation of the
promoter of GST DNA, or decreased levels of GST RNA, as compared
with the level of GST RNA in a normal cell, is indicative of a
GST-associated cell proliferative disorder in hepatic tissue.
[0014] In yet another embodiment, the inventin provides a method
for detecting a hepatic cell proliferative disorder associated with
a glutathione-S-transferase (GST) nucleic acid in a subject. The
method includes contacting a target cellular component containing a
GST nucleic acid with a reagent which reacts with the GST nucleic
acid and detecting hypermethylation of the GST nucleic acid,
wherein hypermethylation of the GST nucleic acid is indicative of a
hepatic cell proliferative disorder.
[0015] In another embodiment, the invention provides a method for
detecting a hepatic cell proliferative disorder associated with a
glutathione-S-transferase (GST) in a subject. The method includes
contacting a sample from the subject with a reagent that detects
GST polypeptide and comparing the level of GST polypeptide in the
sample to a control sample wherein a reduced level in the sample is
indicative of a hepatic cell proliferative disorder.
[0016] In yet another embodiment, the invention provides a method
for treating a hepatic cell proliferative disorder. The method
includes contacting a subject in need of such treatment with an
agent which increases the expression of a glutathione-S-transferase
(GST), thereby treating the hepatic cell proliferative
disorder.
[0017] In another embodiment, the invention provides a kit useful
for the detection of a methylated CpG-containing nucleic acid in a
GSTP1 promoter. The kit includes carrier means containing one or
more containers having a first container containing a reagent which
modifies unmethylated cytosine and a second container containing
primers for amplification of the CpG-containing nucleic acid,
wherein the primers distinguish between modified methylated and
nonmethylated nucleic acid.
[0018] In yet another embodiment, the invention provides isolated
oligonucleotide primer(s) for detection of a methylated
CpG-containing nucleic acid wherein the primer hybridizes with a
target polynucleotide sequence having the sequence in the region
from about -539 to -239 upstream from GSTP1 transcription start
site.
BRIEF DESCRIPTION OF THE FIGURE
[0019] FIG. 1A shows a southern blot analysis of DNA from Hep3B
cells treated for 72 hours with different concentrations of
5-azadeoxycytidine (aza-dC).
[0020] FIG. 1B shows a northern analysis of GSTP1 mRNA expression
by Hep3B cells treated with 5-azadeoxycytidine.
[0021] FIG. 2 shows the detection of GSTP1 `CpG island` methylation
changes in HCC DNA using a PCR assay capable of discriminating CpG
dinucleotide methylation changes affection maternal and paternal
GSTP1 alleles. PCR primers (arrows); U, untreated; H, HpaII
treated; M, MspI treated.
[0022] FIG. 3 shows the detection of hepatitis B virus DNA in DNA
from HCC and DNA from tissues adjacent to HCC. The presence of HBV
DNA sequences was monitored as the appearance of the predicted PCR
product (arrow).
[0023] FIG. 4 shows the heterogeneity of GSTP1 "CpG island" DNA
methylation changes in HCC DNA and in DNA from tissues adjacent to
HCC revealed by bisulfite genomic sequencing. Results of bisulfite
genomic sequencing analyses for .sup.5-mCpG dinucleotides located
between -195 and +35 of the GSTP1 transcription start site using
DNA prepared from HCC tissues and tissues adjacent to HCC are
displayed. Two sets of PCR primers, one set specific for target
sequences containing CpG dinucleotides and the other set specific
for target sequences containing .sup.5-mCpG dincucleotides, were
used to amplify bisulfite-treated DNA for DNA sequence analysis.
Open circles designate CpG dinucleotides; closed circles designate
.sup.5-mCpG dinucleotides. The absence of circles for some cases
indicates failure of the PCR reaction to generate amplification
products.
[0024] FIG. 5 shows the accumulation of GSTP1 "CpG island" DNA
methylation changes during human hepatocarcinogenesis. The
percentage of the 30 CpG dinucleotides located between nucleotides
-195 and +35 of the transcriptional start site carrying .sup.5-mC
instead of C was computed for each of the DNA specimens analyzed by
bisulfite genomic sequencing in FIG. 4. For each CpG dinucleotide
from each DNA specimen, the C nucleotide was scored as .sup.5-mC if
the nucleotide appeared as .sup.5-mC in either of the 2 PCR
reactions performed (using either .sup.5-mCpG-specific-primers or
CpG-specific primers). For HCC tissues and for liver tissues
adjacent to HCC, the percentage of .sup.5-mCpG dinucleotides at the
GSTP1 "CpG island" is displayed as the mean (for the 12
cases).+-.the standard error of the mean. Results of genomic
sequencing analyses for normal white blood cells (WBC; 0.3%) and
for Hep3B HCC cells (Hep3B; 100%) are also displayed.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention is based upon the observation that
human liver carcinogenesis proceeds via an accumulation of "CpG
island" hypermethylation changes at GSTP1 one or both alleles. The
discovery that "CpG island" DNA hypermethylation changes affecting
GSTP1, located on chromosome 11, are present in at least 85% of HCC
cases studied supports the basis of the invention. As described
more fully below, liver cancer cells failed to express either GSTP1
mRNA or GSTP1 polypeptides. Similarly, in 19 of 20 HCC cases
assessed, HCC cells were devoid of GSTP1 polypeptides detected by
immuno-histochemical staining using specific antiserum. DNA
isolated from liver cancer cells and from the majority of HCC
specimens displayed somatic GSTP1 "CpG island" hypermethylation.
Genomic sequencing analyses, undertaken to map
5-methyldeoxycytidine (.sup.5-mC) nucleotides located at the GSTP1
transcriptional regulatory region, indicated that the somatic DNA
hypermethylation changes present in HCC DNA consistently affected
the gene promoter. These GSTP1 DNA hypermethylation changes
contributed to GSTP1 inactivation in the HCC cells. In addition,
treatment of Hep3B HCC cells in vitro with a DNA-methyltransferase
inhibitor (e.g., 5-aza-deoxycytidine (5-aza-dC)) both reversed
GSTP1 "CpG island" hypermethylation and restored GSTP1 expression.
The methods described in the present invention allow detection of
GSTP1 CpG island hypermethylation affecting one or both maternal
and paternal alleles.
[0026] GSTs are dimeric enzymes with subunit polypeptides encoded
by an array of genes organized into several gene families: .alpha.,
.mu., .pi., and .theta.. In rat models of HCC, after exposure to an
initiating carcinogen, hyperplastic nodules containing liver cells
displaying increased expression of the .pi.-class GST (GST-P)
stereotypically appear. This increase in GST-P expression,
accompanied by increases in the expression of other carcinogen
detoxification enzymes, has been proposed to afford the
preneoplastic hepatocytes some protection against ongoing exposure
to hepatic carcinogens. Many of the HCCs which ultimately arise in
the carcinogen-treated rats continue to express high levels of
GST-P. Maintenance of high level expression of GST-P and other
carcinogen detoxification enzymes by HCC cells arising in
carcinogen-treated rats has been offered as evidence for selection
of a carcinogen-resistance phenotype during hepatic carcinogenesis
appear. In this model, increases in the expression of carcinogen
detoxification enzymes enable transformed hepatocytes to attempt to
elude the cytotoxic effects of chronic oxidant or electrophile
exposure. Alternatively, inadequate GST expression by normal and
preneoplastic hepatocytes has been proposed to render such cells
vulnerable to the promutagenic effects of carcinogen exposure that
promote neoplastic transformation. In support of this alternative
model, most hyperplastic nodules in carcinogen-treated rats fail to
progress to HCC, suggesting that increased GST-P expression in
preneoplastic hepatocytes may pose a barrier to hepatocellular
transformation. Fish species, such as the white suckers (Catostomus
commersoni), exposed to carcinogenic water pollutants in Lake
Ontario develop HCC containing low levels of GSTs. Hepatocytes
comprising abnormal liver lesions in rainbow trout (Onchorhynchus
mykiss) which fail to induce GST expression in response to
aflatoxin B1 or 1,2-dimethylbenzanthracene exposure appear prone to
develop larger and more expansive liver neoplasms than hepatocytes
in abnormal liver lesions with increased GST levels. The concept
that lack of GST expression might result in increased vulnerability
to carcinogen-induced tumorigenesis is supported by the recent
finding that mice carrying disrupted Gstp genes display an
increased number of skin tumors following topical exposure to 7,12
dimethylbenzanthracene.
[0027] Analyses of GST expression in human HCC cells have not been
entirely consistent with the findings of rat HCC model. In three
different HCC case studies, high level expression of GSTP1, the
human .pi.-class GST, was reported in only 3 of 12, 9 of 16, and 12
of 31 HCC cases. Moreover, a recent study of the expression of
several different GST isoenzymes in HCC specimens from China
revealed an overall reduction GST levels in HCC tissues. The
decrease in GST levels in HCC tissues was particularly marked in
specimens that were found to contain HBV DNA. New insights into the
molecular pathogenesis of human prostate cancer (PCA) may provide a
clue. During human prostatic carcinogenesis, prostatic carcinoma
(PCA) cells characteristically fail to express GSTP1 as a
consequence of a somatic GSTP1 inactivation. The somatic genome
lesion detected most often, methylation of deoxycytidine
nucleotides comprising a "CpG island" encompassing the gene
promoter, likely causes transcriptional silencing of GSTP1. Somatic
GSTP1 DNA hypermethylation changes have been detected in more than
90% of PCA lesions and some 70% of PCA precursor lesions (prostatic
intraepithelial neoplasia or "PIN" lesions). Somatic GSTP1 DNA
hypermethylation associated with absence of GSTP1 expression has
also been reported for breast and renal carcinomas.
[0028] The present invention reveals that hypermethylation of the
human .pi.-class glutathione-S-transferase structural gene (GSTP1)
positively correlates with hepatic carcinogenesis. Particularly
hypermethylation of the promoter region reduces the expression of
GSTP1 in liver tissue. This unexpected finding now allows the
detection of hepatic tissue cellular proliferative disorders by a
simple assay that detects hypermethylation of
glutathione-S-transferase sequences (e.g., promoter sequences)
either directly, by restriction endonuclease analysis, or
indirectly, by detection of GSTP1 mRNA or GSTP1 gene product. In
addition, methods of treating hepatic cellular cancers are now
possible and include the modulation of hypermethylation of
glutathione-S-transferases in the liver. Methods of treatment which
focus on replacing the hypermethylated promoter with a
non-methylated promoter, for example, are now possible.
[0029] As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are operatively linked to a nucleic acid sequence
when the expression control sequences control and regulate the
transcription and, as appropriate, translation of the nucleic acid
sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
stop codons. The term "control sequences" is intended to include,
at a minimum, components whose presence can influence expression,
and can also include additional components whose presence is
advantageous. Expression control sequences include a promoter.
[0030] By "promoter" is meant a minimal sequence sufficient to
direct transcription, for example, transcription of a
glutathione-S-transferase. In recombinant vectors, for example, a
promoter includes elements which are sufficient to render
promoter-dependent gene expression controllable for cell-type
specific, tissue-specific, or inducible by external signals or
agents; such elements may be located in the 5' or 3' regions of the
of the polynucleotide sequence. Both constitutive and inducible
promoters, are included in the invention (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, 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 nucleic acid sequences of the invention.
[0031] The term "isolated" means altered "by the hand of man" from
its natural state; i.e., if it occurs in nature, it has been
changed or removed from its original environment, or both. For
example, a naturally occurring polynucleotide or a polypeptide
naturally present in a living animal in its natural state is not
"isolated", but the same polynucleotide or polypeptide separated
from the coexisting materials of its natural state is "isolated",
as the term is employed herein. As part of or following isolation,
a polynucleotide can be joined to other polynucleotides, such as
for example DNAs, for mutagenesis studies, to form fusion proteins,
and for propagation or expression of the polynucleotide in a host,
or for gene therapy. The isolated polynucleotides, alone or joined
to other polynucleotides, such as vectors, can be introduced into
host cells, in culture or in whole organisms. Such polynucleotides,
when introduced into host cells in culture or in whole organisms,
still would be isolated, as the term is used herein, because they
would not be in their naturally occurring form or environment.
Similarly, the polynucleotides and polypeptides may occur in a
composition, such as a media formulation (solutions for
introduction of polynucleotides or polypeptides, for example, into
cells or compositions or solutions for chemical or enzymatic
reactions). For example, a polynucleotide encoding a GST protein
(e.g., GSTP1) can be operatively linked to a promoter and delivered
to a subject or cell having a cell proliferative disorder
associated with reduced expression of a GST or GSTP1.
[0032] "Polynucleotide" or "nucleic acid sequence" refers to a
polymeric form of nucleotides. In some instances a polynucleotide
refers to a sequence that is not immediately contiguous with either
of the coding sequences with which it is immediately contiguous
(one on the 5' end and one on the 3' end) in the naturally
occurring genome of the organism from which it is derived. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA)
independent of other sequences. The nucleotides of the invention
can be ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. In addition, the polynucleotide sequence
involved in producing a polypeptide chain can include regions
preceding and following the coding region (leader and trailer) as
well as intervening sequences (introns) between individual coding
segments (exons) depending upon the source of the polynucleotide
sequence.
[0033] The term polynucleotide(s) generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. Thus, for instance,
polynucleotides as used herein refers to, among others, single-and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions.
[0034] In addition, a polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0035] In addition, the polynucleotides or nucleic acid sequences
may contain one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein.
[0036] Nucleic acid sequences can be created which encode a fusion
protein and can be operatively linked to expression control
sequences. "Operatively linked" refers to a juxtaposition wherein
the components so described are in a relationship permitting them
to function in their intended manner. For example, a coding
sequence is "operably linked" to another coding sequence when RNA
polymerase will transcribe the two coding sequences into a single
mRNA, which is then translated into a single polypeptide having
amino acids derived from both coding sequences. The coding
sequences need not be contiguous to one another so long as the
expressed sequences ultimately process to produce the desired
protein. An expression control sequence operatively linked to a
coding sequence is ligated such that expression of the coding
sequence is achieved under conditions compatible with the
expression control sequences.
[0037] The invention provides a method for detecting a cell
expressing GSTP1 or a cell proliferative disorder associated with
GSTP1 in a tissue of a subject. The method includes contacting a
target cell containing a GSTP1 nucleic acid or protein (a target
cell component) and suspectd of having a GSTP1 associated disorder,
with a reagent which binds to the nucleic acid or protein. The
target cell component can be nucleic acid, such as DNA or RNA, or
protein. When the component is nucleic acid, the reagent is a
nucleic acid probe or PCR primer. When the cell component is
protein, the reagent is typically an antibody probe. 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
antibody, or will be able to ascertain such, using routine
experimentation.
[0038] A number of methods exist for detection of methylated
cytosine and can be used in the methods described herein (see, for
example, U.S. Pat. Nos. 5,756,668; 5,786,146; 5,856,094; and
5,922,590, all of which are incorporated herein by reference in
their entirety). For example, traditional methods depend upon
cleavage of the phosphodiester bond alongside cytosine residues,
using either methylation-sensitive restriction enzymes or reactive
chemicals such as hydrazine which differentiate between cytosine
and its 5-methyl derivative. Mapping of methylated regions in DNA
has been performed using Southern hybridization approaches, based
on the inability of methylation-sensitive restriction enzymes to
cleave sequences which contain one or more methylated CpG sites.
This method provides an assessment of the overall methylation
status of CpG islands, including some quantitative analysis. A more
sensitive method of detecting methylation patterns combines the use
of methylation-sensitive enzymes and the polymerase chain reaction
(PCR). After digestion of DNA with the enzyme, PCR will amplify
from primers flanking the restriction site only if DNA cleavage was
prevented by methylation. Another method that avoids the use of
restriction endonucleases utilizes bisulfite treatment of DNA to
convert all unmethylated cytosines to uracil. The altered DNA is
amplified and sequenced to show the methylation status of all CpG
sites.
[0039] Exemplary target regions to which PCR primers of the
invention are designed include primers which flank the region that
lies approximately -539 to -239 bp from the transcription start
site of GSTP1, as described herein. As shown in Example 2 and
Example 5, such primers can be designed to be specific for
methylated regions of DNA if desired. For example, PCR primers
(upstream primer, 5'-AGCCTGGGCCACAGCGTGAGACTACGT-3' (SEQ ID NO:1);
downstream primer, 5'-GGAGTAAACAGACAGCAGGAAGAGGAC-3' (SEQ ID NO:2))
may be used to target a sequence approximately -539 to -239 bp from
the transcription start site of GSTP1.
[0040] To selectively amplify GSTP1 promoter DNA containing
.sup.5-mC in the "sense" strand, primers N-F1 (GenBank position
816-835, 5'-GTAATTTTTTTTTTTT TAAG-3' (SEQ ID NO:7)) and M-R1
(position 1405-1420, 5'-TAAAAACCGCTAACGA-3' (SEQ ID NO:8)) were
included in the PCR reaction mixture; to amplify GSTP1 promoter DNA
containing C in the "sense" strand, primers N-F1 and U-R1 (position
1406-1422 5'-CCTAAAAACCACTAACA-3' (SEQ ID NO:9)) were used. After
heating to 94.degree. C. for 2 min, PCR was conducted by incubation
at 94.degree. C. for 1 min, 44.degree. C. for 2 min, and 72.degree.
C. for 3 min for 5 cycles, followed by incubation 94.degree. C. for
30 sec, 44.degree. C. for 2 min, and 72.degree. C. for 1.5 min for
25 cycles before a final extension at 72.degree. C. for 6 min.
Products from the first PCR reaction mixtures were subjected to a
second round of "nested" PCR. The second PCR reaction mixtures
contained 1 .mu.M of primers, 250 .mu.M of deoxynucleotide
triphosphates, and 1.25 units Taq polymerase in OptiPrime buffer #6
(Stratagene). To amplify GSTP1 promoter DNA containing .sup.5-mC,
primers M-F2 (position 897-918, 5'-TTTTAGGGAATTTTTTTTCGCG-3' (SEQ
ID NO: 10)) and M-R2 (position 1327-1345, 5'-CCCTACCGA
AAACCCGAAC-3' (SEQ ID NO: 11)) were added to PCR reaction mixture;
to amplify GSTP1 promoter DNA containing C, primers U-F2 (position
895-917, 5'-GGTTTTAGGGAATTTTTTTTTGT-3' (SEQ ID NO:12)) and U-R2
(position, 1326-1346, 5'-ACCCTACCAAAAACCCAAAC-3' (SEQ ID NO:13))
were used. It should be understood that one of skill in the art can
design primers to other regions of GSTP1, and the promoter region
in particular.
[0041] Another method for detecting a methylated CpG-containing
nucleic acid, includes contacting a nucleic acid-containing
specimen with an agent that modifies unmethylated cytosine;
amplifying the CpG-containing nucleic acid in the specimen by means
of CpG-specific oligonucleotide primers; and detecting the
methylated nucleic acid. It is understood that while the
amplification step is optional, it is desirable.
[0042] The term "modifies" as used herein means the conversion of
an unmethylated cytosine to another nucleotide which will
distinguish the unmethylated from the methylated cytosine.
Preferably, the agent modifies unmethylated cytosine to uracil.
Preferably, the agent used for modifying unmethylated cytosine is
sodium bisulfite, however, other agents that similarly modify
unmethylated cytosine, but not methylated cytosine can also be used
in the method of the invention. Sodium bisulfite (NaHSO.sub.3)
reacts readily with the 5,6-double bond of cytosine, but poorly
with methylated cytosine. Cytosine reacts with the bisulfite ion to
form a sulfonated cytosine reaction intermediate which is
susceptible to deamination, giving rise to a sulfonated uracil. The
sulfonate group can be removed under alkaline conditions, resulting
in the formation of uracil. Uracil is recognized as a thymine by
Taq polymerase and therefore upon PCR, the resultant product
contains cytosine only at the position where 5-methylcytosine
occurs in the starting template DNA.
[0043] The primers used in the invention for amplification of a
CpG-containing nucleic acid in the specimen, after bisulfite
modification, specifically distinguish between untreated DNA,
methylated, and non-methylated DNA. Methylation specific PCR (MSP)
primers for the non-methylated DNA preferably have a T in the 3' CG
pair to distinguish it from the C retained in methylated DNA, and
the compliment is designed for the antisense primer. MSP primers
usually contain relatively few Cs or Gs in the sequence since the
Cs will be absent in the sense primer and the Gs absent in the
antisense primer (C becomes modified to U (uracil) which is
amplified as T (thymidine) in the amplification product).
[0044] The primers of the invention embrace oligonucleotides of
sufficient length and appropriate sequence so as to provide
specific initiation of polymerization on a significant number of
nucleic acids in the polymorphic locus. Specifically, the term
"primer" as used herein refers to a sequence comprising two or more
deoxyribonucleotides or ribonucleotides, preferably more than
three, and most preferably more than 8, which sequence is capable
of initiating synthesis of a primer extension product, which is
substantially complementary to a polymorphic locus strand.
Environmental conditions conducive to synthesis include the
presence of nucleoside triphosphates and an agent for
polymerization, such as DNA polymerase, and a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency in amplification, but may be double stranded. If double
stranded, the primer is first treated to separate its strands
before being used to prepare extension products. Preferably, the
primer is an oligodeoxy ribonucleotide. The primer must be
sufficiently long to prime the synthesis of extension products in
the presence of the inducing agent for polymerization. The exact
length of primer will depend on many factors, including
temperature, buffer, and nucleotide composition. The
oligonucleotide primer typically contains 12-20 or more
nucleotides, although it may contain fewer nucleotides.
[0045] Primers of the invention are designed to be "substantially"
complementary to each strand of the genomic locus to be amplified
and include the appropriate G or C nucleotides as discussed above.
This means that the primers must be sufficiently complementary to
hybridize with their respective strands under conditions which
allow the agent for polymerization to perform. In other words, the
primers should have sufficient complementarity with the 5' and 3'
flanking sequences to hybridize therewith and permit amplification
of the genomic locus.
[0046] Oligonucleotide primers of the invention are employed in the
amplification process which is an enzymatic chain reaction that
produces exponential quantities of target locus relative to the
number of reaction steps involved. Typically, one primer is
complementary to the negative (-) strand of the locus and the other
is complementary to the positive (+) strand. Annealing the primers
to denatured nucleic acid followed by extension with an enzyme,
such as the large fragment of DNA Polymerase I (Klenow) and
nucleotides, results in newly synthesized + and - strands
containing the target locus sequence. Because these newly
synthesized sequences are also templates, repeated cycles of
denaturing, primer annealing, and extension results in exponential
production of the region (i.e., the target locus sequence) defined
by the primer. The product of the chain reaction is a discrete
nucleic acid duplex with termini corresponding to the ends of the
specific primers employed.
[0047] The oligonucleotide primers of the invention may be prepared
using any suitable method, such as conventional phosphotriester and
phosphodiester methods or automated embodiments thereof In one such
automated embodiment, diethylphosphoramidites are used as starting
materials and may be synthesized as described by Beaucage, et al.
(Tetrahedron Letters, 22:1859-1862, 1981). One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066.
[0048] Any nucleic acid specimen, in purified or non-purified form,
can be utilized as the starting nucleic acid or acids, provided it
contains, or is suspected of containing, the specific nucleic acid
sequence containing the target locus (e.g., CpG). Thus, the process
may employ, for example, DNA or RNA, including messenger RNA,
wherein DNA or RNA may be single stranded or double stranded. In
the event that RNA is to be used as a template, enzymes, and/or
conditions optimal for reverse transcribing the template to DNA
would be utilized. In addition, a DNA-RNA hybrid which contains one
strand of each may be utilized. A mixture of nucleic acids may also
be employed, or the nucleic acids produced in a previous
amplification reaction herein, using the same or different primers
may be so utilized. The specific nucleic acid sequence to be
amplified, i.e., the target locus, may be a fraction of a larger
molecule or can be present initially as a discrete molecule, so
that the specific sequence constitutes the entire nucleic acid. It
is not necessary that the sequence to be amplified be present
initially in a pure form; it may be a minor fraction of a complex
mixture, such as contained in whole human DNA.
[0049] The nucleic acid-containing specimen used for detection of
methylated CpG may be from any source including colon, blood,
lypmthatic and hepatic tissue and may be extracted by a variety of
techniques such as that described by Maniatis, et al. (Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp 280,
281, 1982).
[0050] If the extracted sample is impure (such as plasma, serum, or
blood or a sample embedded in parrafin), it may be treated before
amplification with an amount of a reagent effective to open the
cells, fluids, tissues, or animal cell membranes of the sample, and
to expose and/or separate the strand(s) of the nucleic acid(s).
This lysing and nucleic acid denaturing step to expose and separate
the strands will allow amplification to occur much more
readily.
[0051] Where the target nucleic acid sequence of the sample
contains two strands, it is necessary to separate the strands of
the nucleic acid before it can be used as the template. Strand
separation can be effected either as a separate step or
simultaneously with the synthesis of the primer extension products.
This strand separation can be accomplished using various suitable
denaturing conditions, including physical, chemical, or enzymatic
means, the word "denaturing" includes all such means. One physical
method of separating nucleic acid strands involves heating the
nucleic acid until it is denatured. Typical heat denaturation may
involve temperatures ranging from about 80.degree. to 105.degree.
C. for times ranging from about 1 to 10 minutes. Strand separation
may also be induced by an enzyme from the class of enzymes known as
helicases or by the enzyme RecA, which has helicase activity, and
in the presence of riboATP, is known to denature DNA. The reaction
conditions suitable for strand separation of nucleic acids with
helicases are described by Kuhn Hoffmann-Berling (CSH-Quantitative
Biology, 43:63, 1978) and techniques for using RecA are reviewed in
C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
[0052] When complementary strands of nucleic acid or acids are
separated, regardless of whether the nucleic acid was originally
double or single stranded, the separated strands are ready to be
used as a template for the synthesis of additional nucleic acid
strands. This synthesis is performed under conditions allowing
hybridization of primers to templates to occur. Generally synthesis
occurs in a buffered aqueous solution, preferably at a pH of 7-9,
most preferably about 8. Preferably, a molar excess (for genomic
nucleic acid, usually about 10.sup.8:1 primer:template) of the two
oligonucleotide primers is added to the buffer containing the
separated template strands. It is understood, however, that the
amount of complementary strand may not be known if the process of
the invention is used for diagnostic applications, so that the
amount of primer relative to the amount of complementary strand
cannot be determined with certainty. As a practical matter,
however, the amount of primer added will generally be in molar
excess over the amount of complementary strand (template) when the
sequence to be amplified is contained in a mixture of complicated
long-chain nucleic acid strands. A large molar excess is preferred
to improve the efficiency of the process.
[0053] The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and
dTTP are added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90.degree.-100.degree. C. from about 1
to 10 minutes, preferably from 1 to 4 minutes. After this heating
period, the solution is allowed to cool to room temperature, which
is preferable for the primer hybridization. To the cooled mixture
is added an appropriate agent for effecting the primer extension
reaction (called herein "agent for polymerization"), and the
reaction is allowed to occur under conditions known in the art. The
agent for polymerization may also be added together with the other
reagents if it is heat stable. This synthesis (or amplification)
reaction may occur at room temperature up to a temperature above
which the agent for polymerization no longer functions. Thus, for
example, if DNA polymerase is used as the agent, the temperature is
generally no greater than about 40.degree. C. Most conveniently the
reaction occurs at room temperature.
[0054] The agent for polymerization may be any compound or system
which will function to accomplish the synthesis of primer extension
products, including enzymes. Suitable enzymes for this purpose
include, for example, E. coli DNA polymerase I, Klenow fragment of
E. coli DNA polymerase I, T4 DNA polymerase, other available DNA
polymerases, polymerase muteins, reverse transcriptase, and other
enzymes, including heat-stable enzymes (e.g., those enzymes which
perform primer extension after being subjected to temperatures
sufficiently elevated to cause denaturation). Suitable enzymes will
facilitate combination of the nucleotides in the proper manner to
form the primer extension products which are complementary to each
locus nucleic acid strand. Generally, the synthesis will be
initiated at the 3' end of each primer and proceed in the 5'
direction along the template strand, until synthesis terminates,
producing molecules of different lengths. There may be agents for
polymerization, however, which initiate synthesis at the 5' end and
proceed in the other direction, using the same process as described
above.
[0055] Typically, the method of amplifying is by PCR, as described
herein and as is commonly used by those of ordinary skill in the
art. Alternative methods of amplification have been described and
can also be employed as long as the methylated and non-methylated
loci amplified by PCR using the primers of the invention is
similarly amplified by the alternative means.
[0056] The amplified products are preferably identified as
methylated or non-methylated by sequencing. Sequences amplified by
the methods of the invention can be further evaluated, detected,
cloned, sequenced, and the like, either in solution or after
binding to a solid support, by any method usually applied to the
detection of a specific DNA sequence such as PCR, oligomer
restriction (Saiki, et al., BiolTechnology, 3:1008-1012, 1985),
allele-specific oligonucleotide (ASO) probe analysis (Conner, et
al., Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide
ligation assays (OLAs) (Landegren, et al., Science, 241:1077,
1988), and the like. Molecular techniques for DNA analysis have
been reviewed (Landegren, et al., Science, 242:229-237, 1988).
[0057] Optionally, the methylation pattern of the nucleic acid can
be confirmed by restriction enzyme digestion and Southern blot
analysis. Examples of methylation sensitive restriction
endonucleases which can be used to detect 5'CpG methylation include
SmaI, SacII, EagI, MspI, HpaII, BstUI and BssHII, for example.
[0058] Since the present invention shows that a decreased level of
GSTP1 transcription is often the result of hypermethylation of the
GSTP1 polynucleotide sequence and/or expression control sequences
(e.g., the promoter sequence), it may be desirable to determine
whether the promoter is hypermethylated. Accordinlgy, the invention
provides methods of detecting or diagnosing a cell proliferative
disorder of hepatic tissue or cells by detecting methylation of the
expression control or promoter region of GSTP1. Probes useful for
detecting methylation of the promoter region of GSTP1 are useful in
such diagnositic or prognostic methods. In addition, primers that
flank or amplify the promoter nucleic acid sequence of GSTP1 are
useful in detecting methylation of the promoter and thus the risk
or occurrence of cell proliferative disorders. Probes and primers
useful in the invention for the detection of hypermethylation of
the expresson control or promoter sequences of GSTP1 include, for
example, nucleic acids having a sequence as set forth in SEQ ID
Nos: 1, 2, 7, 8, 9, 10, 11, 13 and combinations thereof.
[0059] Actively transcribed genes generally contain fewer
methylated CGs than the average number in DNA. Hypermethylation can
be detected by, for example, restriction endonuclease treatment and
Southern blot analysis among others. Therefore, in a method of the
invention, when the cellular component detected is DNA, restriction
endonuclease analysis can be used to detect hypermethylation of the
GSTP1 expression control sequence. Any restriction endonuclease
that includes CG as part of its recognition site and that is
inhibited when the C is methylated, can be utilized. Typically, the
methylation sensitive restriction endonuclease is BssHII, MspI, or
HpaII, used alone or in combination. Other methylation sensitive
restriction endonucleases will be known to those of skill in the
art.
[0060] For purposes of the invention, an antibody or nucleic acid
probe specific for GSTP1 may be used to detect the presence of
GSTP1 polypeptide (using antibody) or polynucleotide (using nucleic
acid probe) in biological fluids or tissues. Oligonucleotide
primers based on any coding sequence region in the GSTP1 sequence
are useful for amplifying DNA, for example by PCR. Any specimen
containing a detectable amount of polynucleotide or antigen can be
used. A preferred sample of this invention is tissue of hepatic
origin, for example, liver tissue. Preferably the sample contains
hepatic cells. Alternatively, biological fluids such as bile, lymph
fluid or blood may be used which may contain cells indicative of an
GSTP1-associated cell proliferative disorder. The subject can be
any animal having a hepatic organ including, for example, mice,
rat, fish, bovine, porcine, canine, feline, equine, and primate
species. Preferably the subject is human.
[0061] Another technique which may also result in greater
sensitivity consists of coupling the antibodies to low molecular
weight haptens. These haptens can then be specifically detected by
means of a second reaction. For example, it is common to use such
haptens as biotin, which reacts with avidin, or dinitrophenyl,
pyridoxal, and fluorescein, which can react with specific
antihapten antibodies.
[0062] The method for detecting a cell expressing GSTP1 of the
invention or a cell proliferative disorder associated with an
GSTP1, described above, can be utilized for detection of residual
hepatic cancer or other malignancies in a subject in a state of
clinical remission. Additionally, the method for detecting GSTP1
polypeptide in cells is useful for detecting a cell proliferative
disorder by measuring the level of GSTP1 in cells expressing GSTP1
in a suspect tissue in comparison with GSTP1 expressed in normal
cells or tissue. In addition, the methods of the invention can also
be used in staging of a cell proliferative disorder. Using the
method of the invention, GSTP1 expression can be identified in a
cell and the appropriate course of treatment can be employed (e.g.,
sense gene therapy or drug therapy). The expression pattern of the
GSTP1 of the invention may vary with the stage of malignancy of a
cell, for example as seen with prostatic intraepithelial neoplasia
(PIN) (McNeal, et al., Human Pathol., 17:64, 1986) therefore, a
sample such as liver tissue can be screened with a panel of
GSTP1-specific reagents (i.e., nucleic acid probes or antibodies to
GSTP1) to detect GSTP1 expression and diagnose the stage of
malignancy of the cell.
[0063] 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.
[0064] 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.
[0065] Monoclonal antibodies can be bound to many different
carriers and used to detect the presence of GSTP1. 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.
[0066] For purposes of the invention, GSTP1 may be detected by the
monoclonal antibodies when present in biological fluids and
tissues. Any sample containing a detectable amount of GSTP1 can be
used. A sample can be a liquid such as bile, blood, or lymph and
the like, or a solid or semi-solid such as tissues, feces, and the
like, or, alternatively, a solid tissue such as those commonly used
in histological diagnosis.
[0067] 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 antiheterophilic
immunoglobulins to anti-GSTP1 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.
[0068] It has been found that a number of nonrelevant (e.g.,
nonspecific) antibodies of the same class or subclass (isotype) as
those used in the assays (e.g., IgG1, IgG2a, IgM, and the like) 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.
[0069] 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 GSTP1 antigen for which the monoclonal
antibodies are specific.
[0070] The concentration of detectably labeled monoclonal antibody
which is administered should be sufficient such that the binding to
those cells having GSTP1 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.
[0071] As a general 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.
[0072] 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.
[0073] 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.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72AS, .sup.89Zr, and .sup.201TI.
[0074] 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.157Gd, .sup.55M, .sup.162Dy,
.sup.52Cr, and .sup.56Fe.
[0075] Monoclonal antibodies used in the method of the invention
can be used to monitor the course of amelioration of GSTP1
associated cell proliferative disorder. Thus, by measuring the
increase or decrease in the number of cells expressing GSTP1 or
changes in GSTP1 present in various body fluids, such as bile or
blood, it would be possible to determine whether a particular
therapeutic regiment aimed at ameliorating the disorder is
effective.
[0076] Various antibody types and derivatives are applicable to the
diagnostic and treatment methods of the invention. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA, 86:3833 (1989), which is hereby incorporated in its
entirety by reference. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature, 321:522 (1986); Riechmann et al., Nature, 332:323 (1988);
Verhoeyen et al., Science, 239:1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev. Biotech.,
12:437 (1992); and Singer et al., J. Immunol., 150:2844 (1993),
which are hereby incorporated by reference.
[0077] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12:433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0078] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet., 7:13 (1994); Lonberg et
al., Nature, 368:856 (1994); and Taylor et al., Int. Immunol.,
6:579 (1994), which are hereby incorporated by reference.
[0079] Antibody fragments of the invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E. coli
of DNA encoding the fragment. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfydryl groups resulting from cleavage of disulfide linkages,
to produce 3.5S Fab' monovalent fragments. Alternatively, an
enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat.
No. 4,331,647, and references contained therein. These patents are
hereby incorporated by reference in their entireties. See also
Nisonhoff et al., Arch. Biochem. Biophys,. 89:230 (1960); Porter,
Biochem. J., 73:119 (1959); Edelman et al., Methods in Enzymology,
Vol. 1, page 422 (Academic Press 1967); and Coligan et al. at
sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0080] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0081] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA, 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the F.sub.v
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow et al.,
Methods: A Companion to Methods in Enzymology, Vol. 2, page 97
(1991); Bird et al., Science, 242:423 (1988); Ladner et al., U.S.
Pat. No. 4,946,778; Pack et al., Bio/Technology, 11:1271 (1993);
and Sandhu, supra.
[0082] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
[0083] The present invention also provides a method for treating a
subject with an GSTP1-associated cell proliferative disorder. In
hepatic cancer, the GSTP1 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 GSTP1 associated with malignancy,
nucleic acid sequences that modulate GSTP1 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 GSTP1, for example, nucleic
acid sequences encoding GSTP1 (sense) could be administered to the
subject with the disorder.
[0084] 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 GSTP1. Essentially, any disorder which is
etiologically linked to expression of GSTP1 could be considered
susceptible to treatment with a reagent of the invention which
modulates GSTP1 expression.
[0085] The term "modulate" envisions the suppression of methylation
of GSTP1 promoter or augmentation of other GST gene expression when
GSTP1 is under-expressed. When a cell proliferative disorder is
associated with GSTP1 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 GSTP1 polypeptide, a sense polynucleotide
sequence (the DNA coding strand) encoding the promoter region or
the promoter operably linked to the structural gene, or GSTP1
polypeptide can be introduced into the cell.
[0086] The present invention also provides gene therapy for the
treatment of cell proliferative disorders which are mediated by
GSTP1. Such therapy would achieve its therapeutic effect by
introduction of the appropriate GSTP1 polynucleotide which contains
either a normal GSTP1 regulatory region alone or in combination
with a GSTP1 structural gene (sense), into cells of subjects having
the proliferative disorder. Alternatively, the GSTP1 structural
gene could be introduced operably linked to a heterologous
promoter, such as the GSTM, GSTA or other promoter. Delivery of
sense GSTP promoter polynucleotide constructs can be achieved using
a recombinant expression vector such as a chimeric virus or a
colloidal dispersion system.
[0087] In some instances it may be advantageous to deliver and
express a GST sequence locally (e.g., within a particular tissue or
cell type). For example, local expression of a GST (e.g., GSTP1)
within a hepatic cell or tissue can be performed to treat, modulate
or ameliorate a cell proliferative disorder within a hepatic cell
or tissue. The nucleic sequence may be directly delivered to the
tissue or cells, for example. Such delivery methods are known in
the art and include, for example, electroporation, viral vector
delivery systems and direct DNA uptake.
[0088] For example, a nucleic acid constructs of the present
invention will comprise nucleic acid molecules in a form suitable
for uptake into target cells within a host tissue. The nucleic
acids may be in the form of bare DNA or RNA molecules, where the
molecules may comprise one or more structural genes, one or more
regulatory genes, antisense strands, strands capable of triplex
formation, or the like. Commonly, the nucleic acid construct will
include at least one structural gene under the transcriptional and
translational control of a suitable regulatory region. More
usually, nucleic acid constructs of the present invention will
comprise nucleic acids incorporated in a delivery vehicle to
improve transfection efficiency.
[0089] One such delivery vehicles comprises viral vectors, such as
retroviruses, adenoviruses, and adeno-associated viruses, which
have been inactivated to prevent self-replication but which
maintain the native viral ability to bind a target host cell,
deliver genetic material into the cytoplasm of the target host
cell, and promote expression of structural or other genes which
have been incorporated in the particle. Suitable retrovirus vectors
for mediated gene transfer are described in Kahn et al. CIRC. RES.
71:1508-1517, 1992, the disclosure of which is incorporated herein
by reference. A suitable adenovirus gene delivery is described in
Rosenfeld et al. Science 252:431-434, 1991, the disclosure of which
is incorporated herein by reference. Both retroviral and adenovirus
delivery systems are described in Friedman Science 244:1275-1281,
1989, and "The Development Of Human Gene Therapy," Ed. Theodore
Friedmann, Cold Spring Harbor Laboratory Press, New York, 1999, the
disclosures of which are also incorporated herein by reference.
[0090] A second type of nucleic acid delivery vehicle comprises
liposomal transfection vesicles, including both anionic and
cationic liposomal constructs. The use of anionic liposomes
requires that the nucleic acids be entrapped within the liposome.
Cationic liposomes do not require nucleic acid entrapment and
instead may be formed by simple mixing of the nucleic acids and
liposomes. The cationic liposomes avidly bind to the negatively
charged nucleic acid molecules, including both DNA and RNA, to
yield complexes which give reasonable transfection efficiency in
many cell types. See, Farhood et al. Biochem. Biophys. Acta. 1111
:239-246, 1992, the disclosure of which is incorporated herein by
reference. A typical material for forming liposomal vesicles is
lipofectin which is composed of an equimolar mixture of
dioleylphosphatidyl ethanolamine (DOPE) and
dioleyloxypropyl-triethylammonium (DOTMA), as described in Felgner
and Ringold, Nature 337:387-388, 1989, the disclosure of which is
incorporated herein by reference.
[0091] It is also possible to combine these two types of delivery
systems. For example, Kahn et al. (1992), supra., teaches that a
retrovirus vector may be combined in a cationic DEAE-dextran
vesicle to further enhance transformation efficiency. It is also
possible to incorporate nuclear proteins into viral and/or
liposomal delivery vesicles to even further improve transfection
efficiencies. See, Kaneda et al. Science 243:375-378, 1989, the
disclosure of which is incorporated herein by reference.
[0092] The promoter 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 can be administered to a subject as
drug therapy, alone or simultaneously with a sense promoter for
GSTP1 or a sense promoter operably linked with the structural gene
for GSTP1.
[0093] In another embodiment, a GSTP1 structural gene is operably
linked to a tissue specific heterologous promoter and used for gene
therapy. For example, a GSTP1 gene can be ligated to liver specific
promoters (e.g., albumin promoters, .alpha.1 antitrypsin
promoters), for expression of GSTP1 in hepatic tissue.
Alternatively, the promoter for another GST gene can be linked to
the GSTP1 structural gene and used for gene therapy.
[0094] 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). 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. By inserting a GSTP1 sequence (including promoter
region) of interest into the viral vector, along with another gene
which encodes the ligand for a receptor on a specific target cell,
for example, to render the vector target specific. 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 GSTP1 sense or antisense polynucleotide. Target
specific retroviral vectors can include a combination targeting
proteins on the surface of the viral particle as well as tissue
specific promoters to further allow only expression of the
retroviral vector in the desired tissue.
[0095] 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 that 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, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0096] The vectors of the invention can be used to transform a host
cell or a cell derived from a subject (e.g., ex vivo therapy). By
transform or transformation is meant a permanent or transient
genetic change induced in a cell following incorporation of new DNA
(i.e., DNA exogenous to the cell). Where the cell is a mammalian
cell, a permanent genetic change is generally achieved by
introduction of the DNA into the genome of the cell.
[0097] A transformed cell or host cell generally refers to a cell
(e.g., prokaryotic or eukaryotic) into which (or into an ancestor
of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding a GST polypeptide or a fragment
thereof or which contains an expression control element of
GSTP1.
[0098] 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 by 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 or by electroporation.
[0099] When the host is a eukaryote, methods of transfection or
transformation with DNA include calcium phosphate co-precipitates,
conventional mechanical procedures such as microinjection,
electroporation, insertion of a plasmid encased in liposomes, or
virus vectors, as well as others known in the art, may be used.
Eukaryotic cells can also be cotransfected with DNA sequences
encoding a GST polypeptide and a second foreign DNA molecule
encoding a selectable marker, 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. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,
Gluzman ed., 1982). Typically, a eukaryotic host will be utilized
as the host cell. The eukaryotic cell may be a yeast cell (e.g.,
Saccharomyces cerevisiae), an insect cell (e.g., Drosophila sp.) or
may be a mammalian cell, including a human cell.
[0100] Eukaryotic systems, and mammalian expression systems, allow
for post-translational modifications of expressed mammalian
proteins to occur. Eukaryotic cells which possess the cellular
machinery for processing of the primary transcript, glycosylation,
phosphorylation, and, advantageously secretion of the gene product
should be used. Such host cell lines may include, but are not
limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and
WI38.
[0101] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, a polynucleotide encoding
a GST polypeptide may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric sequence 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 a GST polypeptide or a
fragment thereof 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., Proc. Natl. Acad. Sci. USA, 79:7415-7419, 1982; 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, 198 1). 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
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of a GST (e.g., a GSTP1) 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.
[0102] 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 cDNA encoding a GST polypeptide
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 vector 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-, hgprt- or aprt-cells respectively. Also,
anti-metabolite resistance can be used as the basis of selection
for dhfr, which confers resistance to methotrexate (Wigler, et al.,
Proc. Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc.
Natl. Acad. Sci. USA, 8:1527, 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
(ornithine decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue L., In: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory, ed., 1987).
[0103] Accordingly, the methods of the invention have applicability
to the treatement hepatic cell proliferative disorders in
veterinary applications in addition to applicability in human
subjects. The vectors or delivery vehicles can be optimized by the
skilled artisan for application to various animals and species.
[0104] Another targeted delivery system for GSTP1 polynucleotide is
a colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelies,
mixed micelies, 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).
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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 GSTP1 antibody-containing liposomes
directly to the malignant tumor. Since the GSTP1 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 hepatic tissue. A number of procedures can be used to
covalently attach either polyclonal or monoclonal antibodies to a
liposome bilayer. Antibody-targeted iiposomes can include
monoclonal or polyclonal antibodies or fragments thereof such as
Fab, or F(ab').sub.2, so 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.
[0111] In yet another embodiment, the invention envisions treating
a subject with low levels of GSTP1 expression with a
glutathione-S-transferase inducing agent. Stimulation of the other
classes of GSTs may compensate for the deficiency in GSTP1. Such
inducers include sulfofurain, oltipraz, as well as other substances
known in the art (Prochaska, et al., Proc. Nat'l. Acad. Sci.,
U.S.A., 89:2394, 1992; Zhang, et al., Proc. Nat'l. Acad. Sci.,
U.S.A., 89:2399, 1992; Prestera, et al., Proc. Nat'l. Acad. Sci.,
U.S.A., 90:2965, 1993). Methylation of GSTP1 promoter
polynucleotide can be inhibited in vitro or in vivo by treatment
with 5-aza-cytidine, 5-aza-deoxycytidine or procainamide, for
example. Other similar agents will be known to those of skill in
the art.
[0112] The invention also relates to a medicament or pharmaceutical
composition comprising a GSTP1 promoter polynucleotide or a GSTP1
or other GST promoter polynucleotide or GST polynucleotide. Where
the polynucleotide is an expression control element (e.g., a
promoter), the expression control element is operably linked to the
GSTP1 or GST structural gene in a pharmaceutically acceptable
excipient or medium wherein the medicament is used for therapy of
GSTP1 associated cell proliferative disorders. In this embodiment,
the expression of GST or GSTP1 overcomes the deficiencies of
expression in the target cell or tissue.
[0113] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Such a kit may
comprise a carrier means containing 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.
One of the container means may comprise a probe which is or can be
detectably labeled. Such probe may be a nucleic acid sequence
specific for a GSTP1 promoter region. For example, oligonucleotide
probes of the invention can be included in a kit and used for
examining the presence of hypermethylated nucleic acid sequences in
a sample containing a GST nucleic acid sequence. The kit may also
contain a container comprising a reporter-means, such as an
enzymatic, fluorescent, or radionucleotide label to identify the
detectably labeled oligonucleotide probe.
[0114] 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. When it is desirable to amplify the mutant target
sequence, this can be accomplished using oligonucleotide(s) that
are primers for amplification. These oligonucleotide primers are
based upon identification of the flanking regions contiguous with
the target nucleotide sequence. Accordingly, the kit may contain
primers useful to amplify and screening a promoter region of a GST
(e.g., the pomoter region of GSTP1). Such primers include, for
example, SEQ ID Nos.: 1, 2, 7, 8, 9, 10, 11, 12, 13 and
combinations thereof. The invention will now be described in
greater detail by reference to the following non-limiting
examples.
EXAMPLES
[0115] Hep3B HCC cells and HCC tissue specimens. Human Hep3B HCC
cells (Aden D P. et al., Nature 282:615-616, 1979) were propagated
in vitro in MEM growth medium (Mediatech) supplemented with 1.0 mM
sodium pyruvate and 10% fetal calf serum (Gibco-BRL Life
Technologies). Human Tsu-Pr1 PCA cells (Iizumi et al., J Urol
137:1304-1306, 1987) were cultivated in RPMI 1640 medium
(Mediatech) with 10% fetal calf serum. Treatment of Hep3B and
Tsu-Pr1 cells with the DNA methyltransferase inhibitor
5-aza-deoxycytidine (5-aza-dC; Sigma Chemical Company) was
accomplished by incubation of growing cell cultures in complete
growth medium containing the inhibitor. Human HCC tissue specimens
were recovered from partial liver resection procedures for HCC
performed at the National Cancer Center Hospital in Tokyo, Japan,
with the approval of the institutional ethics committee. The
specimens used for study were residual materials present after
appropriate pathological diagnostic evaluations were completed.
Clinical data for HCC cases, including serum studies for hepatitis
virus infection, were abstracted from case records. Genomic DNA was
isolated from Hep3B HCC cells, from HCC tissues and adjacent
non-cancerous tissues, and from normal white blood cells as
described in Lee et al., Cancer Epidemiol Biomarkers Prev
6:443-450, 1997; and Lee et al., Proc Natl Acad Sci USA
91:11733-11737, 1994.
Example 1
[0116] Assessment of GSTP1 expression. To detect GSTP1 polypeptides
in HCC tissues, formalin-fixed, paraffin-embedded HCC tissue
specimens were cut into tissue sections, deparaffinized, hydrated,
and stained for the presence of GSTP1 polypeptides with specific
rabbit antiserum (Oncor) using an immunoperoxidase technique
(Vector Laboratories). For human cancer cell lines propagated in
vitro, the expression of GSTP1 mRNA and GSTP1 polypeptides were
assessed using Northern blot and immunoblot analyses.
[0117] Increased expression of the rat .pi.-class GST, GST-P,
stereotypically accompanies HCC pathogenesis in rat chemical
carcinogenesis models. In contrast, human HCC specimens contain
neoplastic cells apparently devoid of the human .pi.-class GST,
GSTP1. To determine whether diminished or absent GSTP1 expression
by human HCC cells might be the result of somatic alterations
affecting the GSTP1 gene, human Hep3B HCC cells propagated in vitro
were assessed for GSTP1 polypeptide expression by immununoblot
analysis using anti-GSTP1 antiserum and for GSTP1 mRNA expression
by Northern blot analysis using a GSTP1 cDNA probe (FIG. 1B). Hep3B
HCC cells failed to express either GSTP1 polypeptides or GSTP1 mRNA
(FIG. 1). LNCaP PCA cells and MCF-7 breast carcinoma (BCA) cells,
which also fail to express GSTP1 polypeptides, contain GSTP1 genes
with abnormally hypermethylated "CpG islands". In contrast, "CpG
island" sequences in GSTP1 genes present in a variety of normal
tissues, including normal liver, are characteristically not
hypermethylated, regardless of whether cells comprising the normal
tissues express GSTP1 or not. By Southern blot analysis of Hep3B
HCC DNA digested with the .sup.5-mC-sensitive restriction
endonuclease BssHII, abnormal GSTP1 "CpG island" hypermethylation
was detected (FIG. 1A) reminiscent of the abnormal GSTP1 DNA
hypermethylation present in LNCaP PCA cell DNA and in MCF-7 BCA
cell DNA. In fact, all GSTP1 promoter alleles present in Hep3B HCC
cells manifested abnormal DNA hypermethylation (FIG. 1A).
[0118] To ascertain whether the GSTP1 "CpG island" hypermethylation
changes present in Hep3B HCC cell DNA were associated with
transcriptional silencing of the GSTP1 gene, Hep3B HCC cells were
exposed to 5-aza-dC, an inhibitor of DNA methyltransferases
(DNMTs), for as long as 72 hours. Experiments demonstrated that
GSTP1 mRNA expression by the Hep3B cells began to appear within 24
hours, but increased in abundance by 72 hours. As expected, after
72 hours, Southern blot analysis of DNA from 5-aza-dC-treated Hep3B
cells disclosed the appearance of unmethylated GSTP1 promoter
alleles (FIG. 1A). In addition, Northern blot analysis of RNA from
Hep3B cells treated with 5-aza-dC for 72 hours revealed a
reactivation of GSTP1 mRNA expression (FIG. 1B). Although the
applicants are under no duty or obligation to explain the mechanism
by which the invention works it is suspected that 5-aza-dC
treatment most likely triggered GSTP1 expression in Hep3B HCC cells
by undermining the maintenance of the abnormal GSTP1 "CpG island"
methylation changes. When Tsu-Pr1 PCA cells, which contain
unmethylated GSTP1 promoter alleles and express abundant GSTP1
mRNA, were treated with 5-aza-dC in a manner similar to that used
to reactivate GSTP1 expression in Hep3B HCC cells, no modulation of
GSTP1 expression was evident. These data indicated that GSTP1 "CpG
island" hypermethylation changes present in Hep3B HCC cells result
in an absence of GSTP1 expression.
Example 2
[0119] Detection of somatic GSTP1 "CpG island" DNA hypermethylation
changes. DNA isolated from Hep3B cells before and after exposure to
5-aza-dC in vitro was assessed for GSTP1 "CpG island" DNA
hypermethylation using Southern blot analysis. Purified DNAs were
digested first with HindIII and EcoRI and then extensively with the
.sup.5-mC-sensitive restriction endonuclease BssHII. Digested DNAs
were then electrophoresed on agarose gels, transferred to
Zeta-Probe (BioRad) membranes, and then hybridized with
.sup.32P-labeled GSTP1 cDNA. DNA isolated from Hep3B HCC cells and
from HCC and adjacent non-neoplastic tissues was also subjected to
analysis for GSTP1 "CpG island" DNA hypermethylation using a
.sup.5-mC-sensitive restriction endonuclease-polymerase chain
reaction (PCR) strategy that permitted both detection of CpG
dinucleotide methylation and simultaneous discrimination of
maternal and paternal alleles in informative cases. PCR primers
(upstream primer, 5'-AGCCTGGGCCACAGCGTGAGACTACGT-3' (SEQ ID NO:1);
downstream primer, 5'-GGAGTAAACAGACAGCAGGAAGAGGAC-3' (SEQ ID NO:2))
targeting a sequence (approximately -539 to -239 bp from the
transcription start site; see GenBank accession #X08058 (which is
incorporated herein by reference in its entirety) in the 5'
regulatory region of GSTP1, which included a polymorphic
(ATAAA).sub.n (SEQ ID NO:3) repeat sequence and two recognition
sites for the .sup.5-mC-sensitive restriction endonuclease HpaII
and its isoschizomer MspI, were used to amplify DNA samples that
had been left undigested, or had been extensively digested with
HpaII or MspI. A previous report suggested that among genomic DNA
from healthy Japanese, (ATAAA).sub.n (SEQ ID NO:3) sequences of
between 18-27 repeats in the 5' regulatory region of GSTP1 could be
detected, with 23 (27.2%) and 24 (24.1%) repeats representing the
most common GSTP1 alleles (Harada S et al., Hum Genet 93:223-224,
1994). In all, some 81.8% of healthy Japanese were found to be
polymorphic for GSTP1 (ATAAA).sub.n repeats. For the GSTP1
allele-specific "CpG island" methylation assay, DNA isolated from
HCC and adjacent non-HCC tissues were left undigested, or were
extensively digested with HpaII or MspI before being subjected to
PCR. The 25 .mu.l PCR mixture contained 20-100 ng sample DNA, 1.25
units Taq polymerase (Perkin-Elmer Corporation), 1 .mu.M of each
oligonucleotide primer, 200 .mu.M deoxynucleotide triphosphates,
and 15% glycerol in OptiPrime buffer #10 (Stratagene). To
facilitate detection of the PCR amplification products, the
downstream primer was end-labeled with [.gamma.-.sup.32P]ATP
(Amersham) using T4 polynucleotide kinase (New England BioLabs).
PCR was conducted by incubation at 95.degree. C. for 1 min,
63.degree. C. for 3 min, and 72.degree. C. for 1.5 min, for 30
cycles followed by a final extension at 72.degree. C. for 8 min.
PCR amplification products, ranging in size from 290 bp to 335 bp,
were then subjected to electrophoresis on 6% polyacrylamide DNA
sequencing gels containing 8 M urea at 60 W for 2.5 h. Gels were
subsequently mounted on filter paper (Whatman), dried, and then
exposed to X-OMAT film (Kodak).
Example 3
[0120] Analysis of GSTP1 expression and GSTP1 "CpG island" DNA
hypermethylation in human HCC cells in vivo. To discover whether
GSTP1 "CpG island" hypermethylation changes were responsible for
absence of GSTP1 expression in human HCC cells in vivo, a series of
20 HCC tissue specimens were subjected to analysis for GSTP1
polypeptide expression by immunohistochemical staining using
anti-GSTP1 antiserum and to analysis for GSTP1 "CpG island" DNA
hypermethylation. In each tissue specimen, liver tissue adjacent to
HCC lesions displayed characteristic morphological changes of
hepatitis and cirrhosis. Although bile duct cells in normal liver
tissues tend to express abundant GSTP1, normal hepatocytes
generally fail to express GSTP1. In the HCC case series, even
though the liver tissues adjacent to HCC displayed morphological
changes of hepatitis and cirrhosis, the bile duct cells stained
positively for GSTP1 while the hepatocytes failed to stain
positively. In 19 of 20 HCC cases examined, HCC cells appeared
devoid of GSTP1 expression (Table I). One of the HCC cases (case 1)
appeared to contain rare cells which stained positively for GSTP1
amongst a larger number (>95%) of HCC cells which stained
negatively for GSTP1.
1TABLE I pGSTP1 expression and GSTP1 "CG island" methylation in 20
HCC cases. case anti-GSTP1 GSTP1 "CG island" number age sex
immunostaining.sup.a DNA methylation.sup.b 1 49 F negative/rare
positive.sup.c 2 of 2 alleles 2 61 M negative 2 of 2 alleles 3 61 M
negative 1 of 2 alleles 4 59 M negative yes 5 65 M negative 2 of 2
alleles 6 62 M negative 2 of 2 alleles 7 60 M negative 2 of 2
alleles 8 71 M negative 1 of 2 alleles 9 59 M negative no 10 64 M
negative yes 11 44 M negative no.sup.d 12 60 M negative 1 of 2
alleles 13 73 F negative 1 of 2 alleles 14 69 M negative 2 of 2
alleles 15 72 M negative 1 of 2 alleles 16 68 F negative yes 17 68
M negative 2 of 2 alleles 18 72 M negative 2 of 2 alleles 19 49 M
negative no 20 24 F negative yes .sup.aImmunohistochemical staining
for GSTP1 polypeptides was accomplished as described for FIG. 1.
.sup.bThe presence of GSTP1 "CpG island" methylation was assayed
using PCR as described for FIG. 2. For cases 4, 10, 16, and 20,
only one GSTP1 allele could be discriminated. Among cases with two
GSTP1 alleles discriminated, no allelic losses were evident.
.sup.cFor case 1, GSTP1 polypeptides were detected in a small
fraction of the cells (<5%) comprising the HCC. .sup.dFor case
11, GSTP1 "CpG island" DNA methylation was not detected HCC DNA,
but was detected in 1 of 2 alleles in DNA from adjacent liver
tissue. GSTP1 "CpG island" DNA methylation was not evident in DNA
from liver tissue adjacent to HCC for any of the other cases.
[0121] To determine whether the absence of GSTP1 polypeptide
expression in HCC cells in vivo might be accompanied by somatic
GSTP1 "CpG island" hypermethylation, the methylation status of CpG
dinucleotides present in HpaII/MspI sites located at -343 bp and
-301 bp upstream of the transcription start site of GSTP1 in DNA
from the 20 HCC specimens were surveyed using a PCR approach
targeting a region (from -535 bp to -239 bp) encompassing a
polymorphic (ATAAA).sub.n repeat sequence. In informative cases,
this assay allowed detection of GSTP1 "CpG island" hypermethylation
affecting both the maternal and paternal alleles. Representative
results of the application of this assay to the analysis of somatic
GSTP1 DNA hypermethylation are displayed in FIG. 2. The appearance
of PCR products following HpaII digestion of HCC DNA, but not of
DNA from adjacent non-cancerous tissues, indicated that somatic
GSTP1 "CpG island" DNA hypermethylation changes were likely present
(FIG. 2). The absence of PCR products following MspI digestion of
HCC DNA provided further evidence that the somatic alterations
likely involved CpG dinucleotide hypermethylation at the HpaII/MspI
recognition sites and not mutation. The results obtained from
analysis of each of the 20 HCC cases studied are presented in Table
I. None of the HCC cases for which maternal and paternal GSTP1
alleles could be discriminated showed loss of GSTP1 alleles. In one
case (case 11), non-cancerous liver tissue DNA appeared to manifest
CpG dinucleotide hypermethylation at the two HpaII/MspI sites in
the GSTP1 "CpG island" targeted by the PCR assay used. DNA from 17
of 20 HCC specimens (85%) showed somatic hypermethylation changes
present in at least 1 GSTP1 allele. DNA from 8 HCC specimens
contained somatic hypermethylation changes in both paternal and
maternal GSTP1 alleles (8 of 17 informative cases or 47%); DNA from
6 HCC specimens appeared to contain abnormal hypermethylation
affecting one GSTP1 allele but not the other (6 of 17 informative
cases or 35%).
Example 4
[0122] HBV DNA detection. HBV DNA (HBV complete genome; GenBank
accession #X98077) was detected as described in Zhou et al., Cancer
Res 57:2749-2753, 1997. PCR primers (upstream primer, position
3073-3089, 5'-GGGTGGAGCCCTCAGGCTCAGGGC-3' (SEQ ID NO:4); downstream
primer, position 410-433, 5'-GAAGATGAGGCATAGCAGAC GGAT-3' (SEQ ID
NO:5)) were used to amplify HBV DNA sequences in reaction mixtures
containing 50 ng sample DNA, 1.25 units Taq polymerase, 1 .mu.M
each primer, 250 .mu.M deoxynucleotide triphosphates. After
initially heating the reaction mixtures to 95.degree. C. for 5 min,
PCR was conducted by incubation at 94.degree. C. for 30 sec,
53.degree. C. 35 sec, and 72.degree. C. for 65 sec, 30 cycles. PCR
products were then separated by electrophoresis on 1% agarose gels,
transferred to Zeta-Probe (Biorad) membranes, and hybridized with
.sup.32P-end-labeled oligonucleotide HBV DNA probes
2 (SEQ ID NO:6)) (position 54-69, 5'-TTCCTGCTGGTGGCTC-3'.
[0123] Zhou et al. reported that GST expression was significantly
reduced in HCC cells when HBV DNA was present. Serum studies for
HBV and HCV infection for each of the HCC cases are summarized in
Table II. For all but one of the cases (case 6), serum studies
indicated a history of HBV infection (12 cases), of HCV infection
(11 cases), or of infection with both HBV and HCV (4 cases). Of
cases with a history of HBV infection, HCC DNA displayed evidence
of GSTP1 CG island methylation in 9 of 12 cases (75%). For cases
with a history of HCV infection, GSTP1 CG island DNA methylation
was detected in HCC DNA in 10 of 11 cases (91%). (To discern
whether active HBV infection might contribute to the absence
ofGSTPI expression in the 20 HCC cases examined in the study, HCC
DNA and matched DNA from adjacent liver tissues was subjected to
analysis for HBV infection using PCR technique. Representative
results are shown in FIG. 3. Hep3B HCC cells are known to harbor
HBV DNA. Thus, as expected, HBV DNA was readily detected by PCR as
a single 572 bp product in Hep3B HCC DNA (FIG. 3, lane 1). HBV DNA
was not detected in either HCC DNA or DNA from adjacent liver
tissue in one representative HCC case (case 7; FIG. 3, lanes 2 and
3). HBV DNA was clearly present in DNA from both HCC and adjacent
tissue in another representative HCC case (case 8; FIG. 3, lanes 4
and 5). Assay results for each of the 20 HCC cases are summarized
in Table 2. 10 of 20 HCC cases had detectable HBV DNA among genomic
DNA from either HCC tissue or from adjacent tissue. HBV DNA was
occasionally detected in DNA from HCC tissue but not in DNA from
adjacent tissue (cases 6 and 9) or vice versa (cases 4 and 13). HBV
infection did not appear to cause GSTP1 "CpG island"
hypermethylation. Abnormal GSTP1 promoter DNA hypermethylation was
present in HCC DNA in 7 of 10 cases (70%) in which HBV DNA was
present and in 10 of 10 cases (100%) in which HBV DNA was not
detected.
3TABLE II Hepatits virus infection in the 20 HCC cases. case number
HBsAg HBsAb HBcAb HBeAg HBeAb HCVAb HBV DNAa 1 + - + - + - + (HCC
and adj. tissue) 2 - - - - - + - 3 - - + - + + - 4 - - + - + - +
(adj. tissue only) 5 - - - - 6 - - + + (HCC only) 7 - - - - - + - 8
+ - + - + - + (HCC and adj. tissue) 9 - + + - + + + (HCC only) 10 -
- - - - + - 11 - + + - + - + (HCC and adj. tissue) 12 - - - - - + -
13 + - - - + - + (adj. tissue only) 14 - - + - + + - 15 - - + - + -
- 16 + - + - + - + (HCC and adj. tissue) 17 - - - - + - 18 - - + -
19 - - + - + - + (HCC and adj. tissue) 20 + - + - + - + (HCC and
adj. tissue) .sup.aThe presence of HB virus DNA in DNA isolated
from HCC tissue and adjacent liver tissue was assayed by PCR as
described for FIG. 2.
Example 5
[0124] Mapping of somatic GSTP1 "CpG Island" DNA hyperethylation
changes by genomic sequencing after bisulfite treatment. For 12 of
the HCC cases, sufficient genomic DNA was available from HCC
tissues and from tissues adjacent to HCC to permit an attempt at
fine mapping of GSTP1 "CpG island" DNA hypermethylation changes.
The use of the bisulfite reaction and PCR to discriminate .sup.5-mC
from C in genomic DNA was described by Clark et al. (Nucleic Acids
Res 22:2990-2997, 1994). To map .sup.5-mC nucleotides in the GSTP1
gene promoter in HCC DNA, the procedure described by Clark et al.
was employed with only minor modifications. 200 ng of genomic DNA
isolated from normal and neoplastic liver cells and tissues were
treated with EcoRI, mixed with 2 .mu.g salmon sperm DNA (Sigma
Chemical Company), and then treated with sodium bisulfite.
Bisulfite-treated DNA was then subjected to 2 rounds of PCR. The
first PCR reaction mixtures contained 100 ng of bisulfite-treated
DNA, 1 .mu.M of primers, 250 .mu.M of deoxynucleotide
triphosphates, and 1.25 units Taq polymerase in OptiPrime buffer #1
(Stratagene). To selectively amplify GSTP1 promoter DNA containing
.sup.5-mC in the "sense" strand, primers N-F1 (GenBank position
816-835, 5'-GTAATTTTTTTTTTTT TAAG-3' (SEQ ID NO:7)) and M-R1
(position 1405-1420, 5'-TAAAAACCGCTAACGA-3' (SEQ ID NO:8)) were
included in the PCR reaction mixture, to amplify GSTP1 promoter DNA
containing C in the "sense" strand, primers N-F1 and U-R1 (position
1406-1422 5'-CCTAAAAACCACTAACA-3' (SEQ ID NO:9)) were used. After
heating to 94.degree. C. for 2 min, PCR was conducted by incubation
at 94.degree. C. for 1 min, 44.degree. C. for 2 min, and 72.degree.
C. for 3 min for 5 cycles, followed by incubation at 94.degree. C.
for 30 sec, 44.degree. C. for 2 min, and 72.degree. C. for 1.5 min
for 25 cycles before a final extension at 72.degree. C. for 6 min.
Products from the first PCR reaction mixtures were subjected to a
second round of "nested" PCR. The second PCR reaction mixtures
contained 1 .mu.M of primers, 250 .mu.M of deoxynucleotide
triphosphates, and 1.25 units Taq polymerase in OptiPrime buffer #6
(Stratagene). To amplify GSTP1 promoter DNA containing .sup.5-mC,
primers M-F2 (position 897-918, 5'-TTTTAGGGAATTTTTTTTCGCG-3' (SEQ
ID NO:10)) and M-R2 (position 1327-1345, 5'-CCCTACCGA AAACCCGAAC-3'
(SEQ ID NO:11)) were added to PCR reaction mixture; to amplify
GSTP1 promoter DNA containing C, primers U-F2 (position 895-917,
5'-GGTTTTAGGGAATTTTTTTTTGT-3' (SEQ ID NO:12)) and U-R2 (position,
1326-1346, 5'-ACCCTACCAAAAACCCAAAC-3' (SEQ ID NO:13)) were used.
Following heating to 94.degree. C. for 3 min, PCR was conducted by
incubation at 94.degree. C. for 30 sec, 58.degree. C. for 2 min,
and 72.degree. C. for 1.5 min for 30 cycles with a final extension
at 72.degree. C. for 6 min. To permit DNA sequencing, PCR products
were purified by separation using low melting temperature agarose
gel electrophoresis, isolated from the agarose (using a QIAquick
gel extraction kit; Qiagen), and then recovered by ethanol
precipitation with linear acrylamide (Ambion) as a carrier.
Purified PCR products were subjected to direct DNA sequence
analysis using a cycle sequencing approach with dye-labeled
terminators (ABI PRISM.TM. Dye Terminator Cycle Sequencing Ready
Reaction Kit, Perkin Elmer). DNA sequence ladders were analyzed
using an ABI automated sequencer. The forward sequencing primer
used was (position 1005-1021) 5'-TGGGAAAGAGGGAAAGG-3' (SEQ ID
NO:14). The reverse sequencing primer used was (position 1280-1295)
5'-CTCTAAACCCCATCCC-3' (SEQ ID NO:15).
[0125] Although diminished or absent GSTP1 polypeptide expression
was found for nearly all of HCC cases surveyed in the study, HCC
DNA from only 50% of informative cases (8 of 16) displayed CpG
dinucleotide hypermethylation at both maternal and paternal GSTP1
alleles at HpaII/MspI sites located -343 bp and -301 bp upstream of
the GSTP1 transcription start site (see Table I). The other cases
likely had GSTP1 alleles displaying hypermethylation at CpG
dinucleotides at other sites. To ascertain whether different
patterns of GSTP1 "CpG island" DNA hypermethylation changes might
be present in different HCC cases, genomic sequencing analyses of
bisulfite-treated DNA specimens were performed on DNA from Hep3B
HCC cells and 13 HCC cases. The genomic sequencing strategy
employed involved bisulfite treatment of genomic DNA followed by 2
rounds of PCR. For PCR amplifications, oligonucleotide primers
specific for either the bisulfite reaction products of methylated
GSTP1 target DNA sequences or for the bisulfite reaction products
of unmethylated sequences were used. GSTP1 "DNA
methylation-specific" primers generated PCR products using DNA from
Hep3B HCC cells and from 9 of 12 (75%) HCC specimens (see FIGS. 4
and 5). No PCR products were generated using DNA from normal white
blood cells. Examination of the distribution of methylated CpG
dinucleotides throughout the GSTP1 promoter region in Hep3B cells
indicated that all CpG dinucleotides located between -195 bp and
+35 bp of the transcription start site contained .sup.5-mC. DNA
from the HCC cases exhibited a significant heterogeneity of CpG
dinucleotide methylation patterns. Unfortunately, unlike the GSTP1
"CpG island" methylation assay featured in FIG. 2 and Table 1, the
GSTP1 "CpG island" bisulfite genomic sequencing strategy used in
FIGS. 4 and 5 did not permit selective assessment of CpG
dinucleotide methylation patterns on maternal and paternal alleles.
Rather, the GSTP1 "CpG island" bisulfite genomic sequencing assay,
which subjected GSTP1 "CpG island" PCR products to direct DNA
sequence analysis, was biased to detect the most prevalent CpG
dinucleotide patterns in each DNA specimen. Nonetheless, more
extensive CpG dinucleotide hypermethylation was present in HCC DNA
than in DNA from adjacent liver tissues (FIG. 5). Of the 9 HCC DNA
specimens that generated "DNA methylation-specific" PCR products,
methylation of greater than 50% of the CpG dinucleotides between
-195 bp and +35 bp of the transcription start site were seen in 7
HCC cases. Methylated CpG dinucleotides present in the region -80
bp to +35 bp containing the core transcriptional promoter for GSTP1
were evident in each of the 9 (100%) HCC cases. In contrast,
methylated CpG dinucleotides located at -140 bp and at -100 bp were
detected in only 3 of 9 (33%) HCC cases. GSTP1 "DNA
methylation-specific" PCR products were also detected in DNA
isolated from tissues adjacent to HCC tissue in 3 of 11 (27%) HCC
cases (see FIGS. 4 and 5). For one case (case 15), the pattern of
CpG dinucleotide hypermethylation in the GSTP1 regulatory region in
DNA from tissue adjacent to HCC resembled the CpG dinucleotide
methylation pattern discerned for DNA from HCC tissue. These CpG
dinucleotide hypermethylation changes in the DNA from adjacent
tissue may have been present in non-neoplastic hepatocytes, or may
have been present in HCC cells infiltrating the adjacent tissue. In
another case (case 10), the GSTP1 regulatory region CpG
dinucleotide hypermethylation patterns in HCC DNA and in adjacent
tissue DNA were substantially different, suggesting that
significant CpG dinucleotide hypermethylation changes were likely
present in non-neoplastic hepatocytes. Of interest, in this case
each of the CpG dinucleotide hypermethylation changes in DNA from
tissue adjacent to HCC was also present in HCC DNA; however, the
HCC DNA exhibited more extensive CpG hypermethylation changes. In a
third case (case 11), extensive GSTP1 "CpG island" hypermethylation
changes were detected in DNA from non-neoplastic tissue but not in
DNA from the adjacent HCC tissue.
[0126] PCR primers specific for unmethylated GSTP1 target sequences
generated PCR products using DNA from normal white blood cells and
from each of the HCC specimens (see FIGS. 4 and 5). No PCR products
were generated using DNA from Hep3B HCC cells. The distribution of
methylated CpG dinucleotides in the GSTP1 promoter region in normal
white blood cells appeared restricted to a single CpG dinucleotide
located at -15 bp from the transcriptional start site (FIG. 5).
Similar CpG dinucleotide methylation patterns were detected in 4 of
11(36%) DNA specimens prepared from tissues adjacent to HCC (FIG.
5). For the remaining 7 of 11 (64%) cases, DNA isolated from
tissues adjacent to HCC displayed abnormal CpG methylation
patterns. PCR products generated from HCC DNA also displayed
abnormal CpG dinucleotide methylation patterns in the GSTP1
regulatory region in 5 of 11 cases (46%; FIG. 5). For one such case
(case 18), the abnormal CpG dinucleotide methylation patterns
discriminated using PCR primers specific for methylated target
sequences versus unmethylated target sequences were different (FIG.
4). Analysis of HCC DNA from this case using the HpaII-PCR assay
capable of monitoring DNA hypermethylation in both maternal and
paternal alleles had suggested that both alleles carried somatic
DNA hypermethylation changes affecting GSTP1 regulatory region
(Table I). The two CpG dinucleotide hypermethylation patterns
discriminated using genomic sequence analysis of bisulfite-treated
DNA are likely reflective of different somatic hypermethylation
changes present in maternal versus paternal GSTP1 promoter
alleles.
[0127] The genomic sequence analyses undertaken following bisulfite
treatment of DNA from normal white blood cells, from Hep3B HCC
cells, from HCC tissues, and from tissues adjacent to HCC,
disclosed the presence of .sup.5-mCCG and .sup.5-mC.sup.5-mCG
trinucleotides at some sites in the GSTP1 regulatory region in
addition to .sup.5-mCG dinucleotides. .sup.5-mC.sup.5-mCG
trinucleotides were present at -16 of the transcriptional start
site in almost all normal and neoplastic DNA specimens examined.
.sup.5-mC.sup.5-mCG trinucleotides were present at -148 bp and -77
bp of the transcription start site in Hep3B DNA and in 5 of 9 (56%)
and 3 of 9 (33%) HCC cases, respectively, but were absent from
normal white blood cell DNA and from DNA isolated from tissues
adjacent to HCC in 10 of 12 cases (83%) evaluable. No 5-m CAG or
5-mCTG trinucleotides were detected in any of the DNA specimens
studied.
[0128] For a somatic genome alteration to target a critical gene
for cancer pathogenesis, the alteration must be heritable through
mitosis and affect gene function in a manner that permits cells
containing the alteration to enjoy a selective growth advantage.
CpG dinucleotide methylation patterns can be maintained through
mitosis via DNA-MT action, and "CpG island" hypermethylation
stereotypically affects gene function by preventing gene
transcription. In general, in the absence of"CpG island"
hypermethylation, genes may be transcribed or not transcribed
subject to trans regulatory effects. In the presence of"CpG island"
hypermethylation, genes can not be transcribed independent of trans
regulatory influences. The invention demonstrates an absence of
GSTP1 polypeptides in HCC cells in nearly all HCC cases evaluated.
For many of the HCC cases analyzed, GSTP1 "CpG island"
hypermethylation changes appeared to be present at both GSTP1
alleles, perhaps resulting in an absence of inducible GSTP1
activity.
[0129] Hep3B HCC cells propagated in vitro failed to express GSTP1
mRNA and contained only hypermethylated GSTP1 promoter alleles.
When HCC cells were treated with the DNMT inhibitor 5-aza-dC, the
appearance of unmethylated GSTP1 promoter alleles was accompanied
by the appearance of detectable GSTP1 mRNA. This result
demonstrates that the GSTP1 "CpG island" hypermethylation changes
were associated with GSTP1 silencing in Hep3B HCC cells in vitro
and that similar GSTP1 DNA hypermethylation changes are associated
with GSTP1 silencing in HCC cells in vivo. Studies of DNA
methylation effects on the transcriptional regulation of several
different genes have identified direct promoter silencing effects,
resulting from interference of transcription factor binding to cis
regulatory sequences (Watt F and Molloy P L, Genes Dev 2:1136-1143,
1988; Bednarik D P et al., New Biol 3: 969-976, 1991; Comb M and
Goodman H M, Nucleic Acids Res 18:3975-3982, 1990; Singal R et al.,
Proc Natl Acad Sci USA 94:13724-13729, 1997; Prendergast G C et
al., Cell 65:395-407, 1991; Prendergast G C and Ziff E B, Science
251:186-189, 1991), and indirect promoter repression effects,
mediated through .sup.5-mC binding proteins (Keshet I et al., Cell
44:535-543, 1986; Meehan R R et al., Cell 58: 499-507, 1989; Boyes
J and Bird A, Cell 64: 1123-1134, 1991; Boyes J and Bird A, Embo J
11: 327-333, 1992; Meehan R R et al., Nucleic Acids Res
20:5085-5092, 1992; Lewis J D et al., Cell 69: 905-914, 1992; Nan X
et al., Cell 88:471-481, 1997; Kudo S, Mol Cell Biol 18:5492-5499,
1998; Nan X, Nature 393: 386-389, 1998; Jones P L et al., Nat Genet
19:187-191, 1998). Indirect promoter repression effects have been
reported to depend both on the density of CpG dinucleotide
methylation and on promoter strength. Preliminary results of an
analysis of the consequences of CpG dinucleotide methylation on
GSTP1 promoter activity in MCF-7 BCA cells in vitro have indicated
that direct promoter silencing effects may be sufficient for
transcriptional inhibition (unpublished data). Whether similar
mechanisms contribute to GSTP1 gene silencing in HCC cells has not
been tested. Nonetheless, genome sequencing analyses of "CpG
island" methylation changes in DNA from Hep3B HCC cells in vitro
and from HCC tissues in vivo revealed fairly consistent CpG
dinucleotide hypermethylation changes directly affecting the core
GSTP1 promoter, despite a general heterogeneity in CpG dinucleotide
hypermethylation changes throughout the GSTP1 "CpG island."
Detailed promoter analyses may prove necessary to ascertain whether
GSTP1 "CpG island" hypermethylation changes of the kind present in
HCC tissues in vivo result in transcriptional inactivation via a
direct or via an indirect promoter silencing mechanism.
[0130] GSTP1 "CpG island" hypermethylation, the most somatic common
genome alteration yet reported in human PCA cells, appears to
result in a crippling of inducible enzyme defenses against oxidant
and electrophilic carcinogens. GSTP1 DNA hypermethylation changes
have also been detected in the majority of PIN lesions thought to
represent PCA precursors. These findings have formed the basis for
a new model of prostatic carcinogenesis, in which prostatic cells
with defective GSTP1 genes become vulnerable to oxidants and
electrophiles tending to promote neoplastic transformation and PCA
cells with defective GSTP1 genes remain vulnerable to similar
stresses tending to promote malignant. If the pathogenesis of human
HCC proceeds via a similar mechanism, a new model for human
hepatocarcinogenesis can be proposed. In this model, normal
hepatocytes do not express GSTP1, but when cells are exposed to
electrophilic or oxidant carcinogens, GSTP1 expression can be
induced as a defense against genome damage. Hepatocytes that
contain inactivated GSTP1 genes will be incapable of GSTP1
induction and will become vulnerable to genome damage inflicted by
carcinogen exposure. Hepatocytes that acquire alterations in
critical genes will be prone to undergo neoplastic transformation
and tumor formation.
[0131] Our new model for human HCC pathogenesis can also be
considered in comparison to previous mechanistic models and
previous observations derived from studies of chemical
hepatocarcinogenesis in rodents and other species. The hypothesis
that a somatic deficiency in inducible GSTP1 activity in some human
hepatocytes may confer carcinogen sensitivity is most reminiscent
of hepatocarcinogenesis in different fish species, where liver
tumor development may be facilitated by inadequate GST expression
(Hayes MA et al., Sci Total Environ 94:105-123, 1990; Kirby GM et
al., Carcinogenesis 11: 2255-2257, 1990). In addition, a possible
reasons for the differences between GST-P expression during
hepatocarcinogenesis in rodents and GSTP1 expression during
hepatocarcinogenesis in humans may be (i) that hepatitis virus
exposure and cirrhosis, which may be associated with diminished GST
expression, constitute major etiological factors in human HCC
development in the case series provided here while chemical
carcinogen exposure constitutes the major etiological factor in HCC
development in the various rodent model systems, (ii) that human
GSTP1 gene may be regulated differently in hepatocytes than the
rodent GST-P gene, and (iii) that the human GSTP1 gene may be more
prone to suffer somatic de novo "CpG island" DNA hypermethylation
during hepatocarcinogenesis than the rodent GST-P gene. In fact, a
recent report suggests that hypomethylation at the GST-P gene may
be more characteristic of rodent hepatocarcinogenesis (Steinmetz K
L et al., Carcinogenesis 19:1487-1494, 1998). Thus, although a
dysregulation of the fidelity of DNA methylation pattern
maintenance may be characteristic of both human and rodent
hepatocarcinogenesis, the resultant DNA methylation pattern changes
appear distinct, complex, and likely affected by different
selection pressures.
[0132] The findings of somatic GSTP1 defects associated with the
pathogenesis of human HCC has significant implications both for the
diagnosis and staging of HCC. PCR-based strategies targeting GSTP1
"CpG island" DNA hypermethylation changes, such as the assays
presented in above or other assays useful for the detection of
other cancer cells with similar GSTP1 "CpG island" hypermethylation
changes, can be used to detect HCC DNA in liver biopsy tissues and
in serum or plasma (78). GSTP1 "CpG island" hypermethylation
detection provides a valuable molecular biomarker for HCC with
clinical applications. In the present study, abnormal GSTP1 "CpG
island" hypermethylation was detected in DNA from the majority of
HCC cases regardless of the assay used (17 of 20 cases or 85% for
the HpaII-PCR assay (see Table I) and a total of 10 of 12 cases or
83% for the bisulfite genomic sequencing assay (see FIG. 5)). HCC
most commonly arises in the setting of chronic hepatitis and
cirrhosis. Thus, the one strategy for detecting GSTP1 "CpG island"
hypermethylation as a biomarker for HCC DNA will be an assay
targeting the specific region of the GSTP1 "CpG island" most
selectively hypermethylated in HCC DNA relative to DNA from liver
tissue displaying hepatitis and cirrhosis.
[0133] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
15 1 27 DNA Artificial sequence upstream primer targeting a piece
of GenBank #X08058 1 agcctgggcc acagcgtgag actacgt 27 2 27 DNA
Artificial sequence downstream primer targeting a piece of GenBank
#X08058 2 ggagtaaaca gacagcagga agaggac 27 3 5 DNA Artificial
sequence 5' regulatory region of GSTP1, polymorphic repeat sequence
3 ataaa 5 4 24 DNA Artificial sequence upstream primer for piece of
GenBank # X98077 4 gggtggagcc ctcaggctca gggc 24 5 24 DNA
Artificial sequence downstream primer for piece of GenBank # X98077
5 gaagatgagg catagcagac ggat 24 6 16 DNA Artificial sequence HBV
DNA probes 6 ttcctgctgg tggctc 16 7 20 DNA Artificial sequence
primer N-F1 7 gtaatttttt tttttttaag 20 8 16 DNA Artificial sequence
Primer M-R1 8 taaaaaccgc taacga 16 9 17 DNA Artificial sequence
Primer U-R1 9 cctaaaaacc actaaca 17 10 22 DNA Artificial sequence
Primer M-F2 10 ttttagggaa ttttttttcg cg 22 11 19 DNA Artificial
sequence Primer M-R2 11 ccctaccgaa aacccgaac 19 12 23 DNA
Artificial sequence Primer U-F2 12 ggttttaggg aatttttttt tgt 23 13
20 DNA Artificial sequence Primer U-R2 13 accctaccaa aaacccaaac 20
14 17 DNA Artificial sequence forward sequencing primer 14
tgggaaagag ggaaagg 17 15 16 DNA Artificial sequence reverse
sequencing primer 15 ctctaaaccc catccc 16
* * * * *