Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-12
2004-03-23
Benzion, Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06709818
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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.
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.
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.
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.
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′ CpG island.
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
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.
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 n family GST (e.g., GSTP
1
).
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 norm
Bakker Jila
Lin Xiaohui
Nelson William G.
Tchou Julia C.
Benzion Gary
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Souaya Jehanne E
The Johns Hopkins University School of Medicine
LandOfFree
Methods of diagnosing and treating hepatic cell... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods of diagnosing and treating hepatic cell..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of diagnosing and treating hepatic cell... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3207049