Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-06
2001-07-24
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024310, C536S024330
Reexamination Certificate
active
06265161
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to new polynucleotides or new combinations of polynucleotides useful as diagnostic tools for predicting the occurrence of a human hepatocellular carcinoma disease. The invention is also directed to polynucleotides that consist in candidate tumor suppressor genes the alteration of which is involved in the occurrence of hepatocellar carcinoma in a patient, as well as to polynucletides derived from such new candidate tumor suppressor genes and to the corresponding expressed polypeptides. The invention also concerns diagnostic methods using said polynucleotides as diagnostic tools.
BACKGROUND OF THE INVENTION
Hepatocellular carcinoma (HCC) is the most common primary liver cancer in the world, with 251,000 new cases each year (Bosh et al., 1991) and, to date, this pathology carries a very poor prognosis. Epidemiological evidence has shown the predominant role of hepatitis B virus (HBV) as a major causal agent of liver cancer. Other risk factors include chronic infection with hepatitis C virus (HCV), alcohol abuse, environmental exposure to hepatocarcinogens such as aflatoxin B1, and several genetic diseases (Reviewed in Buendia et al., 1995, and described also in Bosch et al., 1991; Wogan, 1992). More particularly, epidemiologic studies indicate that more than 50% of HCCs are attributable to chronic hepatitis B virus (HBV) infection (Bosch et al., 1991). However, the role of hepatotropic viral agents and the molecular events leading to liver carcinogenesis remain unknown. A mutagenic role of HBV DNA integration in the host genome, that occurs frequently at early stages of HBV-associated tumorigenesis, was conclusively established only in rare cases (Dejean et al., 1986; De Thé et al., 1987; Wang et al., 1990), suggesting more indirect transformation pathways (Matsubara, 1991). Viral DNA integrated into hepatocyte DNA can be detected in about 80% of chronic HBV carriers (Chen et al., 1986).
A common feature in chronic viral hepatitis and liver cirrhosis is long lasting inflammation of the liver associated with chronic regenerative conditions, which might enhance the susceptibility of liver cells to genetic changes. HCC usually develops after a 20-50 year period of HBV chronic infection, often subsequent to cirrhosis (Lok et al., 1991). The long latent period before the establishment of carcinomas indicates that they are the result of a multistep process, and several studies have been directed toward the identification of common genetic alterations (Sugimura et al., 1992). Both activation of cellular oncogenes and inactivation of tumor suppressor genes have been implicated (Okuda et al., 1992; Sugimura et al., 1992).
Generally, the development of human cancer results from clonal expansion of gentically modified cells, that acquired selective growth advantage through accumulated alterations of photo-oncogenes and tumor suppressor genes (Weinberg, 1991). Somatic inactivation of tumor suppressor genes is usually achieved by intragenic mutations in one allele of the gene and by the loss of a chromosomal region spanning the second allele.
The steps that lead to homozygosity of a mutant suppressor allele usually involve the flanking chromosomal regions as well. Accordingly, anonymous DNA markers mapping to nearby chromosomal sites, which may have shown heterozygosity prior to tumor progression, will suffer a parallel reduction to homozygosity (or loss of heterozygosity—LOH). Indeed the repeated observation of LOH of a specific chromosomal marker in cells from a particular type suggests the presence of a closely mapping tumor suppressor gene, the loss of which is involved in tumor pathogenesis (Hansen et al., 1987). The recessive action of mutant suppressor gene alleles permits any resulting phenotypic effects to be delayed for long periods of time after conception. These alleles are effectively latent until they are exposed by a reduction to homozygosity in one or another cell.
Thus, a tumor suppressor gene is a genetic element whose loss or inactivation allows a cell to display one or another phenotype of neoplastic growth deregulation. Such a definition exclude genes that are cytostatic or cytotoxic when introduced into a cell and inappropriately overexpressed. The arena of action of tumor suppressor genes may thus be defined: biochemically, these genes serve as transducers of anti-proliferative signals; biologically, they serve as part of the response machinery that enables a cell to stop progression through the cell cycle, to differentiate, to senesce, or to die (Weinberg, 1991).
Chromosomal analysis using polymorphic DNA markers that distinguish different alleles has revealed loss of hereozygosity (LOH) of specific chromosomal regions in various types of cancers and the mapping of regions with a high frequency of LOH has been critical for identifying negative regulators of tumor growth (Call et al., 1990; Fearon et al., 1990; Friend et al., 1986). The recent development of microsatellite polymorphic markers has allowed positional cloning of several tumor suppressor genes such as the BRCA1, BRCA2 and DPC4 genes (Hahn et al., 1996; Miki et al., 1994; Wooster et al., 1995).
Previous studies, mainly relying upon either restriction fragment length polymorphism (RFLP) markers or microsatellite markers restricted to specific chromosome arms, have defined a number of chromosomal regions of LOH in liver cancer. One of the most frequent allelic deletions in HCC has been found at chromosome 17p where the tumor suppressor gene p53 is located (Fujimori et al., 1991; Murakami et al., 1991; Slagle et al., 1991). The frequency of p53 mutations varies largely among HCC samples, depending on the geographic location in the world, and a hot spot mutation at codon 249 was observed in HCCs from regions with high levels of dietary aflatoxins and high prevalence of HBV infection (Bressac et al., 1991; Buetow et al., 1992; Hsu et al., 1991). Regional deletions spanning the RB locus on chromosome 13q have also been described, but in this case, a low mutation rate was found in the remaining allele (Murakami et al., 1991; Wang and Rogler., 1988; Zhang et al., 1994). The most frequent chromosome arm deletion is observed in 13q (53% of informative tumors). Deletions were encompassing a large region of 13q (13q12-q32) which harbors the RB and BRCA2 tumor suppressor genes (Friend et al., 1986; Wooster et al., 1995; Zhang et al., 1994). Other frequent LOH was reported on chromosome arms 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q and 22q (Buetow et al., 1989; De Souza et al., 1995; Emi et al., 1992; Fujimori et al., 1991; Takahashi et al., 1993, Tsuda et al., 1990; Wang and Rogler, 1988; Yeh et al., 1994). Candidate tumor suppressor genes in these regions include the mannose 6-phosphate/insulin-like growth factor II receptor gene (M6P/IGF2R) on 6q26-q27 (De Souza et al., 1995), the PDGF-receptor beta-like tumor suppressor gene (PRLTS) on 8p21-p22 (Fujiwara et al., 1995) and the E-Cadherin gene on 16q22 (Slagle et al., 1993).
Yeh et al. (1994) have performed a genetic analysis of HCC cell lines and 30 primary HCC tissues. Using 8 Polymorphic DNA markers for RFLP experiments and also microsatellites markers spanning 12 loci in chromosome 1p, these authors have shown that many chromosomal abnormalities seemed to cluster at the distal part of chromosome 1p, with a common region mapped to 1p35-36, which is also the region with frequent loss of heterozygosity in neuroblastoma and colorectal as well as breast cancers.
Tsuda et al. (1990) have studied allele loss on chromosme 16 by performing RFLP analysis of 70 surgically resected tumors by using 15 polymorphic DNA markers distributed overall both the short arm and the long arm of said chromosome. They detected LOH in 52% of informative cases (i.e. 36 cases), the common region of allele loss being located between the HP locus (16q22.1) and the CTRB locus (16q22.3-q23.2).
Fujimori et al. (1991) have realized an allelotype study of HCC by examining LOH with 44 RFLP markers in 46 cases of HCC. The markers used by Fujimori et al
Buendia Marie Annick
DeJean Anne
Nagai Hisaki
Pineau Pascal
Arthur Lisa B.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Goldberg Jeanine
Institut Pasteur and Institut Nationale de la Sante et de la Rec
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