Differentially methylated sequences in pancreatic cancer

#The present invention relates generally to the regulation of gene expression and more specifically to a method of determining the DNA methylation status of CpG sites in a given locus and correlating the methylation status with the presence of a cell proliferative disorder.

#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 for methylated DNA is the protection of 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 that are 5′ neighbors of guanine (CpG). This modification of cytosine residues 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.

#Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R. D. eds. in DNA Methylation Biochemistry and Biological Significance, Springer-Verlag, New York, 1984). In eukaryotic cells, methylation of cytosine residues that are immediately 5′ to a guanosine, occurs predominantly in CG poor regions (Bird, A., Nature, 321:209, 1986). In contrast, CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation (Migeon, et al., supra) and parental specific imprinting (Li, et al., Nature, 366:362, 1993) 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 (Sakai, et al., Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700, 1994). Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated CpG island (Issa, et al., Nature Genet., 7:536, 1994; Herman, et al., supra; Merlo, et al., Nature Med., 1:686, 1995; Herman, et al., Cancer Res., 56:722, 1996; Graff, et al., Cancer Res., 55:5195, 1995; Herman, et al., Cancer Res., 55:4525, 1995).

#FIG. 1 shows the representative results of MCIPs isolated by MCA/RDA. Dot blot analysis. 1A, An example of kinetic enrichment of methylated sequences by RDA. MCA products from the driver and the tester (PL8) and the PCR products from first (1st SA), second (2nd SA) and third (3rd SA) competitive hybridization/selective amplification were blotted onto the membrane and hybridized with a labeled Cyclin G probe. 1B, Dot blot analysis using MICPs isolated from MCA/RDA as probes. First three MICPs were hybridized only to the tester (either PL3 or PL8), whereas the next three MICPs were weakly hybridized to the driver as well as the tester.

#FIG. 2 shows a CpG plot across the 5′ CpG islands of Cyclin G and ppENK showing the relation between isolated clones and their corresponding genes. The positions of MSP primers are also indicated. The black boxes represent exons.

#FIG. 3 shows a summary of the average level of methylation of selected MCA/RDA MICPs by bisulfite-modified genomic sequencing. The specimen number and the age of the patients are in the left column. Empty oval, 0-10% methylation; white oval with black dots, 11-30% methylation; black oval with white dots, 31-70% methylation; black oval, 71-100% methylation; ., not determined, NP, normal pancrease; PCa, primary pancreatic adenocarcinonma.

#It has been determined that an aberrant methylation state of nucleic acids in certain genes, particularly regulatory sequences, is diagnostic for the presence or potential development of a cellular proliferative disorder in subjects bearing the aberrantly methylated nucleic acids. More particularly, the hypermethylation of certain nucleotides localized in CpG islands has been shown to affect the expression of genes associated with the CpG islands; typically such hypermethylated genes have reduced or abolished expression, primarily due to down-regulated transcription. Using a recently developed PCR-based technique called methylated CpG island amplification (MCA), several nucleic acid molecules aberrantly methylated in pancreatic cancer cells line were identified.

#Of the 42 unique clones isolated using MCA/RDA, seven (Cyclin G, ppENK, MICP20, 23, 33, 35 and 36) were CpG islands aberrantly methylated in pancreatic carcinoma compared to normal pancreas. Indeed, none of these 7 clones, were methylated in a panel of normal pancreata using the highly sensitive method of MSP. Two of these seven clones corresponded to the 5′ CpG island of the genes ppENK and Cyclin G. ppENK encodes opioid growth factor, also known as [Met5]-enkephalin. This opioid peptide induces apoptosis in lung cancer cell lines (Maneckjee & Minna, Cell Growth Differ. 5:1033-1040, 1994) and several studies have demonstrated that this peptide has a negative growth regulatory effect on various kinds of cancers, including pancreatic cancer (Zagon, et al., Int. J. Oncol. 14:577-584, 1999). Aberrant methylation of the 5′ CpG island of ppENK was found in 87% of primary pancreatic carcinomas. Hypermethylation was associated with transcriptional silencing of this gene in pancreatic cancer cell lines. Although a low level of methylation in other normal mucosae by MSP was also observed, these results indicate that de novo methylation of 5′ CpG islands and transcriptional repression of ppENK might contribute to pancreatic carcinogenesis.

#Cyclin G is a target for transcriptional activation by p53 (Okamoto, et al., Embo. J. 13:48616-4822, 1994). Conflicting functions for Cyclin G have been reported. When overexpressed Cyclin G is associated with cell growth in vitro (Smith, et al., Exp. Cell. Res. 230:61-68, 1997), while increased expression of Cyclin G also augments apoptosis of multiple cancer cell lines in response to different stimuli (Okamoto & Prives, Oncogene 18:4606-4615, 1999). The data demonstrate the association between hypermethylation of the 5′ CpG island of Cyclin G and its transcriptional repression in the pancreatic cancer cell line PL8 (FIG. 4C), suggesting that Cyclin G could have been selected for silencing because of tumor suppressive functions. These data extend previous findings demonstrating aberrant methylation of multiple cancer-related genes including p16 and HMLH1 in pancreatic cancer (Ueki, et al., Cancer Res. 60:1835-1839, 2000).