Klonierung methylierungsabhängiger Gene in Neuroendokrinen Pankreastumorzellen durch cDNA Representational Difference Analysis (cDNA-RDA)

Background: Hypermethylation is an alternative mechanism for early gene inactivation in cancer cells. Reaquisition of p16/CDKN2A tumor suppressor gene expression by 5-Aza-2`deoxycytidine (5-Aza-CdR) results in concurrent growth inhibition of neuroendocrine pancreatic tumor cells. However, the growth suppressive effects of 5-Aza CdR is unlikely to be solely attributable to the restored p16/CDKN2A function but rather a consequence of reexpression of additional genes silenced by de novo methylation. In an effort to validate DNA methylation as a cancer-specific biomarker that could be integrated into prevention we attempted to isolate methylation-specific transcripts in neuroendocrine pancreatic tumor cells. Methods: Differentially expressed methylation-associated genes were identified by cDNA-representational difference analysis (cDNA-RDA). For the generation of representations RNA was extracted from QGP-1 cells cultured in the presence or absence of the methyltransferase-inhibitor 5-Aza-CdR and subjected to cDNA synthesis followed by two PCR-coupled rounds of substractive hybridisation and selective amplification (cDNA-RDA). The RNA of treated cells and untreated controls was used in separate experiments as tester or driver, respectively. The second difference products were cloned into Bluescript SK II for cDNA fragment sequencing. Sequence homology and chromosomal localization resulted from blast-algorithm. Differential expression was confirmed by semi-quantitative RT-PCR using insert specific primers. Results: We identified 48 differently expressed gene fragments and methylation-associated expression was confirmed by semi-quantitative RT-PCR. Sequence analysis of the isolated cDNA fragments revealed homology to 41 known genes. They were genes encoding tumor suppressors, sorting proteins, membrane proteins, proteases, enzymes, proteins involed in tumor invasion and metastases, apoptosis, ubiquitination, signal transduction, nuclear and ion transport, cytoskeleton, translational control and protein trafficking. The remaining 7 fragments showed homology to genes with unknown function. Conclusion: DNA methylation appears an important molecular mechanism in the process of neuroendocrine pancreatic tumorigenesis and the definition of DNA methylation patterns associated with neuroendocrine pancreatic tumors might open up the potential for a new sensitive and specific diagnostic and predictive tool.