Increased cell proliferation and R. Msp l fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism

Abstract

DNA methylation is one of the crucial mechanisms for cellular and tissue differentiation during developmental stages in mammals. 5-aza-2'-deoxycytidine, a specific cytosine DNA Methyltransferase inhibitor, is known to cause DNA hypomethylation in CpG, CpNpG and CCGG sequences. Therefore the present study was designed to determine the effects of 5-aza-2'-deoxycytidine on the germinal cells of the adult rat testicular tissue. Rat testicular tissues from the 5-aza-2'-deoxycytidine treated experimental and non-treated control groups were processed for light microscopy and also for genomic DNA isolation assays. The isolated genomic DNAs were digested with R.Msp1 in order to determine the methyl pattern differences in the enzyme cognate CCGG sequence. Testicular tissues from treated rats showed increased cell proliferation when investigated at the light microscopical level. On the other hand, genomic DNA of these proliferative tissue showed high fragmentation sizes of R.Msp1 digestion when compared to controls. While the R.Msp1 digested control group DNA fragmentation condensed at approximately 4700-5100 bps size, the experimental group DNA fragmentation was condensed at 700-900 bps size. In addition, 5-aza-2'-deoxycytidine has effects on increased cell proliferation via the loss of somatic de novo gene imprinting. These results imply that abnormally imprinted normal somatic cells in mammals are susceptible to epigenetic modification. These results also suggest that the genomic DNA of testicular tissues from control rats is resistant to R.Msp1 while DNA from the experimental group testicular cells demonstrating high proliferation rate could not resist to R.Msp1 digestion due to DNA hypomethylation in CCGG sequence. In conclusion, it could be suggested that the reversal of gene imprinting in germinal cells may cause an increased cellular proliferation and R.Msp1 fragmentation when induced by 5-aza-2'-deoxycytidine.