DNA (cytosine) methyltransferase overexpression is associated with acquired drug resistance of murine neuroblastoma cells

We have previously reported that C-1300 murine neuroblastoma (rMNB) cells made resistant to the nucleoside analogue, (Z)-5'-fluoro-4', 5'-didehydro-5'deoxyadenosine (MDL), an irreversible inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase have an increased expression of the S-adenosylmethionine (AdoMet) synthetase gene. Results of the immunoblot analysis of DNA (cytosine) methyltransferase with anti-human DNA (cytosine) methyltransferase specific polyclonal antibody demonstrated a significant increase (≈2-fold, p<0.01) in expression of DNA (cytosine) methyltransferase protein in rMNB/MDL cells compared to wild-type C1300 MNB (wMNB) cells. To rule out the possibility that multidrug resistance (MDR) genes are involved in development of acquired drug resistance in murine neuroblastoma (rMNB/MDL) cells made resistant to MDL, the expression of Mdr1a, Mdr1b, Mdr2 (multidrug resistance/P-glycoprotein), and Mrp-1 (multidrug resistance associated protein) was examined in rMNB-MDL cells. The analysis of Mdr and Mrp-1 expression was performed by RT-PCR using PCR specific primers to respective genes. No significant difference was observed in the expression of MDR1a, Mdr1b and Mrp-1 genes between wMNB and rMNB-MDL cells, however, a slight decrease was noticed in Mdr1 expression in some samples. Expression of the Mdr2 (human MDR3) gene, which is not associated with the acquired drug resistance phenotype, was significantly decreased in rMNB-MDL cells. These findings were also confirmed by the immunoblot analyses using specific monoclonal antibodies to Mdr1/3 proteins. Expression of N-Myc gene - a prognostic factor in neuroblastoma tumors was also not altered in rMNB-MDL cells. Results of the present study suggest that acquired drug resistance in rMNB-MDL cells to MDL is associated to the overexpression of DNA (cytosine) methyltransferase, and could be due to genetic or epigenetic changes in particular to DNA hypermethylation in response to an increased AdoMet synthetase gene expression.