Inhibition of DNA methyltransferase reverses cisplatin induced drug resistance in murine neuroblastoma cells

Abstract

Background: Acquired drug resistance is a major obstacle to the successful treatment of neuroblastoma by chemotherapy. Recent studies from our laboratory have demonstrated that drug-induced alterations in DNA methylation play an important role in this process.

Methods: The reversal of resistance to cisplatin in murine neuroblastoma (MNB) was induced by inhibition of DNA methyltransferase activity. MNB cells overexpressing DNA methyltransferase activity (Dnmt3a or Dnmt3b) were established by stable co-transfection of wild type MNB cells with plasmids containing Dnmt3a or Dnmt3b cDNA. Cytotoxic response (IC50), total DNA methyltransferase activity and expression of Dnmt3a or Dnmt3b methyltransferase were determined in Dnmt3a or Dnmt3b transfected MNB cells, respectively.

Results: These data demonstrated that total DNA methyltransferase activity was increased to 3-fold above controls (P<0.001) in cisplatin resistant MNB cells, 3-fold in Dnmt3a and 4-fold in Dnmt3b transfected MNB cells. Western blot and RT-PCR data confirmed a corresponding increase in Dnmt3a and 3b expression in cisplatin resistant and transfected cells when compared with control MNB cells (P<0.001). MNB clones overexpressing Dnmt3a or Dnmt3b proteins were resistant to cisplatin treatment (10(-6) M). Incubation of cisplatin resistant, Dnmt3a or Dnmt3b overexpressing MNB cells with 5'-azacytidine (5'-azaC), a methylation inhibitor (2.5 microM) significantly decreased DNA methyltransferase activity, expression of Dnmt3a and Dnmt3b proteins and mRNA levels of cisplatin resistant, Dnmt3a and Dnmt3b transfected MNB cells. Cytotoxicity studies using the MTT assay demonstrated that the sensitivity of cisplatin resistant, Dnmt3a and Dnmt3b overexpressing MNB cells to cisplatin was increased 10-fold (P<0.001) following 5'-azaC treatment.

Conclusions: These findings suggest that the overexpression of DNA methyltransferase is associated with a cisplatin resistant phenotype in MNB cells that may or may not be true in animal studies or in the clinical setup. Thus, DNA methylation plays a central role in onset of drug resistance in cisplatin resistant neuroblastoma cells in vitro.