Published in Cancer Detection and Prevention 2002; 26(6).

Differential expression of DNA-methyltransferases in drug-resistant murine neuroblastoma cells

Y-Yong Qiu, MD, Bernard L. Mirkin, PhD,MD, Rama S. Dwivedi, PhD

Departments of Pediatrics, Molecular Pharmacology and Biological Chemistry, Children's Memorial Hospital, Children's Memorial Institute for Education and Research, Northwestern University, Medical School, 2300 Children's Plaza, Mail Box no. 204, Chicago, IL 60614-3394, USA

Neuroblastoma tumors frequently become drug resistant during the process of chemotherapy resulting in unfavorable clinical outcomes. Development of sustained drug resistance in neuroblastoma is a major problem in successful treatment. To explore the role of DNA-methyltransferases (Dnmt), in acquired drug resistance of neuroblastoma the present investigation was carried out to study the expression of Dnmt1, Dnmt3a, and Dnmt3b in drug resistant murine neuroblastoma cells, in an in vitro model system. We have analyzed the expression of Dnmt1, Dnmt3a, and Dnmt3b methyltransferases in wild type and drug resistant murine neuroblastoma cells by using Western blot, immunofluorescence microscopy, semiquantitative and quantitative real time RT-PCR analyses. The present investigation demonstrates that total Dnmt enzymatic activity was increased to two-fold ( P < 0.001) with a 33% increase in global DNA methylation rate in drug resistant cells. Results of the Western blot, immunofluorescence microscopy, RT-PCR, and quantitative real time RT-PCR analysis demonstrated that Dnmt1 and Dnmt3b expression increased significantly ( P < 0.001) in drug resistant cells when compared with wild type cells. Dnmt3a expression did not reveal any change between wild type and drug resistant cells. These findings suggest that Dnmtases are differentially expressed in drug resistant murine neuroblastoma cells and overexpression of Dnmt1 and Dnmt3b may contribute towards loss of function of the growth regulatory or tumor suppressor genes by methylation of their `CpG' region and subsequently silencing of their expression. The products of these methylated genes may, thus, confer a high level of drug resistant phenotype in drug resistant neuroblastoma cells.

KEY WORDS: DNA-methyltransferases, Neuroblastoma, Drug resistance, DNA methylation, Murine, Cells.