ISPO

DNA methyltransferase-3b is preferentially expressed in drug resistant cells

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

Childrens Memorial Institute for Education and Research, Childrens Memorial Hospital and Department of Pediatrics, Northwestern University Medical School; Chicago, IL 60614, USA

Aim: Development of a sustained drug resistance in neuroblastoma is a major problem in successful treatment. To explore out the role of DNA methyltransferases (DNMT), if any, in acquired drug resistance of neuroblastoma present investigation was carried out to study the expression of DNMT1, DNMT3a, and DNMT3b in drug resistant murine neuroblastoma cells. Methods: We have analyzed the expression of DNMT1, DNMT3a, and DNMT3b methyltransferases in wild type and drug resistant murine neuroblastoma cells by using Western blot, Confocal immunofluorescence microscopy, semiquantitative and quantitative Real Time RT-PCR analyses. Results: The present investigation demonstrates that total Dnmt enzymatic activity was increased to two folds (P<0.001) with a 33% increase in global DNA methylation rate in drug resistant cells. Western blot data revealed a 1.6 fold increases in Dnmt1 and a 1.7 fold increase in DNMT3b expression without any appreciable change in DNMT3a expression in drug resistant cells when compared with wild type cells. Confocal immunofluorescence microscopy showed a 2.1 fold increase in DNMT1 and a 2.3 fold increase in DNMT3b expression in drug resistant cells. RT-PCR analysis has demonstrated a 1.7 fold increase in DNMT1 and a 1.8 fold increase in DNMT3b expression without any change in Dnmt3a expression in drug resistant cells. Quantitative Real Time RT-PCR analysis demonstrated 1.22 cycle difference in relative threshold (CT) value indicating more than two fold increase (P<0.001) in DNMT1 expression and a 5.0 cycle difference with more than ten fold increase (P<0.001) in Dnmt3b expression in drug resistant cells. DNMT3a expression did not reveal any change between wild type and drug resistant cells. Conclusions: These findings suggest that overexpression of DNMT1 and DNMT3b methyltransferases may contribute towards loss of function of the growth regulatory or tumor suppressor genes by methylation of their CpG region and subsequent silencing their expression. The products of these methylated genes may affect the sensitivity of drugs, and thus confer a high level of drug resistance during the chemotherapy.

For more information, contact ramaa@nwu.edu

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Multidrug Resistance - MDR.

http://www.cancerprev.org/Journal/Issues/26/101/1095/4604