Zebularine selectively kills human glioblastoma cells deficient in DNA dependent protein kinase (DNA-PK)

1970

Cancer cells display abnormal DNA methylation patterns leading to epigenetic silencing of many vital genes involved in DNA metabolic activities. Although zebularine, a DNA demethylation agent, is widely used as an effective chemotherapeutic agent to kill cancer cells, the underlying mechanism for zebularine induced killing of tumor cells remains largely unknown. As DNA dependent protein kinase (DNA-PK) plays critical roles in genome surveillance, we have evaluated the role of DNA-PK to gain insights into molecular mechanisms for zebularine induced cell killing. Human glioblastoma cells deficient (MO59J) and proficient (MO59K) in DNA-PK were utilized in this study. Cytotoxicity assay with increasing concentrations of zebularine up to 300 μM for 65 hr demonstrated a marked elevation of cell killing specifically in DNA-PK deficient MO59J cells. The increased cell death in MO59J was confirmed by cell cycle analysis as well as by western blot using antibodies specific for PARP-1, caspases 3, 7, 9, and their cleavage products. Zebularine effectively inhibited DNMT1 and DNMT3b in both soluble and insoluble protein fractions to the same extent in both cell lines suggesting that DNA methyltransferase inhibition per se is not responsible for selective killing of MO59J cells by zebularine. Of interest, DNA-PK deficient MO59J cells showed an elevated proportion of polyploid cells after zebularine treatment suggestive of a molecular deficiency in mitotic checkpoint regulation. In corroboration, nocodazole, a spindle poison known to arrest cells at mitosis, induced a G2/M arrest in MO59K cells whereas DNA-PK deficient MO59J cells showed a significant decline in the proportion of G2/M phase cells with a concomitant increase in polyploidy and apoptosis. To determine whether polyploidy is due to deficient mitotic checkpoint regulation, expression of some of the mitotic checkpoint genes was analyzed. Bub1 expression remained unchanged in MO59K although a significant increase in Bub1 expression was observed in M059J following zebularine treatment. Mad1 was significantly reduced in MO59J but a slight increase in expression was observed with increasing concentration of zebularine. Collectively, our novel study brings out two important findings regarding the mode of action of zebularine induced killing of tumor cells: (I) the functional status of DNA-PK is the major determining factor for zebularine induced apoptotic death in brain tumor cells and (II) cells deficient in mitotic checkpoint regulation are selectively sensitized to zebularine. This study was supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-05ER64055.