Flow cytometric analysis for the mechanism of the new antineoplastic agent temozolomide in glioma cells

Temozolomide (TMZ) has been accepted as a standard antitumor drug for glioma worldwide. Regarding its mechanism of action, there are quite a few analyses. In the present study, we investigated the cell-killing effect and mechanism of action of TMZ with flow cytometry using glioblastoma cell lines. Each cell line was divided into three groups: a control group, a low-dose TMZ group, and a highdose TMZ group. On day 1, TMZ was added to each cell line. Then, we counted the numbers of cells on days 2, 3, 4, and 5; in U87MG, we counted the number of cells on days 8 and 9. Simultaneously, we performed flow cytometric analysis with single- and double-staining methods. Although results varied slightly depending on the cell line, with flow cytometric analysis we identified the G0G1-, S-phase block on days 2 through 4, at the beginning of TMZ administration. After that we identified the deviation of the G2M block over days 3 to 5. Dominant morphological changes observed in U87MG were confined to the nuclei, with positive TUNEL staining. Early S-phase block and then a G2M block were observed; consecutively, we could analyze these blocks with a double-staining method more clearly. The flow cytometric method is very effective in the analysis of the antitumor mechanism of each agent. On the basis of our analysis, more effective combined chemotherapy may be expected.