Novel cellular determinants for reversal of multidrug resistance in cells expressing P170-glycoprotein

The newly synthesized calcium channel blocker, Ro44-5912, significantly potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Box-resistant human ovarian cell line, A2780/DX5, overexpressing P170-glycoprotein (Pgp). Induction of DNA single- and double-strand breaks (ssbs and dsbs) was measured using alkaline elution and constant-field gel electrophoresis (CFGE) assays. The results indicate that potentiation of the cytotoxicity of Box by Ro44-5912 was accompanied by significant increases in both, Box-induced DNA ssbs and dsbs in the resistant cells, Pulsed-field gel electrophoresis (PFGE) analysis showed that Dox induced DNA fragments in the 50-800 kilobase (kb) and 0.8-5.7 megabase (Mb) ranges. The majority of the newly synthesized DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resulted in significant potentiation of DNA fragmentation in the 50-800 kb range with a minor increase in 0.8-5.7 Mb DNA fragments, suggesting that the modulator functions by potentiating nascent DNA fragmentation in the resistant cells. Exposure to Dox with Ro44-5912 was associated with a prolonged blockage of cells in the S-phase. In contrast, exposure to Box alone resulted in temporary blockage of cells in G₂/M phase (~ 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation of BrdUrd by flow cytometric analysis was inhibited by Box in the presence of Ro44-5912, showing that there is a block of DNA replication. An increased damage in newly synthesized DNA could concur with a blocked DNA replication. Moreover. slowing progression through the S-phase in cells exposed to Box in combination with Ro44-5912 is accompanied by increased sensitivity of Box poisons, indicating a correlation of specific S-phase perturbation with the reversal of Dox resistance by Ro34-5912 in cells expressing Pgp. The results suggest that drug-induced augmentation of nascent DNA fragmentation and specific cell-cycle perturbation are potentially important molecular dererminants for reversal of multidrug resistance in addition to restoration of intracellular drug retention.