NOVEL CELLULAR DETERMINANTS FOR REVERSAL OF MULTIDRUG-RESISTANCE IN CELLS EXPRESSING P170-GLYCOPROTEIN

The newly synthesized calcium channel blocker, Ro44-5912, significantl y potentiates doxorubicin (Dox)-induced cytotoxicity at non-cytotoxic concentrations in Dox-resistant human ovarian cell line, A2780/DX5, ov erexpressing P170-glycoprotein (Pgp). Induction of DNA single-and doub le-strand breaks (ssbs and dsbs) was measured using alkaline elution a nd constant-field gel electrophoresis (CFGE) assays. The results indic ate that potentiation of the cytotoxicity of Dox by Ro44-5912 was acco mpanied by significant increases in both, Dox-induced DNA ssbs and dsb s in the resistant cells. Pulsed-field gel electrophoresis (PFGE) anal ysis 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 synthesiz ed DNA fragments were in the 50-800 kb range. Ro44-5912 treatment resu lted 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 t hat 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, exposu re to Dox alone resulted in temporary blockage of cells in G(2)/M phas e (similar to 24 h) followed by restoration of cell proliferation and normal DNA histograms at 48 h after 2 h drug exposure. Incorporation o f BrdUrd by flow cytometric analysis was inhibited by Dox in the prese nce 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 Dox in combination with Ro14-5912 is accompanied by increased sensitivity of Dox poisons, indicating a correlation of spec ific S-phase perturbation with the reversal of Dox resistance by Ro44- 5912 in cells expressing Pgp. The results suggest that drug-induced au gmentation of nascent DNA fragmentation and specific cell-cycle pertur bation are potentially important molecular determinants for reversal o f multidrug resistance in addition to restoration of intracellular dru g retention, (C) 1998 Elsevier Science B.V.