Tumor cell resistance to topoisomerase II poisons - Role for intracellular free calcium in the sensitization by inhibitors of calcium-calmodulin-dependent enzymes

Tumor cell resistance to inhibitors of topoisomerase II (topo II) is associated frequently with the overexpression of P-glycoprotein (PGP), and strategies to overcome resistance are focused on restoring defects in drug accumulation. Inhibitors of calcium calmodulin dependent enzymes sensitize resistant tumor cells to the topo II poison etoposide (VP-16) by enhancing DNA damage and an apoptotic response. In the present study, we have investigated the consequences of buffering intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra(acetoxy-methyl) ester (BAPTA-AM) on the sensitizing effects of the calmodulin-dependent protein kinase II inhibitor 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- piperazine (KN-62) in etoposide-resistant human leukemia HL-60 (HL-60/ADR0.05) cells. In cells pretreated with 20 mu M BAPTA-AM for 2 hr, extracellular ATP failed to trigger intracellular calcium transients, and no effects on the accumulation of VP-16 were apparent. Also, the effect of KN-62 in significantly (P = 0.002 to 0.042) enhancing the accumulation of VP-16 in HL-60/ADR0.05 cells was unaffected due to pretreatment with BAPTA-AM. In contrast, pretreatment with BAPTA-AM reduced the DNA damage induced by VP-16, and significantly (P = 0.038) reversed the enhancement by KN-62 of VP-16-stabilized topo II-mediated DNA cleavable complex formation. The pretreatment of HL-60/ADR0.05 cells with BAPTA-AM was also associated with the hypophosphorylation of topo II alpha. Consistent with the ability of BAPTA-AM to circumvent the potentiation by KN-62 of VP-16-induced DNA damage, survival of cells created with 40 mu M VP-16 in the absence of KN-62 and 10 mu M VP-16 in the presence of KN-62 was significantly (P = 0.026 to 0.031) higher due to BAPTA-AM pretreatment. Results demonstrate that intracellular calcium transients could play a key role in the sensitization of etoposide-resistant tumor cells by inhibitors of calcium-calmodulin-dependent enzymes. BIOCHEM PHARMACOL 56;3:345-349, 1998. (C) 1998 Elsevier Science Inc.