CELLULAR EVENTS INVOLVED IN THE SENSITIZATION OF ETOPOSIDE RESISTANT CELLS BY INHIBITORS OF CALCIUM-CALMODULIN-DEPENDENT PROCESSES - ROLE FOR EFFECTS ON APOPTOSIS, DNA CLEAVABLE COMPLEX, AND PHOSPHORYLATION

Inhibitors of calcium calmodulin dependent processes, 1-[N,O-bis(1,5-i soquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-piperazine KN-62 and trifl uoperazine (TFP), at non-cytotoxic concentrations (2 and 5 mu M, respe ctively) enhanced etoposide (VP-16) cytotoxicity in Adriamycin(R)-resi stant (HL-60/ADR0.05) cells (3- to >50-fold). In contrast to TFP, the inhibitor KN-62 was able to reverse resistance in HL-60/ADR0.05 cells at VP-16 concentrations that produced equivalent cytotoxicity in sensi tive (HL-60/S) cells. Unlike TFP, the cellular accumulation of VP-16 i n the presence of KN-62 was enhanced 1.5- to 2-fold in HL-60/S (MDR1 - ve) and HL-60/ADR0.05 (MDR1 +ve) cells. To achieve equivalent cytotoxi city, levels of VP-16 in the resistant cells were >4-fold lower in the presence of KN-62 compared with treatment with VP-16 alone. The sensi tizing effects of both KN-62 and TFP were due to enhancement (2- to 4- fold) of VP-16 induced topoisomerase II (TOPO II)-mediated DNA cleavab le complex formation, and depletion of the 170 kDa (alpha) TOPO II iso form. The DNA damage induced by VP-16 in the presence of KN-62 or TFP resulted in the rapid induction of apoptosis and depletion of cells in ''S'' phase of the cell cycle. Both 5 mu M TFP and 2 mu M KN-62 enhan ced the phosphorylation of 170 kDa TOPO II 1.6-fold and 1.5-fold, resp ectively. Results suggest that the inhibitory effect of KN-62 or TFP o n calcium-calmodulin-dependene processes may be mechanistically involv ed in sensitizing resistant cells to VP-16 by enhancing TOPO II-mediat ed DNA damage. Copyright (C) 1996 Elsevier Science Inc.