RESISTANCE TO ETOPOSIDE IN HUMAN LEUKEMIA HL-60 CELLS - REDUCTION IN DRUG-INDUCED DNA CLEAVAGE ASSOCIATED WITH HYPOPHOSPHORYLATION OF TOPOISOMERASE-II PHOSPHOPEPTIDES

Tumor cell resistance to anthracyclines and epipodophyllotoxins can be due to reduced drug accumulation and/or alterations in the activity o f topoisomerase II (TOPO II). HL-60 cells selected in 0.05 mu g/ml dox orubicin (DOX) are 10-fold and >20-fold resistant to DOX and etoposide (VP-16), respectively. The accumulation of [H-3]VP-16 was 2-3-fold lo wer in the resistant cells (HL-60/DOX 0.05) than in similarly treated parent-sensitive cells (HL-60/S). However, compared with HL-60/S cells , the HL-60/DOX 0.05 cells required >20-fold higher concentrations of VP-16 to produce equivalent damage to DNA. The reduced formation of VP -16-stabilized DNA cleavable complex in the HL-60/DOX 0.05 cells was n ot due to differences in the amount of 170-kDa TOPO (alpha) II protein or enzyme catalytic activity between HL-60/S and HL-60/DOX 0.05 cells . Metabolic labeling with [P-32]orthophosphoric acid and immunoprecipi tation indicated that the level of phosphorylated 170-kDa TOPO II alph a protein in the HL-60/S cells was 2.2 +/- 0.4-fold higher than that i n HL-60/DOX 0.05 cells. Hypophosphorylation (3-fold) of 170-kDa TOPO I I protein in HL-60/S cells treated with the calcium chelator 2-bis-(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester pro duced a >2-fold reduction in VP-16-induced TOPO II-mediated DNA cleava ble complex formation. Two-dimensional mapping of phosphopeptides in c omplete tryptic digests demonstrated that the reduced phosphorylation of the 170-kDa TOPO II alpha in HL-60/DOX 0.05 cells was due to the hy pophosphorylation of at least three phosphopeptides characteristic of HL-60/S cells. Thus, the attenuated ability of TOPO II to form drug-st abilized DNA cleavable complex is related to the phosphorylated state of 170-kDa TOPO II, and in HL-60/DOX 0.05 cells, resistance may be rel ated to hypophosphorylation of three phosphopeptides characteristic of HL-60/S cells.