Resistance to etoposide in human leukemia HL-60 cells: Reduction in drug-induced DNA cleavage associated with hypophosphorylation of topoisomerase II phosphopeptides

Abstract

Tumor cell resistance to anthracyclines and epipodophyllotoxins can be due to reduced drug accumulation and/or alterations in the activity of topoisomerase II (TOPO II). HL-60 cells selected in 0.05 μg/ml doxorubicin (DOX) are 10-fold and >20-fold resistant to DOX and etoposide (VP-16), respectively. The accumulation of [3H]VP-16 was 2-3-fold lower in the resistant cells (HL-60/DOX 0.05) than in similarly treated parent-sensitive cells (HL-60/s). However, compared with HL-6O/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 not due to differences in the amount of 170-kDa TOPO (α) II protein or enzyme catalytic activity between HL- 60/S and HL-60/DOX 0.05 cells. Metabolic labeling with [32P]orthophosphoric acid and immunoprecipitation indicated that the level of phosphorylated 170-kDa TOPO IIα protein in the HL-60/S cells was 2.2 ± 0.4-fold higher than that in HL-60/DOX 0.05 cells. Hypophosphorylation (3- fold) of 170kDa TOPO II protein in HL-60/S cells treated with the calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester produced a >2-fold reduction in VP-16-induced TOPO II- mediated DNA clearable complex formation. Two-dimensional mapping of phosphopeptides in complete tryptic digests demonstrated that the reduced phosphorylation of the 170-kDa TOPO IIα in HL-60/DOX 0.05 cells was due to the hypophosphorylation of at least three phosphopeptides characteristic of HL-60/S cells. Thus, the attenuated ability of TOPO II to form drug- stabilized 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 related to hypophosphorylation of three phosphopeptides characteristic of HL-60/S cells.