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 micrograms/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-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 not 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 [32P]orthophosphoric acid and immunoprecipitation indicated that the level of phosphorylated 170-kDa TOPO II alpha 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 170-kDa 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 cleavable complex formation. Two-dimensional mapping of phosphopeptides in complete 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 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.
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|