HYPOPHOSPHORYLATION OF TOPOISOMERASE-II IN ETOPOSIDE (VP-16)-RESISTANT HUMAN LEUKEMIA K562 CELLS ASSOCIATED WITH REDUCED LEVELS OF BETA(II)PROTEIN-KINASE-C

We selected and characterized a 30-fold etoposide (VP-16)-resistant su bline of K562 human leukemia cells (K/VP.5) that exhibits quantitative and qualitative changes in topoisomerase II, including hypophosphoryl ation of this drug target. The initial rate of topoisomerase II phosph orylation was reduced 3-fold in K/VP.5 compared with K562 cells, but t he rate of dephosphorylation was similar. Analysis of potential topois omerase II protein kinases revealed a 3-fold reduction in the level of the beta(II) protein kinase C (PKC) in K/VP.5 cells, whereas levels o f alpha- and epsilon PKC, casein kinase II, p42(map) kinase, and p34(c dc2) kinase were comparable for both cell lines. The PKC activator, br yostatin 1, together with K562 nuclear extracts potentiated VP-16-indu ced topoisomerase II/DNA covalent complex formation in nuclei isolated from K/VP.5 cells but not from K562 cells. Bryostatin 1 effects were blocked by the PKC inhibitor 7-O-methyl-hydroxy-staurosporine. Bryosta tin 1 also up-regulated topoisomerase II phosphorylation and potentiat ed VP-16 activity in intact K/VP.5 cells but had no enhancing effect i n K562 cells. 4 beta-Phorbol-12,13-dibutyrate and 12-O-tetradecanoylph orbol- 13-acetate did not potentiate VP-16-induced topoisomerase II/DN A complex formation in intact cells or in isolated K/VP.5 nuclei. Toge ther, our results indicate that beta(II) PKC plays a role in modulatin g the VP-16-induced DNA binding activity of topoisomerase II in resist ant K/VP.5 cells through a mechanism linked to phosphorylation of topo isomerase II.