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 subline of K562 human leukemia cells (K/VP.5) that exhibits quantitative and qualitative changes in topoisomerase II, including hypophosphorylation of this drug target. The initial rate of topoisomerase II phosphorylation was reduced 3-fold in K/VP.5 compared with K562 cells, but the rate of dephosphorylation was similar. Analysis of potential topoisomerase 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 of alpha- and epsilon PKC, casein kinase II, p42map kinase, and p34cdc2 kinase were comparable for both cell lines. The PKC activator, bryostatin 1, together with K562 nuclear extracts potentiated VP-16-induced 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. Bryostatin 1 also up-regulated topoisomerase II phosphorylation and potentiated VP-16 activity in intact K/VP.5 cells but had no enhancing effect in K562 cells. 4 beta-Phorbol-12,13-dibutyrate and 12-O-tetradecanoylphorbol-13-acetate did not potentiate VP-16-induced topoisomerase II/DNA complex formation in intact cells or in isolated K/VP.5 nuclei. Together, our results indicate that beta II PKC plays a role in modulating the VP-16-induced DNA binding activity of topoisomerase II in resistant K/VP.5 cells through a mechanism linked to phosphorylation of topoisomerase II.
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis*; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D004247:DNA; D011493:Protein Kinase C; D004250:DNA Topoisomerases, Type II