Retinoic acid and phorbol ester induced hyperphosphorylation of topoisomerase II-alpha is an early event in HL-60 human leukaemia cell differentiation: effect on topoisomerase activity and etoposide sensitivity.

Treatment of HL-60 with phorbol myristate acetate (PMA) for 30 min, or all-trans retinoic acid (RA) for 60 min, results in hyperphosphorylation (3-5x) of topoisomerase II (p170, topo II) in vivo. RA and PMA activate a coprecipitating kinase, respectively inducing 1.6 and 2.7-fold increases in phosphorylation of topo II in immunoprecipitates. The activity of the co-precipitating kinase is inhibited by heparin and unlabelled GTP suggesting that casein kinase II (CKII) is, at least in part, responsible for the topo II hyperphosphorylation in response to differentiation signals. Although following dephosphorylation of the enzyme with alkaline phosphatase there was virtual abrogation of activity, the differentiation associated hyperphosphorylation had little impact on the decatenation activity of topo II in nuclear extracts. There were, however detectable changes in topo II function in vivo which affected the formation of the etoposide stabilised cleavable complex, but only after PMA treatment. PMA resulted in a rapid reduction in etoposide induced cleavage, 30 min treatment with PMA reducing cleavage by 20%. However, treatment with RA for 1 or 2 h when hyperphosphorylation was maximal did not affect cleavage. Immunoband depletion assays suggested that differentiation associated changes in chromatin structure rather than alterations in the enzyme per se are responsible for the reduction in cleavable complex formation following PMA treatment. Etoposide cytotoxicity was significantly reduced following just 30 min PMA treatment, but not reduced and even possibly enhanced by retinoic acid treatment. These findings are relevant not only to the dissection of the role of topo II in differentiation but also to its exploitation as a therapeutic target.