REDUCED PHOSPHORYLATION OF TOPOISOMERASE-II IN ETOPOSIDE-RESISTANT HUMAN LEUKEMIA K562 CELLS

In this report we examine biochemical and genetic alterations in DNA t opoisomerase II (topoisomerase II) in K562 cells selected for resistan ce in the presence of etoposide (VP-16). Previously, we have demonstra ted that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreas ed stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoiso merase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomera se II, and attenuated ATP stimulation of VP-16-induced topoisomerase I I/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K /VP.5 topoisomerase II gene indicated that no mutations were present i n these domains. In addition, single-strand conformational polymorphis m analysis of restriction fragments encompassing the entire topoisomer ase II cDNA revealed no evidence of mutations in the gene for this enz yme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoiso merase II/DNA covalent complex formation in isolated nuclei from K/VP. 5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5 -fold less phosphorylated, compared with enzyme from K562 cells. Colle ctively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regul ates topoisomerase II phosphorylation and hence drug-induced topoisome rase II/DNA covalent complex formation and stability.