title: Reduced phosphorylation of topoisomerase II in etoposide-resistant human leukemia K562 cells.
abstract: In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase 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 topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/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 in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme 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 topoisomerase 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. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.
authors.affiliation: Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
authors.forename: M K
authors.initials: MK
authors.lastname: Ritke
authors.forename: W P
authors.initials: WP
authors.lastname: Allan
authors.forename: C
authors.initials: C
authors.lastname: Fattman
authors.forename: N N
authors.initials: NN
authors.lastname: Gunduz
authors.forename: J C
authors.initials: JC
authors.lastname: Yalowich
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II