Reduced phosphorylation of topoisomerase II in etoposide-resistant human leukemia K562 cells.
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.
Ritke
M K
MK
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Allan
W P
WP
Fattman
C
C
Gunduz
N N
NN
Yalowich
J C
JC
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
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II