Altered phosphorylation, biosynthesis and degradation of the 170 kDa isoform of topoisomerase II in amsacrine-resistant human leukemia cells.
Resistance to amsacrine in HL-60/AMSA is 50-100 fold compared to the parental HL-60/S cells. Synthesis and phosphorylation of topoisomerase II (TOPO II) were 2-3 fold lower in HL-60/AMSA compared to HL-60/S cells metabolically labelled with [32P]-orthophosphoric acid or [35S]-L-methionine. Incubating cells in radiolabel-free media following metabolic labelling for 4 hr revealed: (a) dephosphorylation of topoisomerase II at 4 hr was 70% and 20% in HL-60/S and HL-60/AMSA cells, respectively; and (b) degradation of topoisomerase II at 4 hr was 40% and 10% in HL-60/S and HL-60/AMSA cells, respectively, while at 8 hr degradation was 80% and 50% in HL-60/S and HL-60/AMSA cells, respectively. The magnitude of topoisomerase II band depletion in immunoprecipitates of amsacrine-treated cells labelled with [35S]-L-methionine or [32P]-orthophosphoric acid, correlated with the differential amsacrine sensitivity of HL-60/S and HL-60/AMSA cells, suggesting that the amount of newly synthesized and phosphorylated topoisomerase II may be contributing to amsacrine resistance. Thus, the attenuated synthesis and phosphorylation of TOPO II in HL-60/AMSA may contribute to the resistance of these cells to amsacrine.
Research Institute, Cleveland Clinic Foundation, Ohio 44195.
R
R
Ganapathi
L
L
Zwelling
A
A
Constantinou
J
J
Ford
D
D
Grabowski
D000677:Amsacrine / Q000494:pharmacology*; D001345:Autoradiography; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes; D000677:Amsacrine; D008715:Methionine; D004250:DNA Topoisomerases, Type II