Altered topoisomerase I expression in two subclones of human CEM leukemia selected for resistance to camptothecin.
Two camptothecin-resistant variants of the CEM human leukemia cell line were developed by stepwise selection in camptothecin (CPT) in vitro. The two lines, named CEM/C1 and CEM/C2, were found to be approximately 31- and 970-fold less sensitive to CPT, respectively, than the CEM parental line and variably cross-resistant to the CPT analogs 9-amino-CPT, 10,11-methylenedioxy-CPT, and topotecan. Levels of DNA-protein complex formation resulting from cell exposure to CPT were found to be progressively reduced in the CPT-resistant cells, despite equivalent CPT accumulation in the drug-sensitive and -resistant cells. Nuclear extracts (1.0 M NaCl) prepared from the CEM/C1 and CEM/C2 lines contained 1.5- to 2-fold less DNA topoisomerase I catalytic activity per microgram of protein than did extracts from the drug-sensitive CEM line, in association with altered sensitivity of the enzyme in the CEM/C1 and CEM/C2 extracts to the inhibitory activity of CPT. Only minor differences were noted in the CPT IC50s for the topoisomerase I activity in extracts from the two CPT-resistant cell lines, however, despite the marked differences in cellular sensitivity to CPT. There were notable differences in the level of CPT-induced cleavage of DNA oligonucleotides by topoisomerase I in nuclear extracts from CEM cells compared with the drug-resistant cell extracts, with very little oligonucleotide cleavage induced by enzyme in either drug-resistant cell type, despite the use of very high (100 microM) CPT concentrations. The alterations in topoisomerase I catalytic activity were associated with reduced cellular levels of both immunoreactive topoisomerase I protein (representing 59 +/- 19% [CEM/C1] and 49 +/- 12% [CEM/C2] of that in CEM, respectively) and mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Kapoor
R
R
Department of Medicine, VA Medical Center, Salt Lake City, UT 84148, USA.
Slade
D L
DL
Fujimori
A
A
Pommier
Y
Y
Harker
W G
WG
D000208:Acute Disease; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004351:Drug Resistance; D006801:Humans; D007942:Leukemia, Experimental; D015458:Leukemia, T-Cell / Q000188:drug therapy* / Q000201:enzymology*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D059004:Topoisomerase I Inhibitors; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin