Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content.
A camptothecin-resistant subline of P388 leukemia (P388/CPT) was developed by repeated transplantation of P388 cells in mice treated with therapeutic doses of camptothecin. In mice bearing the resistant tumor, a maximally tolerated dose of camptothecin produced no net reduction in tumor cell burden, in contrast to a 5-log cell kill in the parental P388 (P388/S). The IC50 of camptothecin, as determined by colony formation assays of cultured cells, was 8 times greater for the cloned P388/CPT cell line than for P388/S. P388/CPT cells were not cross-resistant to other antineoplastic agents, including topoisomerase II inhibitors. The type I topoisomerases purified from P388/CPT and P388/S cells were identical with respect to molecular weight, specific activity, in vitro camptothecin sensitivity, and DNA cleavage specificity. Camptothecin induced fewer protein-associated DNA single-strand breaks in the resistant cells than in the wild-type P388 cells. Topoisomerase I mRNA, immunoreactivity, and extractable enzymatic activity were 2-4 times lower for P388/CPT cells than for P388/S cells. As resistance to camptothecin developed, topoisomerase I extractable activity decreased, concomitant with an increase in topoisomerase II extractable activity. Furthermore, the appearance of camptothecin resistance was associated with specific rearrangements of the topoisomerase I gene. These results suggest that development of resistance to inhibitors of topoisomerase I can occur by down-regulation of the target enzyme, thus reducing the production of lethal enzyme-mediated DNA damage. The enhanced topoisomerase II activity in these cells suggests that resistance to camptothecin may be overcome by co-treatment with topoisomerase II inhibitors.
Eng
W K
WK
Department of Biomolecular Discovery, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
McCabe
F L
FL
Tan
K B
KB
Mattern
M R
MR
Hofmann
G A
GA
Woessner
R D
RD
Hertzberg
R P
RP
Johnson
R K
RK
D000818:Animals; D002166:Camptothecin / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D004250:DNA Topoisomerases, Type II / Q000032:analysis; D004351:Drug Resistance; D015321:Gene Rearrangement; D007941:Leukemia P388 / Q000201:enzymology*; D051379:Mice; D008811:Mice, Inbred DBA; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D004247:DNA; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D002166:Camptothecin