ALTERED TOPOISOMERASE-I EXPRESSION IN 2 SUBCLONES OF HUMAN CEM LEUKEMIA SELECTED FOR RESISTANCE TO CAMPTOTHECIN

Two camptothecin-resistant variants of the CEM human leukemia cell lin e were developed by stepwise selection in camptothecin (CPT) in vitro. The two lines, named CEM/C1 and CEM/C2, were found to be approximatel y 31- and 970-fold less sensitive to CPT, respectively, than the CEM p arental line and variably cross-resistant to the CPT analogs 9-amino-C PT, 10,11-methylenedioxy-CPT, and topotecan. Levels of DNA-protein com plex formation resulting from cell exposure to CPT were found to be pr ogressively reduced in the CPT-resistant cells, despite equivalent CPT accumulation in the drug-sensitive and -resistant cells. Nuclear extr acts (1.0 M NaCl) prepared from the CEM/C1 and CEM/C2 lines contained 1.5- to a-fold less DNA topoisomerase I catalytic activity per microgr am of protein than did extracts from the drug-sensitive CEM line, in a ssociation with altered sensitivity of the enzyme in the CEM/C1 and CE M/C2 extracts to the inhibitory activity of CPT. Only minor difference s were noted in the CPT IC(50)s for the topoisomerase I activity in ex tracts from the two CPT-resistant cell lines, however, despite the mar ked differences in cellular sensitivity to CPT: There were notable dif ferences in the level of CPT-induced cleavage of DNA oligonucleotides by topoisomerase I in nuclear extracts from CEM cells compared with th e drug-resistant cell extracts, with very little oligonucleotide cleav age induced by enzyme in either drug-resistant cell type, despite the use of very high (100 mu M) CPT concentrations. The alterations in top oisomerase 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 GEM, respectively ) and mRNA. In contrast, nuclear and whole cell levels of DNA topoisom erase II catalytic activity and protein were identical in the drug-sen sitive and -resistant cell lines. These results suggest that the relat ively low levels of CPT resistance displayed by the CEM/C1 cell line m ight have resulted from the alterations in DNA topoisomerase I activit y and protein content found in those cells and imply that additional f actors, such as qualitative changes in the topisomerase I enzyme, migh t be responsible for the higher levels of CPT resistance displayed in the CEM/C2 cells.