Elsevier

Biochemical Pharmacology

Volume 45, Issue 2, 26 January 1993, Pages 339-348
Biochemical Pharmacology

Sequential modifications of topoisomerase I activity in a camptothecin-resistant cell line established by progressive adaptation

https://doi.org/10.1016/0006-2952(93)90069-9Get rights and content

Abstract

The DNA-topoisomerase I (Topo I) inhibitor, camptothecin (CPT), is a plant alkaloid with an important antitumor activity. In order to investigate the cellular mechanism leading to the development of the resistance to this agent, we have established by progressive adaptation a P388 subline resistant to CPT. After 5 months of continuous drug exposure, the resistance index reached a value of 20 and the resistant cell line, P388CPT0.3, was maintained in the presence of CPT. CPT-induced single strand breaks measured by alkaline elution were found drastically reduced in the resistant cell line. Topo I activity and CPT-induced DNA cleavage were measured on cells at different steps of resistance. We first observed that the Topo I activity was strongly decreased. In addition, the resistant cells recovered the ATP-independent relaxation activity after 3 months of exposure to CPT, but still kept a reduced CPT-induced DNA cleavage. Further evaluations at the final stage of the resistance induction have indicated that cells presented a CPT-resistant form of Topo I. Rearranged Topo I gene on one allele and a reduced Topo I transcription were also observed in resistant cells. The putative role of the rearrangement was discussed. These data show that the resistance mechanism has evolved from a decreased Topo I activity to an altered form of the enzyme, and suggest that multiple mechanisms of Topo I modifications could contribute to CPT resistance.

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      P388 and P388CPT5 murine leukemia cell lines sensitive and resistant to CPT, respectively, were kindly provided by Dr J.-F. Riou (Rhône-Poulenc Rorer, France). The P388CPT5 cell line resistant to campthotecin was derived from a stable clone of the P388CPT0.3 cell line obtained at the 42nd passage [24]. Both cell lines were grown in RPMI-1640 medium containing 0.01 mM 2-mercaptoethanol, 10 mM l-glutamine, 10% (v/v) fetal bovine serum, 100 IU/mL of penicillin, 2 μg/mL of streptomycin, 50 μg/mL of gentamycin, and 50 μg/mL of nystatin at 37° in a humidified atmosphere containing 5% CO2.

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