Camptothecin induction of a time- and concentration-dependent decrease of topoisomerase I and its implication in camptothecin activity.

Abstract

Camptothecin (CPT) has been shown to induce protein-linked DNA breaks (PLDB), which are stabilized intermediates of topoisomerase I (TOP1) activity. Due to the reversible nature of PLDB and the need for replication fork movement for CPT toxicity, both the time of CPT exposure and TOP1 levels are determinants of CPT toxicity. Therefore, the effects of CPT exposure on TOP1 over time were examined in an established human cell line, KB. Using an in vivo KCl-SDS co-precipitation assay, it was determined that 1 hr of CPT exposure resulted in a concentration-dependent increase in PLDB that reached a maximum at 5 microM CPT. However, prolonged incubations with CPT revealed a concentration- and time-dependent decrease in CPT-induced PLDB formation. The most rapid loss of PLDB was within 6 hr. Neither aphidicolin nor cycloheximide cotreatments altered the PLDB decrease induced by CPT. Immunoblot analysis revealed a reduction in TOP1 protein upon CPT exposure, whereas RNA analysis revealed no changes, which suggested a post-transciptional mechanism of TOP1 down-regulation. The CPT-induced reduction was specific for TOP1, because actin and tubulin levels were unaltered by CPT exposure. Finally, clonogenic assays revealed a small but statistically significant decrease in CPT toxicity throughout the CPT exposure period. Because PLDB formation based on TOP1 levels is an important step in the toxicity of CPT, the CPT-induced TOP1 reduction could be a transient mechanism of resistance for cells to avoid toxic levels of PLDB.