Camptothecin induces DNA strand breaks and is cytotoxic in stimulated normal lymphocytes

Abstract

Camptothecin (CPT), a topoisomerase I inhibitor, forms a cleavable complex with topoisomerase I and single-stranded DNA. When this complex meets a replication fork, the collision generates irreversible double-strand breaks, thereby inducing apoptosis. Based on the mechanism of action, we hypothesized that cycling cells would be more sensitive to CPT than non-cycling cells and that cells stimulated to undergo DNA synthesis would be sensitized to CPT. The study focused on the association between CPT-induced DNA strand breaks and apoptotic cell death, because the induction of DNA strand breaks is indispensable for cytotoxicity. We used the Comet assay to quantitate DNA strand breaks and Annexin V positivity to determine the level of cytotoxicity. Normal lymphocytes were used as a model for quiescent cells. First, the cultured leukemic cell line CCRF-CEM was treated with CPT. CEM cells were sensitive to CPT, and the amount of CPT-induced DNA strand breaks was concentration- and time-dependent. The increase in DNA strand breaks appeared to be correlated to a subsequent increase in apoptosis. When normal lymphocytes were treated with CPT, DNA strand breaks quickly disappeared, and the subsequent induction of apoptosis was minimal. However, when normal lymphocytes were stimulated to undergo DNA synthesis, the lymphocytes were sensitized to CPT with increased DNA strand breaks and enhanced apoptosis. Again, the extent of DNA strand breaks was associated with the magnitude of cytotoxicity. Thus, CPT was cytotoxic to stimulated normal lymphocytes in the context of DNA synthesis.