Department of Hematology and Oncology, Faculty of Medical Sciences, University of Fukui, 23-3 Shimoaizuki, Matsuoka, Eiheiji, Fukui 910-1193, Japan.
Oncol Rep. 2011 Feb;25(2):347-52. doi: 10.3892/or.2010.1100. Epub 2010 Dec 10.
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.
喜树碱（CPT）是一种拓扑异构酶 I 抑制剂，它与拓扑异构酶 I 和单链 DNA 形成可裂解复合物。当这个复合物遇到复制叉时，碰撞会产生不可逆转的双链断裂，从而诱导细胞凋亡。基于作用机制，我们假设处于细胞周期的细胞比非细胞周期的细胞对 CPT 更敏感，并且被刺激进行 DNA 合成的细胞对 CPT 会变得敏感。该研究重点关注 CPT 诱导的 DNA 链断裂与细胞凋亡之间的关系，因为诱导 DNA 链断裂对于细胞毒性是必不可少的。我们使用彗星试验来定量 DNA 链断裂，并用 Annexin V 阳性来确定细胞毒性的水平。正常淋巴细胞被用作静止细胞的模型。首先，用 CPT 处理培养的白血病细胞系 CCRF-CEM。CEM 细胞对 CPT 敏感，CPT 诱导的 DNA 链断裂的数量与浓度和时间呈依赖性。DNA 链断裂的增加似乎与随后的细胞凋亡增加有关。当用 CPT 处理正常淋巴细胞时，DNA 链断裂迅速消失，随后诱导的细胞凋亡最小。然而，当正常淋巴细胞被刺激进行 DNA 合成时，淋巴细胞对 CPT 变得敏感，导致 DNA 链断裂增加和凋亡增强。同样，DNA 链断裂的程度与细胞毒性的大小有关。因此，CPT 在 DNA 合成的背景下对受刺激的正常淋巴细胞具有细胞毒性。
