Elucidating the molecular actions of camptothecin and ara-C using a synthesome-based in vitro replication assayIndiana University Purdue University Indianapolis,IN, USA
Aim: The purpose of this study is to elucidate the molecular actions of camptothecin (CPT) and 1-beta-D-arabinofuranosylcytosine (ara-C) using a multiprotein DNA replication complex (DNA synthesome) purified from human cells. The DNA synthesome is competent to support SV40 origin specific and large T antigen-dependent DNA replication in vitro. Methods: Synthesome-mediated in vitro DNA replication was performed in the absence or presence of increasing concentrations of CPT or ara-CTP and the daughter DNA molecules were analyzed using neutral and alkaline gel electrophoresis. Results: A close correlation was found between the IC50 values of CPT and ara-C required to inhibit intact cancer cell DNA synthesis and those for inhibition of synthesome-mediated in vitro DNA replication. CPT and ara-CTP showed different effects on the DNA replication intermediates produced in the in vitro replication reactions mediated by the synthesome. Using a novel in vitro kinetic assay, we demonstrated that DNA replication mediated by the synthesome is initiated within the SV40 replication origin and proceeds bidirectionally in a manner analogous to that which occurs inside the cell. Ara-CTP inhibited both the initiation and elongation stages, whereas, CPT produced most of its effects through the inhibition of the elongation phase of DNA replication. Conclusions: These data support our contention that the DNA synthesome represents the mammalian cell DNA replication machinery and further strengthens its usefulness as a highly effective and powerful in vitro model system for investigating the mechanism by which anticancer drugs affect the DNA replication process.
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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Prevention.