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[Cancer Research 49, 5077-5082, September 15, 1989]
© 1989 American Association for Cancer Research
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Arrest of Replication Forks by Drug-stabilized Topoisomerase I-DNA Cleavable Complexes as a Mechanism of Cell Killing by Camptothecin1

Yaw-Huei Hsiang, Michelle G. Lihou2 and Leroy F. Liu

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Camptothecin, which induces an unusual type of DNA damage by trapping cellular topoisomerase I on chromosomal DNA in the form of drug-enzyme-DNA cleavable complexes, inhibits DNA synthesis and specifically kills S-phase cells. Cotreatment of L1210 cells with aphidicolin, which is an inhibitor of replicative DNA polymerases, completely abolished camptothecin cytotoxicity, suggesting the involvement of DNA replication in camptothecin cytotoxicity. In order to study the role of DNA replication in drug action, a cell-free SV40 DNA replication system was used in the present study. Camptothecin inhibited SV40 DNA replication in this cell-free system only in the presence of topoisomerase I. Addition of excess purified calf thymus DNA topoisomerase I to this extract system in the presence of camptothecin resulted in severe inhibition of SV40 DNA replication and the accumulation of linearized replication products, which contained covalently bound DNA topoisomerase I. We propose that the collision between moving replication forks and camptothecin-stabilized topoisomerase I-DNA cleavable complexes results in fork arrest and possibly fork breakage, which are lethal to proliferating cells.

1 This work was supported by NIH Grants CA-39962 and CA-40884.

2 Present address: Queensland Institute of Medical Research, Herstin, Queensland, 4004, Australia.

Received 12/15/88. Revised 3/27/89. Revised 5/26/89. Accepted 6/15/89.




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