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Research Articles: Therapeutics, Targets, and Development

Inhibition of topoisomerase II and G₂ cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials

¹ Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center; ² The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas; and ³ Nippon Kayaku Co. Ltd., Tokyo, Japan

Requests for reprints: William Plunkett, Department of Experimental Therapeutics, Box 71, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3335; Fax: 713-794-4316. E-mail: wplunket{at}mdanderson.org

Abstract

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G₂ cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G₂ checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G₂ population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G₂ checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase II to relax supercoiled DNA and trapped topoisomerase II in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase II, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase II, an action that leads to the generation of double-strand DNA breaks, which activate the G₂ DNA damage checkpoint pathway. [Mol Cancer Ther 2007;6(5):1501–8]

Grant support: Grants CA28596 and CA32839 and Cancer Center Support grant P30 CA55164 from the National Cancer Institute, Department of Health and Human Services.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁴ Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

Received 12/15/06; revised 3/ 2/07; accepted 3/26/07.

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