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J. Biol. Chem., Vol. 279, Issue 38, 39584-39592, September 17, 2004

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ATM-dependent CHK2 Activation Induced by Anticancer Agent, Irofulven^*

Jian Wang, Timothy Wiltshire, Yutian Wang, Carmenza Mikell, Julian Burks, Cynthia Cunningham, Emily S. Van Laar¶, Stephen J. Waters¶, Eddie Reed, and Weixin Wang||

From the Mary Babb Randolph Cancer Center, and Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, West Virginia 26506 and ^¶MGI Pharma, Inc., Bloomington, Minnesota 55437

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser¹⁹⁸¹, which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.

Received for publication, January 2, 2004 , and in revised form, July 9, 2004.

^* 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.

^|| To whom correspondence should be addressed. Tel.: 304-293-2243; Fax: 304-293-4667; E-mail: wwang{at}hsc.wvu.edu.

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