Originally published In Press as doi:10.1074/jbc.M313538200 on April 8, 2004
J. Biol. Chem., Vol. 279, Issue 24, 25813-25822, June 11, 2004
Sulforaphane-induced G2/M Phase Cell Cycle Arrest Involves Checkpoint Kinase 2-mediated Phosphorylation of Cell Division Cycle 25C*
Shivendra V. Singh
,
Anna Herman-Antosiewicz,
Ajita V. Singh,
Karen L. Lew,
Sanjay K. Srivastava,
Ravindra Kamath¶,
Kevin D. Brown||,
Lin Zhang, and
Rajasekaran Baskaran¶
From the
Department of Pharmacology and University of Pittsburgh Cancer Institute, and the ¶Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, the ||Department of Biochemistry and Molecular Biology and the Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G2/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3
. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G2/M arrest. HCT116 human colon cancer-derived Chk2-/- cells were significantly more resistant to G2/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G2/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.
Received for publication, December 10, 2003
, and in revised form, April 5, 2004.
* This work was supported by United States Public Health Service Grants CA101753 (to S. V. S.) and CA60945 (to R. B.) awarded by the NCI, National Institutes of Health. 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: Hillman Cancer Center, Research Pavilion Suite 2.32A, University of Pittsburgh Cancer Institute, 5117 Center Ave., Pittsburgh, PA 15213. Tel.: 412-623-3263; Fax: 412-623-7828; E-mail: singhs{at}msx.upmc.edu.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
