p 34 SEI-1 Inhibits Doxorubicin-Induced Senescence through a Pathway Mediated by Protein Kinase C-δ and c-Jun-NH 2-Kinase 1 Activation in Human Breast Cancer MCF 7 Cells

In this study, we describe a novel function of the p34 protein, which is both an oncogenic protein and a positive regulator of the cell cycle. The p34 protein was found to inhibit doxorubicin-induced senescence. We investigated the molecular mechanisms of the inhibitory effect of p34 on senescence. First, we found that the activation of protein kinase C-δ (PKC-δ), which is cleaved into a 38 kDa active form from a 78 kDa pro-form, induced after doxorubicin treatment, was inhibited by p34. Furthermore, p34 induced the ubiquitination of PKC-δ. Yet, there is no interaction between p34 and PKC-δ. We also found that the phosphorylation of c-Jun-NH2-kinase 1 (JNK1) induced after doxorubicin treatment was suppressed by p34, but not in JNK2. Consistently, pharmacologic or genetic inactivation of either PKC-δ or JNK1 was found to inhibit doxorubicin-induced senescence. In addition, the genetic inactivation of PKC-δ by PKC-δ small interfering RNA resulted in an inhibition of JNK1 activation, but PKC-δ expression was not inactivated by JNK1 small interfering RNA, implying that the activation of JNK1 could be dependently induced by PKC-δ. Therefore, p34 inhibits senescence by inducing PKC-δ ubiquitination and preventing PKC-δ–dependent phosphorylation of JNK1. [Mol Cancer Res 2009;7(11):1845–53] Introduction SEI-1 was recently identified as a cyclin-dependent kinase 4–binding protein (1), as well as a protein encoded in the chromosomal region 19q13, a region frequently amplified in patients with ovarian carcinomas, osteosarcomas, lung cancers, and pancreatic carcinomas (2-4), suggesting that SEI-1 is potentially an oncogene. An oncogenic role for p34 has been shown because expression of the protein contributes to neoplastic cell expansion through genomic instability (5). Although the precise biochemical function of p34 is not fully understood, p34 exerts at least some of its oncogenic effect although positive regulation of the cell cycle and the resulting genomic instability (6, 7). Nude mice given p34–transfected NIH3T3 mouse fibroblast cells showed more anchorage-independent growth and tumor formation than did control mice receiving vector-transfected NIH3T3 cells only (5). As with p34, proteins in the Bcl-2 family, the cell death antagonists such as Bcl-2, Bcl-xL, and E1B-19K have oncogenic effects, with the exception of death agonists such as Bax, Bak, or Bid (8, 9). High levels of Bcl-2 expression are found in a wide variety of human cancers (10). Bcl-2 has been shown to protect cells against hydrogen peroxide–induced or thiol depletion–induced death, and to suppress lipid peroxidation (11). Bcl-2 therefore functions to prevent the generation of reactive oxygen species (ROS) induced by various environmental stresses. This ability of Bcl-2 was found to be involved in the inhibition of cellular senescence, in that Bcl-2 protected cells from ROS stress (12). These results suggest that the oncogenic potential of Bcl-2 lies in its ability to inhibit senescence by preventing increases in intracellular ROS levels. Various cancer cells exposed to DNA-damaging agents undergo irreversible growth arrest and acquire a senescence-like phenotype (SLP; ref. 13). Doxorubicin is one of the most important DNA-damaging agents for the treatment of solid tumors (14). Cell death induced by doxorubicin is commonly effective only at high drug doses, with low doses showing merely cytostatic effects. A previous study found that various cancer cells treated with low doses of doxorubicin showed SLP resembling the replicative senescence of normal cells (15, 16). Senescence of cells involves the development of a morphology characterized by cell flattening and enlargement, and an increase in senescence-associated β-galactosidase (SA-β-Gal) activity (17). Received 3/4/09; revised 9/17/09; accepted 9/22/09; published OnlineFirst 11/10/09. Grant support: KOSEF (Research Center for Women's Diseases) and Sookmyung Women's University (2007). 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. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). S.L. O. Lee and S-W. Hong contributed equally. Requests for reprints: Dong-Hoon Jin, Department of Anatomy and Tumor Immunity, Medical Research Center, Seoul National University College of Medicine, Seoul 110-799, Korea. Phone: 82-2-3668-7935; Fax: 82-2-741-8208. E-mail: inno183@snu.ac. kr and Myeong-Sok Lee, Division of Biological Sciences, Sookmyung Women's University, Seoul 140-742, Korea. Phone: 822-2077-9418; Fax: 82-2-2077-7257. E-mail: mslee@sookmyung.ac. kr Copyright © 2009 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-09-0086 Mol Cancer Res 2009;7(11). November 2009 1845 Published Online First on November 10, 2009 Research. on December 29, 2017. © 2009 American Association for Cancer mcr.aacrjournals. org Downloaded from Published OnlineFirst November 10, 2009; DOI: 10.1158/1541-7786.MCR-09-0086