Protein Kinase C Overexpression Enhances Radiation Sensitivity via Extracellular Regulated Protein Kinase 1 / 2 Activation , Abolishing the Radiation-induced G 2M Arrest 1

Protein kinase C (PKC) has been widely implicated in regulation of cell growth/cell cycle progression and apoptosis. However, the role of PKC in radiosensitivity and cell cycle regulation remains unclear. Overexpression of PKC increased Ca independent PKC activity without altering other PKC isoforms (PKC , 1, , and ), and extracellular regulated protein kinase (ERK) 1/2 activity was also increased in PKC -specific manner. A clonogenic survival assay showed that PKC -overexpressed cells had more radiosensitivity and pronounced induction of apoptosis than control cells. Flow cytometric analysis revealed that PKC made the cells escape from radiation-induced G2-M arrest. Moreover, p53 and p21 induction by radiation were higher in PKC overexpressed cells than control cells, and PKC mediated apoptosis was reduced, when radiationinduced ERK1/2 activity was inhibited by PD98059. Furthermore, PKC antisense and rottlerin, PKC inhibitor-abrogated PKC -mediated radiosensitivity and reduced ERK1/2 activity to the control vector level. These results demonstrated that PKC overexpression enhanced radiation-induced apoptosis and radiosensitivity via ERK1/2 activation, thereby abolishing the radiation-induced G2-M arrest and finally apoptosis. Introduction PKC is a multigene family of serine/threonine kinases that are central to many signal transduction pathways, involving cellular growth, transformation, and differentiation. PKCfamily genes encode at least 12 distinct isoforms of lipidregulated serine/threonine kinases, and PKC isoforms can be classified into three groups, based on their structure and cofactor requirement: (a) classical PKCs (, 1, , ), which can be activated by DAG or calcium; (b) novel PKCs (, , , , ), which can be activated by DAG but not by calcium; and (c) atypical PKCs (, ), which are not responsive to either DAG or calcium (1–3). The complexity in the regulation of PKC activity, which includes not only regulation by DAG and tyrosine phosphorylation but also association to tyrosine kinases, suggests an important role of this PKC isozyme in proliferation and cell death (4, 5). Proteolytic cleavage and generation of an active CAT fragment of PKC has been visualized after treatment of DNA-damaging agent (6), and the cleaved CAT fragment of PKC is constitutively activated and is sufficient to induce apoptosis when overexpressed (7). The inhibition of apoptosis by overexpression of Bcl-2 or Bcl-XL is associated with a block of PKC cleavage (8, 9). Therefore, it is likely that PKC -mediated apoptosis may proceed by two distinct mechanisms, namely proteolytic cleavage (after DNA damage) and allosteric activation (after direct activation). Phorbol esters such as TPA and phorbol 12,13-dibutyrate activates the Raf-MEK-ERK pathway. Treatment of cells with TPA results in the activation of c-Raf and MAPK within minutes (10), and DN mutant PKC decreases the total protein level of MAPK (11), which suggests the involvement of PKC in the signaling pathway leading to MAPK activation. Whereas it is generally accepted that the activation of the ERK cascade leads to mitogenic effect (12, 13), MAPK signaling cascades are also involved in many cellular responses, including apoptosis. For example, MAPK/ERK inhibitor PD98059 blocks asbestos-induced apoptosis (14), and a requirement for ERK in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells and ovarian cell lines (15, 16) has also been demonstrated. Moreover, persistent activation of ERK1/2 contributes to glutamateinduced oxidative toxicity (17). Received 9/11/01; revised 3/19/02; accepted 4/1/02. 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. 1 Supported by the Nuclear Research and Development Program from the Ministry of Science and Technology of Korea. 2 S-J. L. and Y-S. L. contributed equally to this work. 3 To whom requests for reprints should be addressed, at Laboratory of Radiation Effect, Korea Cancer Center Hospital, 215-4 Gongneung-Dong, Nowon-Ku, Seoul 139-706, Korea. Phone: 82-2-970-1325; Fax: 82-2-9770381; E-mail: yslee@kcchsun.kcch. re.kr. 4 The abbreviations used are: PKC, protein kinase C; CDK, cyclin-dependent kinase, DAG, diacylglycerol; TPA, 12-O-tetradecanoylphorbol 13-acetate; ERK, extracellular regulated protein kinase; MAP, mitogenactivated protein; MAPK, MAP kinase; MEK, MAP/ERK kinase; JNK, cJun NH2-terminal kinase; DN, dominant-negative; CAT, catalytic. 237 Vol. 13, 237–246, May 2002 Cell Growth & Differentiation