Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21□S

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation. Small interfering RNA (siRNA) to c-Jun was used to interrupt the amplification cycle and was found to be highly effective, reducing vinblastine-induced c-Jun expression at both the mRNA and protein levels by 90%. Apoptosis and caspase-3 activation were significantly inhibited in c-Jun siRNA-treated cells. To uncover potential mechanisms of c-Jun-mediated cell death and protection by c-Jun siRNA, candidate target genes were examined. Chromatin immunoprecipitation revealed preferential association of c-Jun with the p21 (cyclin-dependent kinase inhibitor) gene promoter after vinblastine treatment. In KB-3 cells, which have compromised p53 function, and in p53-null cells but not in p53 wild-type cells, vinblastine caused down-regulation of p21 expression concomitant with increased c-Jun expression, suggesting a role for c-Jun in negative regulation of the p21 promoter independent of p53. These results provide strong evidence that c-Jun induction in response to vinblastine plays a proapoptotic role in part via down-regulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells. The vinca alkaloid vinblastine is an important antitumor agent used for the treatment of testicular cancer and Hodgkin’s and non-Hodgkin’s lymphomas and in combination therapy for carcinomas of the lung, bladder, and for several other cancers (Rowinsky and Donehower, 1998). Vinblastine binds to tubulin subunits and inhibits tubulin polymerization, thus disrupting the dynamic instability of spindle microtubules (Jordan and Wilson, 2004). This leads to mitotic arrest and subsequent cell death by apoptosis, either soon after metaphase arrest, after a delay, or after resumption of an impaired cell cycle entailing DNA endoreduplication (Khan and Wahl, 1998; Casenghi et al., 1999). The mechanisms that trigger apoptosis after metaphase arrest are under active investigation. Antimitotic drugs such as vinblastine activate several signal transduction pathways that may promote apoptosis. Two prominent and perhaps universal responses are those that lead to the activation of c-Jun NH2-terminal kinase (JNK) (Fan and Chambers, 2001) and to phosphorylation of Bcl-2 and Bcl-xL (Ruvolo et al., 2001). Although some evidence has been presented to suggest that JNK catalyzes Bcl-2/Bcl-xL phosphorylation occurring in response to antimitotic drugs (Yamamoto et al., 1999), more recent work has indicated that a distinct kinase is responsible (Du et al., 2005). This work was supported by National Institutes of Health grant CA-5577 (to T.C.C.) and in part through Act 1 of the Arkansas Tobacco Settlement Proceeds Act of 2000 and National Center for Research Resources grants through the Biomedical Research Infrastructure Network Program (P20-RR16460). S.N.K. and A.B. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.039750. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: JNK, c-Jun NH2-terminal protein kinase; AP-1, activating protein 1; DMSO, dimethyl sulfoxide; RT, reverse transcription; siRNA, small interfering RNA; PCR, polymerase chain reaction; VBL, vinblastine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; AMC, amino-4-methyl coumarin; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone. 0026-895X/08/7301-128–136$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 1 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 39750/3285096 Mol Pharmacol 73:128–136, 2008 Printed in U.S.A. 128 http://molpharm.aspetjournals.org/content/suppl/2007/10/05/mol.107.039750.DC1 http://molpharm.aspetjournals.org/content/suppl/2008/06/24/73.1.128.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A ril 4, 2017 m oharm .aspeurnals.org D ow nladed from We have documented previously that vinblastine induces JNK activation, c-Jun expression and phosphorylation, and AP-1 activation in KB-3 carcinoma cells (Berry et al., 2001; Fan et al., 2001; Brantley-Finley et al., 2003). The increase in c-Jun expression is blocked by the JNK inhibitor SP600125, suggesting that c-Jun induction occurs via a JNK-dependent pathway (Brantley-Finley et al., 2003). Various approaches have been used to decipher the role of this pathway in the cellular response to this drug, but these have not produced a consistent answer. For example, whereas a dominant-negative to c-Jun rendered KB-3 cells more vinblastine-resistant (Fan et al., 2001), suggesting a proapoptotic role, c-Jun knockout fibroblasts, predicted to be more resistant, were equally as sensitive as wild-type cells (Obey et al., 2005). In addition, stable or inducible overexpression of c-Jun renders cells markedly resistant to vinblastine (Obey et al., 2005; Duan et al., 2007). These results may reflect cell type-specific differences in c-Jun function and probably reflect the fact that c-Jun plays a role in both cell proliferation and cell death and can functionally participate in many different protein complexes. The mechanism(s) underlying the robust induction of c-Jun protein observed in vinblastine-treated cells has not been established. Several possibilities exist. One is through transcriptional regulation. The human c-Jun promoter has several regulatory elements responsive to different signal transduction pathways. An AP-1 site at 71 to 64 is particularly responsive to c-Jun/ATF2 heterodimers and confers the ability of the c-Jun promoter to be autoregulated by its product (Angel et al., 1988). Another is through enhanced protein stability. For example, c-Jun is targeted for ubiquitin-mediated degradation by inactive JNK, and JNK-mediated phosphorylation protects against degradation (Fuchs et al., 1998). In this article, we show that the increase in c-Jun expression in response to vinblastine is through a transcriptional autoamplification mechanism involving the proximal AP-1 site in the c-Jun promoter. This suggested the use of small interfering RNA (siRNA) to block the amplification cycle, and this approach effectively eliminated c-Jun mRNA and protein induction in response to vinblastine. Apoptosis was significantly inhibited in c-Jun siRNA-treated cells compared with control cells, indicating that c-Jun induction plays an important proapoptotic role, and we present evidence implicating the cyclin-dependent kinase inhibitor, p21, as a key target. Materials and Methods Materials. ATF2, JNK1, JNK2, and actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); c-Jun (NH2 terminus) antibody was from Transduction Laboratories (San Jose, CA); c-Jun (Ser-63) and ATF2 (Thr-71) phosphospecific antibodies and p53 antibody were from Cell Signaling Technology (Waltham, MA); p21 antibody was from Calbiochem (San Diego, CA); and GAPDH monoclonal antibody was from Ambion (Austin, TX). The dual luciferase reporter assay system was from Promega (Madison, WI). Fetal bovine serum was from Hyclone (Logan, UT), and other cell culture reagents were from Invitrogen (Carlsbad, CA). c-Jun promoter luciferase reporter plasmids were kindly provided by Dr. X.-F. Wang (Duke University Medical Center, Durham, NC). Unless otherwise stated, other reagents were from Sigma Chemical Co. (St.