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Clusterin-Mediated Apoptosis Is Regulated by Adenomatous Polyposis Coli and Is P21 Dependent but P53 Independent

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Clusterin-Mediated Apoptosis Is Regulated by Adenomatous Polyposis Coli and Is P21 Dependent but P53 Independent [CANCER RESEARCH 64, 7412–7419, October 15, 2004] Clusterin-Mediated Apoptosis Is Regulated by Adenomatous Polyposis Coli and Is p21 Dependent but p53 Independent Tingan Chen,1 Joel Turner,1 Susan McCarthy,1 Maurizio Scaltriti,2 Saverio Bettuzzi,2 and Timothy J. Yeatman1 1Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida; and 2Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell’Esercizio Fisico, Parma, Italy ABSTRACT ptosis (15). Additional data suggest that a secreted form of clusterin acts as a molecular chaperone, scavenging denatured proteins outside Clusterin is a widely expressed glycoprotein that has been paradoxi- cells following specific stress-induced injury such as heat shock. cally observed to have both pro- and antiapoptotic functions. Recent Other data, show that overexpression of a specific nuclear form of reports suggest this apparent dichotomy of function may be related to two different isoforms, one secreted and cytoplasmic, the other nuclear. To clusterin acts as a prodeath signal (16). Furthermore, studies of human clarify the functional role of clusterin in regulating apoptosis, we exam- colon cancer suggest a conversion from the nuclear form of clusterin ined its expression in human colon cancer tissues and in human colon to the cytoplasmic form, which may promote tumor progression (17). cancer cell lines. We additionally explored its expression and activity using Recently, a link between the accumulation of the nuclear form of models of adenomatous polyposis coli (APC)- and chemotherapy-induced clusterin and anoikis induction in prostate epithelial cells was shown apoptosis. Clusterin RNA and protein levels were decreased in colon (18). cancer tissues largely devoid of wild-type APC when compared with To better elucidate the functional role of clusterin, we examined its matched normal tissue controls, suggesting a means for invasive cancers to expression and localization in a panel of normal and neoplastic human avoid apoptosis. Conversely, induction of apoptosis by expression of wild- colon cancer tissues, as well as a panel of human colon cancer cell type APC or by treatment with chemotherapy led to increased clusterin lines. We then evaluated the effect of APC expression and chemo- RNA and protein levels localizing to apoptotic nuclei. We found that transient transfection of clusterin to colon cancer cell lines directly en- therapy, both known to induce apoptosis, on clusterin expression and hanced basal and chemotherapy-induced apoptosis. Clusterin-induced associated apoptotic events. The effect of clusterin transfection, or apoptosis was inhibited by antisense clusterin and was found to be highly alternatively, antisense clusterin transfection, on basal and chemother- dependent on p21 but not p53 expression, yet a deficit in p21 can be apy-induced apoptosis was investigated. Because both p21 and p53 subverted by clusterin transfection. Collectively, these data support the have been lined to regulation of apoptotic events, clusterin expression hypothesis that nuclear clusterin function is proapoptotic when induced and induction of apoptosis was evaluated in the context of their by APC or chemotherapy in the context of p21 expression. Absent of p21, expression using targeted knockout cell line models. clusterin in not induced, and apoptosis is significantly inhibited. These data support a potential therapeutic role for clusterin in enhancing che- motherapy-induced apoptosis and in promoting apoptosis in cells deficient MATERIALS AND METHODS in p21. Human Tissues. Snap-frozen human colon cancers (Dukes’ stage C) and INTRODUCTION their cognate normal mucosal controls were obtained from the Moffitt Cancer Center tissue bank in a de-identified fashion with an Institution Review Clusterin is a widely expressed glycoprotein whose function re- Board-approved protocol. Tissues were microdissected for purity and then mains somewhat enigmatic. Also known as apolipoprotein J, clusterin exposed to cellular lysis buffers and Trizol for RNA extraction. was initially characterized as an apoptosis-associated transcript after it Cell Lines. Four different human colon cancer cell lines were obtained was identified as testosterone-repressed prostate message 2 (1) and from the American Type Culture Collection (HT29, KM12C, HCT116, and sulfated glycoprotein 2 (2) that is overexpressed in the epithelial cells SW620), and two targeted knockout cell lines were a kind gift of Dr. B. Ϫ Ϫ Ϫ Ϫ of the regressing rat ventral prostate (3). Increases in clusterin mRNA Vogelstein (HCT116p21 / and HCT116p53 / ). The HT29 cell line de- void of APC but engineered with a zinc-inducible wild-type APC vector and protein levels have since been consistently detected in apoptotic (HT29APC) was also a gift of Dr. Bert Vogelstein. HT29, KM12C, HCT116, heart, brain, lung, liver, kidney, pancreas, and retinal tissue both in and SW620 cells were cultured in RPMI medium with 10% fetal bovine serum. vivo and in vitro (1, 4–11) Moreover, clusterin-knockout mice show HT29APC, HCT116p21Ϫ/Ϫ, HCT116p53Ϫ/Ϫ, and their parental cells were reductions of cell death in hypoxia-ischemia–induced brain damage cultured in McCoy’s 5A medium with 10% fetal bovine serum. Cells were (12–14). Clusterin function, however, has also been reported to be treated with Zn2ϩ (120 ␮mol/L) 4, 8, and 16 hours or with 5-flurouracil (5-FU; antiapoptotic and has been found to be overexpressed in progressing 5 ␮mol/L) and anti-Fas antibody (50 ng/mL) or with irinotecan (6 ␮mol/L) 24, breast carcinomas, where its expression correlated inversely with the 48, and 72 hours to induce apoptosis. apoptotic index. Similarly, clusterin-knockout mice show an increase in Transient Transfection of Clusterin. The plasmids p4 empty vector and autoimmune myocardial damage, suggesting an antiapoptotic function. p4/SGP2 (containing human clusterin full-length cDNA) were obtained from Recent studies have shed some light on the dichotomous functions Dr. Saverio Bettuzzi (Parma, Italy). Transient transfection of p4 empty vector (as a mock) or p4/SGP2 were performed using FuGENE 6 Transfection of clusterin, suggesting pro- and antiapoptotic functions may be Reagent (Boehringer Mannheim, Mannheim, Germany) according to the man- related to nuclear and secreted isoforms, respectively, and that the ufacturer’s protocol. After transfection with mock or clusterin 24 to 48 hours, function of clusterin may be context dependent. Overexpression of the cells were either harvested or treated by apoptotic stimuli for 48 hours, then secreted form of clusterin in human cancer cells caused drug resist- harvested to detect clusterin protein expression by Western blotting or to ance and protection against certain cytotoxic agents that induce apo- perform apoptosis assays. Each transfection experiment was done in triplicate. Data are presented as a mean Ϯ SE. Received 6/11/04; revised 8/4/04; accepted 8/19/04. Western Blot Analyses. Cells were harvested in protein lysis buffer [20 The costs of publication of this article were defrayed in part by the payment of page mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% NP40, charges. This article must therefore be hereby marked advertisement in accordance with 1 mmol/L DTT, 5 ␮mol/L trichostatin A, 1 mmol/L sodium orthovanadate, 1 18 U.S.C. Section 1734 solely to indicate this fact. mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L NaF, and complete protease Requests for reprints: Timothy J. Yeatman, 12902 Magnolia Drive, SRB-2, Tampa, FL 33612-9497. Phone: (813) 979-7292; Fax: (813) 632-1433; E-mail: [email protected]. inhibitors (Roche Applied Science, Indianapolis, IN)], and Western blotting ©2004 American Association for Cancer Research. was performed as described previously (19). All blots were performed in 7412 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. CLUSTERIN-MEDIATED APOPTOSIS triplicate. Representative examples are shown in the figures. Antibodies used labeling (TUNEL) staining with the In Situ Cell Death Detection Kit (TMR include clusterin (Alexis, San Diego, CA) and p53 and p21 (BD Biosciences, red; Roche Applied Science) according to the manufacturer’s protocol and San Jose, CA). immunoblotting with human clusterin antibodies (Alexis). Antisense Clusterin Oligonucleotide Transfection. Preattached ϳ1 ϫ 105 Immunocytofluorescence and TUNEL Assay. Preattached ϳ1 ϫ 104 HT29, HCT11,6 and HT29APC cells in 100-mm culture dishes, respectively, cells/well were exposed to treatment conditions as planned (some cells were for overnight. They were transfected with antisense clusterin oligonucleotides treated 48 hours by apoptotic stimuli, some cells were transiently transfected or mismatch clusterin oligonucleotides, respectively. Phosphorothioate oligo- with clusterin 24 hours, and then treated another 48 hours with apoptotic nucleotides used in this study were purchased from Integrated DNA Technol- stimuli). Cells were fixed with acetone-methanol (1:1; Sigma, St. Louis, MO) ogies, Inc. (Coralville, IA). The sequence of the clusterin antisense clusterin for 3 minutes, then were blocked with PBS ϩ10% normal goat serum for 20 oligonucleotide used corresponded to the human clusterin translation initiation minutes. Cells were incubated with clusterin antibody (1:200; Alexis) in PBS site (5Ј-CAGCAGCAGAGTCTTCATCAT-3Ј). A 2-base clusterin mismatch ϩ10% normal goat serum for 2 hours,
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