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• W2051570559 abstract "Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation- and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation. Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation- and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation. The regulatory protein antizyme (AZ) 1The abbreviations used are: AZ, antizyme; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; GST, glutathione S-transferase. has been studied primarily in the context of its role in facilitating degradation of the enzyme ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine synthesis (reviewed in Refs. 1Coffino P. Biochimie (Paris). 2001; 83: 319-323Crossref PubMed Scopus (69) Google Scholar, 2Coffino P. Nat. Rev. Mol. Cell. Biol. 2001; 2: 188-194Crossref PubMed Scopus (303) Google Scholar, 3Hayashi S. Murakami Y. Matsufuji S. Trends Biochem. Sci. 1996; 21: 27-30Abstract Full Text PDF PubMed Scopus (238) Google Scholar). AZ is therefore thought to be dedicated principally to the feedback regulation of polyamine levels. AZ synthesis is controlled via an unusual mechanism of translational frameshifting. The ribosomal frameshift required for the translation of full-length AZ is directly induced by polyamines when their levels rise (4Rom E. Kahana C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3959-3963Crossref PubMed Scopus (149) Google Scholar, 5Matsufuji S. Matsufuji T. Miyazaki Y. Murakami Y. Atkins J.F. Gesteland R.F. Hayashi S. Cell. 1995; 80: 51-60Abstract Full Text PDF PubMed Scopus (414) Google Scholar). Polyamines can thus inhibit their own synthesis via AZ-mediated down-regulation of ODC. This mechanism serves to prevent extreme fluctuations in polyamine levels, which are thought to be toxic (6Webber M.M. Chaproniere-Rickenberg D. Cell Biol. Int. Rep. 1980; 4: 185-193Crossref PubMed Scopus (19) Google Scholar, 7Brunson V.G. Grant M.H. Wallace H.M. Biochem. J. 1991; 280: 193-198Crossref PubMed Scopus (89) Google Scholar, 8Allen R.D. Roberts T.K. Am. J. Reprod. Immunol. 1987; 13: 4-8Crossref PubMed Scopus (32) Google Scholar). Despite this homeostatic mechanism, the levels of polyamines and ODC vary markedly during the cell cycle, indicating that additional factors control polyamine levels and suggesting a role for this pathway in the regulation of cell proliferation (9Oredsson S.M. Biochem. Soc. Trans. 2003; 31: 366-370Crossref PubMed Scopus (118) Google Scholar). Overproduction of AZ in a variety of cell types, including malignant oral keratinocytes, hepatoma cell lines, and prostate cancer cells, coincides with growth inhibition (10Murakami Y. Matsufuji S. Miyazaki Y. Hayashi S. Biochem. J. 1994; 304: 183-187Crossref PubMed Scopus (43) Google Scholar, 11Iwata S. Sato Y. Asada M. Takagi M. Tsujimoto A. Inaba T. Yamada T. Sakamoto S. Yata J. Shimogori T. Igarashi K. Mizutani S. Oncogene. 1999; 18: 165-172Crossref PubMed Scopus (74) Google Scholar, 12Mitchell J.L. Leyser A. Holtorff M.S. Bates J.S. Frydman B. Reddy V.K. Marton L.J. Biochem. J. 2002; 366: 663-671Crossref PubMed Scopus (57) Google Scholar) and cell cycle arrest in the G1 phase (13Tsuji T. Usui S. Aida T. Tachikawa T. Hu G.F. Sasaki A. Matsumura T. Todd R. Wong D.T. Oncogene. 2001; 20: 24-33Crossref PubMed Scopus (41) Google Scholar, 14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). Furthermore, overexpression of antizyme in both mouse skin cancer and gastric epithelia models has been shown to result in tumor suppression (15Feith D.J. Shantz L.M. Pegg A.E. Cancer Res. 2001; 61: 6073-6081PubMed Google Scholar, 16Fong L.Y. Feith D.J. Pegg A.E. Cancer Res. 2003; 63: 3945-3954PubMed Google Scholar). These and other observations of antiproliferative effects of antizyme prompted us to test whether antizyme may have a specific role in cell cycle regulation, thereby accounting for its potential role as a tumor suppressor. The cell cycle arrest previously seen in prostate carcinoma and malignant oral keratinocyte models and the growth inhibition seen in a variety of cell types upon antizyme overexpression could be explained if antizyme modulated intracellular levels of cell cycle regulatory proteins such as cyclins, as it does for ODC. We previously showed that treatment of cells with the polyamine spermine resulted in both antizyme up-regulation and cell cycle arrest (14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). In the current study, we have studied the ability of antizyme to complex with and degrade cyclin D1. We report that antizyme binds to cyclin D1 and facilitates its proteasomal degradation. Using purified components, we find that antizyme-mediated degradation of cyclin D1 proceeds in the absence of ubiquitin. Since the degradation of this cyclin has been previously characterized and found to be dependent on the SCF family of ubiquitin-protein ligases (17Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar, 18Tyers M. Jorgensen P. Curr. Opin. Genet. Dev. 2000; 10: 54-64Crossref PubMed Scopus (270) Google Scholar), our data indicate that the same protein may be delivered to the proteasome via two distinct targeting mechanisms. More generally, our results suggest that antizyme may indeed act as a tumor suppressor by controlling progression through the G1 phase of the cell cycle by a novel mechanism. Plasmid Construction—The rat antizyme 1 gene was PCR-amplified from plasmid GST-AZΔT (gift of T. Tsuji) using primers AZHis-FWD (5′-gcgtggacatatggtgaaatcctcc-3′) and AZHis-REV (5′-gcacaatcatgactcgaggacaaaccc-3′). The PCR product was digested with NdeI and XhoI enzymes and ligated into plasmid pET-33b(+) (Novagen) digested with the same. The AZ PCR product was also cloned into pCMV-FLAG using the same restriction enzymes. To construct plasmids HKT-D1 and HKT-ODC, cDNAs encoding human cyclin D1 and ODC were PCR-amplified from plasmids pRcCMVD1wt and pODC10/2H, respectively (gifts of M. Ewen and T. Tsuji) using primers D1-FWD (5′-cgaggatccaatggaacaccagctcctgtgctg-3′) and D1-REVB (5′-cgaagctttcagatgtccacgtcccgcacgtc-3′) for cyclin D1 and primers RM-ODC1 (5′-ggaacagagctcaatcatgaacaac-3′) and RM-ODC2 (5′-cagctactcgagtgctatctacac-3′) for ODC. The cyclin D1 PCR product was digested with BamHI and HindIII and subcloned into plasmid pET-33b(+), whereas the ODC PCR product was digested with SacI and XhoI prior to subcloning into the same vector. Human antizyme 1 cDNA was amplified from a human cDNA library using primers AZHis-FWD and AZHis-REV and cloned into plasmid pQE30 (Qiagen). The cyclin D1 mutant T286A was generated by site-directed mutagenesis using the Stratagene QuikChange mutagenesis kit according to the manufacturer's protocol. As template, the pcDNA3-HA-CD1 construct (human cyclin D1 with C-terminal HA tag cloned into pcDNA3) was used. The mutagenic primers were 5′-acctggcttgcgcacccaccgacg-3′ (T286A.For) and 5′-cgtcggtgggtgcgcaagccaggt-3′ (T286A.Rev). Cell Culture and Transfection—The AT2.1 cell line derived from the Dunning rat prostate carcinoma (obtained from Dr. John Isaacs) was maintained as described previously (19Isaacs J.T. Isaacs W.B. Feitz W.F. Scheres J. Prostate. 1986; 9: 261-281Crossref PubMed Scopus (385) Google Scholar). For spermine treatment, cells were plated at a density of 2 × 105 cells/10-cm2 dish in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 250 nm dexamethasone (Sigma) and then incubated overnight. Cells were subsequently washed twice in serum-free RPMI 1640 containing 5 ng/ml human recombinant epidermal growth factor, 5 μg/ml bovine insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (Sigma). Cells were then incubated in the above medium without serum in the presence or absence of 10 μm spermine tetrahydrochloride (Sigma). For immunoblot analysis, 2.5 × 105 AT2.1 cells were plated in 60-mm dishes. After 16–24 h, cells were transiently transfected with 1.5 μg of pcDNA3, pcDNA3-HA-CD1, and pcDNA3-HA-CD1 (T286A), respectively using Fugene 6 (Roche Applied Science). After an additional 16–24 h, cells were treated with 10 μm spermine for 8 h. The rat hepatoma HTC cell line (obtained from Dr. John Mitchell) was maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For transient transfection for pulse-chase experiments, cells were plated at a density of 2 × 105 cells/10-cm2 dish 1 day prior to transfection. On the day of transfection, cells were transfected with 7 μg of either FLAG vector or FLAG-AZ plasmid DNA using LipofectAMINE Plus (Invitrogen) transfection reagent according to the manufacturer's recommendations. For additional pulse-chase experiments, HTC cells were transiently transfected with either 7 μg of FLAG vector or FLAG-AZ plasmid DNA in combination with 7 μg of D1(T286A) plasmid DNA. For transient transfection for immunoblot analysis, 2.5 × 105 AT2.1 cells were plated in 60-mm dishes. After 16–24 h, cells were transiently transfected with 1.5 μg of pcDNA3, pcDNA3-HA-CD1, and pcDNA3-HA-CD1 (T286A), respectively, using Fugene 6 (Roche Applied Science). After an additional 16–24 h, cells were treated with 10 μm spermine for 8 h. Stable transfectants of the hamster malignant oral keratinocyte cell line HCPC containing either pcDNA3 vector alone or pcDNA3/hamster AZ were obtained from Dr. Takanori Tsuji. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 200 μg/ml G418. Immunoblotting—Cells were washed twice in phosphate-buffered saline (PBS) and lysed in lysis buffer A (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 50 mm Tris-HCl (pH 8.0)). Equal amounts of total protein were separated by SDS-PAGE; electrotransferred to nitrocellulose membrane (Schleicher and Schuell); and probed with either cyclin D1 (HD-11; 1:500), cyclin A (C19; 1:500), or Cdk2 (M2; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), pRb (1:500; Pharmingen), ODC (1:500 (gift from P. Coffino) or 1:1000 (Progen)), actin (C4; 1:2500; Roche Applied Science), or antizyme (polyclonal, 1:2000; gift from J. Mitchell). Immunoprecipitation—For immunoprecipitation of in vitro translated proteins, human cyclin D1 and rat antizyme were individually transcribed and translated in vitro from plasmids pD1HA (gift of M. Ewen) and pHKTAZ, respectively, in the presence of [35S]methionine, using the reticulocyte-based TNT transcription/translation system (Promega). The transcription/translation reaction was stopped by the addition of 20 mm EDTA, which also served to prevent protein degradation by inhibiting any remaining ATP-dependent proteasome activity in the reaction mixture. Extracts were mixed in proportions yielding equivalent radioactivity among the in vitro translated proteins. Proteins were then allowed to interact in immunoprecipitation buffer (20 mm HEPES (pH 7.5), 5 mm KCl, 1.5 mm MgCl2, 1 mm dithiothreitol) for 60 min at 4 °C prior to the addition of antibody against antizyme or cyclin D1. After an additional 60-min incubation at 4 °C, immune complexes were isolated by the addition of agarose beads conjugated to either protein A or protein G (Santa Cruz Biotechnology, Inc.) and additional incubation for 60 min at 4 °C. The beads were harvested by centrifugation at 8,000 × g for 5 min, resuspended in sample buffer, and boiled prior to separation of proteins by SDS-PAGE. For immunoprecipitation-Western blots of FLAG-hAZ or HA-human cyclin D1, 3.5 × 105 293 cells were transfected with 1 μg of each plasmid (pcDNA3HA-D1, pCMVFLAG-AZ1, or empty vector) using Fugene 6 transfection reagent according to the manufacturer's instructions. Cells were harvested 36 h after transfection and lysed by vortexing on ice. The lysis buffer contained 50 mm Tris (pH 7.4), 50 mm NaCl, 5 mm MgCl2, 0.1% Triton X-100 supplemented with protease inhibitor mixture (Roche Applied Science). The cell lysate (1 ml) was incubated with 4 μg of antibody against the FLAG epitope of AZ (M2, mouse monoclonal; Sigma) for 2 h at 4 °C. The antibody was precipitated at 4 °C for an additional 2 h using 30 μl of protein A beads (Pierce). The beads were washed three times in lysis buffer, resuspended in 60 μl of Laemmli loading buffer, and boiled. Samples were applied to SDS-PAGE and analyzed by immunoblot using antibody against the HA tag of cyclin D1. 10 μl of preimmunoprecipitation cell lysate was also analyzed by SDS-PAGE and immunoblot as a transfection control. Antisense Inhibition of Antizyme Up-regulation in Vivo—AT2.1 cells were cultured for spermine treatment as described above. Prior to treatment, 0.5 mm standard control (5′-cctcttacctcagttacaatttata-3′) or antizyme-specific (5′-gggtccgaaaccagcaaaaacctcc-3′) fluorescein isothiocyanate-conjugated antisense morpholino oligonucleotides (GeneTools) were delivered into cells using Special Delivery reagent (GeneTools) according to the manufacturer's protocol. 24 h after morpholino delivery, cells were treated with spermine as described above for an additional 24 h prior to lysis and immunoblotting. To silence AZ gene expression in HCPC-1 or HTC cells, a 21-nucleotide sequence was designed to target the mRNA sequence 5′-aaccuucagcuuucuuggcuu-3′. The AZ-specific siRNA and negative control siRNA (Scramble II duplex) were from Dharmacon Research. 2 × 105 (HCPC-1) or 3.5 × 105 (HTC) cells were plated per 35-mm well. For each well, 6 μl of LipofectAMINE 2000 (Invitrogen) was used as transfection reagent to deliver 400 pmol of siRNA duplex. Cells were washed first with serum-free, antibiotic-free medium. The mix containing RNAi and LF2000 was then added. After 4 h of incubation, serum was added to the medium. Cell lysates were collected 2 days after transfection. 35S Labeling and Pulse-Chase—For short metabolic labeling, AT2.1 cells were treated with spermine as described above. 1, 4, 8, and 12 h after spermine addition, cells were washed and starved for 30 min in methionine-free, cysteine-free RPMI (Invitrogen). Subsequently, cells were washed twice in PBS and then incubated for 10 min in the above medium containing 10% dialyzed fetal bovine serum (Invitrogen) and 100 mCi/ml 35S EXPRESS labeling mix (PerkinElmer Life Sciences). After labeling, cells were washed four times in PBS and lysed in lysis buffer A (see above). Subsequently, lysates were used for immunoprecipitation using antibody against cyclin D1. Purified complexes were separated by SDS-PAGE and quantified using a phosphorimaging system (ImageQuant). For pulse-chase analysis, HTC cells were cultured and transiently transfected as described above. Cells were then starved for 30 min in methionine-free, cysteine-free Dulbecco's modified Eagle's medium (Invitrogen). Subsequently, cells were washed twice with PBS and then incubated for 30 min in the above medium containing 10% dialyzed fetal bovine serum and 100 μCi/ml 35S EXPRESS labeling mix. After labeling, cells were washed twice in PBS. The medium was then replaced with growth medium containing 50 μg/ml cysteine (Invitrogen), and 50 μg/ml methionine (Invitrogen). After rinsing with PBS, cells were lysed in lysis buffer A (see above) for 0, 10, 20, 40, 60, and 80 min after the addition of the chase medium. The lysates were then used for immunoprecipitation as described above using antibody against the FLAG epitope of AZ, cyclin D1, or β-actin. Proteins were separated by SDS-PAGE, visualized, and quantitated using a phosphorimaging system. The signal intensity of each band was normalized against that of β-actin. Complex Formation Using Purified Proteins—His-tagged human cyclin D1 and ODC proteins were expressed in Escherichia coli strain BL21(DE3). Proteins were purified by nickel chelate affinity chromatography (Qiagen) following the manufacturer's instructions. Rat GST-AZ was expressed in E. coli strain BL21(DE3) and purified with glutathione-Sepharose resin (Amersham Biosciences) according to manufacturer's instructions. To assay complex formation using GSH resin (Amersham Biosciences), 31 μg of GST or 56 μg of rat GST-AZ proteins were incubated with a 3-fold molar excess of cyclin D1, ODC, or citrate synthase (Roche Applied Science) in binding buffer (50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 5 mm MgCl2, 10% glycerol, 4 mg/ml bovine serum albumin) for 45 min at 30 °C in a volume of 1 ml. To show competition between cyclin D1 and ODC for AZ binding, AZ was preincubated with a 2- or 10-fold molar excess of ODC for 15 min. The reaction mixture was then applied to a GSH resin column at 4 °C for 1 h and subsequently washed with binding buffer lacking bovine serum albumin. Bound proteins were eluted with buffer containing 50 mm Tris-HCl (pH 7.8), 150 mm NaCl, 10% glycerol, and 20 mm reduced glutathione. Proteins were separated by SDS-PAGE and analyzed by Coomassie Brilliant Blue staining or immunoblotting. In Vitro Protein Degradation in Reticulocyte Lysates—Human cyclin D1 and rat antizyme were synthesized in vitro using reticulocyte lysates as described above. Equal amounts of 35S-labeled cyclin D1 were incubated with antizyme in ATP-regenerating buffer (30 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 2 mm dithiothreitol, 1 mm ATP, 10 mm creatine phosphate, 1.6 mg/ml phosphocreatine kinase) for 90 min at 37 °C. The reaction was stopped by the addition of an equal volume of 2× SDS-PAGE sample buffer (100 mm Tris-HCl (pH 6.8), 4% SDS, 0.2% bromphenol blue, 10% glycerol, 100 mm dithiothreitol) and then boiled prior to analysis by SDS-PAGE. In Vitro Degradation Assay Using Purified Proteasomes—Human 26 S proteasomes were purified from red blood cells as described previously (20Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.M. Finley D. Schmidt M. Nat. Cell Biol. 1999; 1: 221-226Crossref PubMed Scopus (388) Google Scholar). Purified protein substrates, rat antizyme, and purified proteasomes were incubated in degradation buffer (50 mm Tris (pH 7.2), 0.01% Tween 20, 1 mm dithiothreitol, 400 μg/ml bovine serum albumin, 1 mm EDTA, 5 mm MgCl2, 100 mm NaCl, 10% glycerol) and an ATP-regenerating system containing phosphocreatine (10 mm), ATP (2 mm), and creatine phosphokinase (3 units) for 0, 2, and 4 h at 30 °C. Benzyloxycarbonyl-Ile-Glu-(OBut)-Ala-Leu-H-aldehyde (0.2 mm; Peptide Institute) and apyrase (0.6 units; Sigma) were added where indicated. The reaction volume was 25 μl. After incubation, the reaction was stopped by the addition of SDS-PAGE sample buffer and then boiled prior to analysis by SDS-PAGE. Up-regulation of Endogenous Antizyme in Prostate Cancer Cells Results in Degradation of Cell Cycle Regulatory Proteins—The addition of exogenous polyamines has been shown to up-regulate endogenous antizyme levels in a variety of different cell lines. We previously showed that induction of antizyme within 2 h after the addition of the polyamine spermine is followed by G1 growth arrest in prostate carcinoma cell lines (14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). Since cyclins are key determinants of cell cycle transitions, we investigated whether growth arrest was due to a change in the levels of specific cyclins responsible for the G1-S transition. Cyclin levels were measured in cell lines grown in defined media in the presence or absence of 10 μm spermine. After 24 h, we observed a marked decrease in the levels of cyclin D1 in spermine-treated cells relative to untreated controls (Fig. 1a). Under the conditions of this experiment, antizyme is efficiently induced, as previously shown (14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). As expected, ODC levels are reduced following antizyme induction (Fig. 1a). Levels of other cell cycle regulatory proteins, such as Cdk2 and cyclin A, remained unchanged, as did the level of actin (Fig. 1a). Transcriptional regulation could not account for the decrease in cyclin D1 levels in these cell lines, since treated and untreated cells contained equivalent levels of mRNA encoding this protein (Fig. 1b). Furthermore, loss of cyclin D1 could not be attributed to a block in translation, since synthesis of cyclin D1 protein remained constant during spermine treatment in these cell lines (Fig. 1c). A critical function of the cyclin D1-cyclin-dependent kinase complex is phosphorylation of the retinoblastoma protein (pRb), which is required for entry into the S phase of the cell cycle (21Ewen M.E. Sluss H.K. Sherr C.J. Matsushime H. Kato J. Livingston D.M. Cell. 1993; 73: 487-497Abstract Full Text PDF PubMed Scopus (917) Google Scholar, 22Dowdy S.F. Hinds P.W. Louie K. Reed S.I. Arnold A. Weinberg R.A. Cell. 1993; 73: 499-511Abstract Full Text PDF PubMed Scopus (691) Google Scholar, 23Kato J. Matsushime H. Hiebert S.W. Ewen M.E. Sherr C.J. Genes Dev. 1993; 7: 331-342Crossref PubMed Scopus (1092) Google Scholar). After spermine-induced loss of cyclin D1, pRb was found in a hypophosphorylated form (Fig. 1d). By this functional criterion, it is likely that cyclin D1-cyclin-dependent kinase complexes are effectively inhibited by spermine treatment. The loss of G1-specific cyclin D1-cyclin-dependent kinase complexes and the resulting decrease in Rb phosphorylation may account for the predominant G1 arrest seen in these cells after spermine treatment (14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). Antizyme Is Required for the Destruction of Cyclin D1—The loss of cyclin D1 seen in spermine-treated prostate carcinoma cells could be a consequence of spermine-induced cell cycle arrest rather than a consequence of antizyme up-regulation. To determine whether antizyme alone is sufficient to promote degradation of cyclin D1, we looked at the effect of blocking antizyme synthesis on the levels of cyclin D1 following spermine treatment. Nonspecific and antizyme-specific antisense morpholino oligonucleotides were introduced into rat prostatic carcinoma cells and incubated for 24 h. Cells were then incubated in the presence or absence of spermine prior to analysis of antizyme and cyclin D1 levels. As expected, after 24 h in the presence of spermine, we observed a marked increase in the levels of antizyme in those cells containing the control morpholino (Fig. 2a). In contrast, the addition of the antizyme-specific antisense morpholino inhibited antizyme up-regulation relative to the control. Coincident with this decrease in antizyme levels, we observe increased levels of cyclin D1 in spermine-treated cells in the presence of the anti-antizyme morpholino. Levels of Cdk2 were unaffected under all conditions tested. These results suggest that degradation of cyclin D1 in these cells is not a nonspecific effect of spermine treatment but does depend on the presence of increased levels of antizyme. Whereas our data indicate that spermine-mediated induction of antizyme leads to degradation of cyclin D1, levels of polyamines and antizyme have been shown to fluctuate even in cells growing under normal conditions. To determine whether antizyme has a role in the degradation of cyclin D1 in the absence of spermine treatment, we looked at the effect of blocking endogenous antizyme synthesis on the levels of cyclin D1 in untreated cells. HTC hepatoma cells were grown in routine cell culture medium and were transfected with either scrambled or AZ-specific siRNA and incubated for 24 h. Analysis of the endogenous levels of AZ along with cell cycle regulators indicated that suppression of antizyme expression again coincided with stabilization of cyclin D1 (Fig. 2b). Levels of other cell cycle regulators such as Cdk4 or cyclin A were not affected by ablation of AZ, indicating that antizyme specifically promotes degradation of cyclin D1 even in cells grown under normal cell culture conditions. As expected, levels of ODC, a known target of AZ-mediated degradation, were increased in HCPC malignant oral keratinocytes transfected with AZ-specific siRNA (Fig. 2c). Antizyme Overexpression Leads to Loss of Cyclin D1—Similar to the effects seen in prostate carcinoma cells, stimulation of antizyme expression in rat hepatoma cells has been shown to result in growth inhibition (12Mitchell J.L. Leyser A. Holtorff M.S. Bates J.S. Frydman B. Reddy V.K. Marton L.J. Biochem. J. 2002; 366: 663-671Crossref PubMed Scopus (57) Google Scholar). Additionally, overexpression of antizyme in malignant oral keratinocytes and prostate carcinoma has previously been shown to lead to G1 growth arrest and a reversal of the malignant phenotype (13Tsuji T. Usui S. Aida T. Tachikawa T. Hu G.F. Sasaki A. Matsumura T. Todd R. Wong D.T. Oncogene. 2001; 20: 24-33Crossref PubMed Scopus (41) Google Scholar, 14Koike C. Chao D.T. Zetter B.R. Cancer Res. 1999; 59: 6109-6112PubMed Google Scholar). We overexpressed antizyme in rat hepatoma cells (Fig. 3a) and malignant oral keratinocytes (Fig. 3b) to investigate whether the growth inhibition previously seen in these cell types could be correlated with a change in cyclin D1 levels. Full-length antizyme is efficiently expressed in these transfectants. We observed a marked decrease in the levels of cyclin D1 in cell lines overexpressing AZ as compared with control cells. Levels of another cell cycle regulatory protein, Cdk2, remained unchanged, as did the level of actin. These results show that overexpression of antizyme alone is sufficient to promote specific degradation of cyclin D1 in these cell types. To further investigate the ability of AZ to degrade cyclin D1 and to dissociate this process from the well studied ubiquitin-dependent degradation pathway, we employed a mutant form of cyclin D1 (T286A) that cannot be degraded via the ubiquitin pathway (24Diehl J.A. Zindy F. Sherr C.J. Genes Dev. 1997; 11: 957-972Crossref PubMed Scopus (648) Google Scholar, 25Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1861) Google Scholar). As shown in Fig. 4, levels of this mutant are stable in control AT2.1 cells but fell significantly following spermine-induced up-regulation of antizyme. These results add support for the hypothesis of an alternate pathway for a ubiquitin-independent pathway of cyclin D1 turnover. Taken together, our antizyme ablation and overexpression results show that alteration of antizyme levels leads to changes in cyclin D1 levels, consistent with the hypothesis that antizyme influences cyclin D1 levels in vivo whether levels of AZ are modulated by overexpression, by gene silencing, or by polyamine-mediated induction. Our results in untreated HTC cells confirm a role for endogenous AZ in modulating cyclin D1 levels. During normal cell cycle progression, similar results were obtained using a variety of cell types. This indicates that even low, endogenous levels of antizyme have some capacity to affect cyclin D1 levels in the cell types tested. The Proteasome Mediates Down-regulation of Cyclin D1 via Accelerated Breakdown in a Ubiquitin-independent Manner—To show directly that enhanced protein degradation mediates the decreased levels of cyclin D1 in a ubiquitin-independent manner, we used AZ-overexpressing cells to determine the half-life of the mutant form of cyclin D1 (T286A) that cannot be degraded via the ubiquitin pathway. Hepatoma cells transiently transfected with a plasmid vector expressing the T286A cyclin D1 mutant along with either empty vector or full-length AZ were pulsed with [35S]methionine and then chased with medium contai" @default.
• W2051570559 created "2016-06-24" @default.
• W2051570559 creator A5011006647 @default.
• W2051570559 creator A5011693258 @default.
• W2051570559 creator A5028446388 @default.
• W2051570559 creator A5032162260 @default.
• W2051570559 creator A5032324140 @default.
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• W2051570559 date "2004-10-01" @default.
• W2051570559 modified "2023-09-30" @default.
• W2051570559 title "Antizyme Targets Cyclin D1 for Degradation" @default.
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