p53-dependent Down-regulation of Telomerase Is Mediated by p21
2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês
10.1074/jbc.m402502200
ISSN1083-351X
AutoresIgor Shats, Michael Milyavsky, Xiaohu Tang, Perry Stambolsky, Neta Erez, Ran Brosh, Ira Kogan, Ilana Braunstein, Maty Tzukerman, Doron Ginsberg, Varda Rotter,
Tópico(s)RNA Interference and Gene Delivery
ResumoInactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F. Inactivation of p53 and activation of telomerase occur in the majority of human cancers, raising the possibility of a link between these two pathways. Overexpression of wild-type p53 down-regulates the enzymatic activity of telomerase in various cancer cell lines through transcriptional repression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In this study, we re-evaluated the role of p53 in telomerase regulation using isogenic cell lines expressing physiological levels of p53. We demonstrate that endogenous wild-type p53 was able to down-regulate telomerase activity, hTERT mRNA levels, and promoter activity; however, the ability to repress hTERT expression was found to be cell type-specific. The integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains of p53 was essential for hTERT promoter repression, whereas the proline-rich domain and the extreme C terminus were not required. Southwestern and chromatin immunoprecipitation experiments demonstrated lack of p53 binding to the hTERT promoter, raising the possibility of an indirect repressive mechanism. The down-regulation of hTERT promoter activity was abolished by a dominant-negative E2F1 mutant. Mutational analysis identified a specific E2F site responsible for p53-mediated repression. Knockdown of the key p53 transcriptional target, p21, was sufficient to eliminate the p53-dependent repression of hTERT. Inactivation of the Rb family using either viral oncoproteins or RNA interference attenuated the repression. Inhibition of histone deacetylases also interfered with the repression of hTERT by p53. Therefore, our results suggest that repression of hTERT by endogenous p53 is mediated by p21 and E2F. Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres at the end of eukaryotic chromosomes, has been implicated in aging, immortalization, and transformation. The human telomerase complex is composed of a catalytic subunit (hTERT) 1The abbreviations used are: hTERT, human telomerase reverse transcriptase; siRNA, small interfering RNA; RNAi, RNA interference; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UTR, untranslated region; PBS, phosphate-buffered saline. with a reverse transcriptase activity (1Nakamura T.M. Morin G.B. Chapman K.B. Weinrich S.L. Andrews W.H. Lingner J. Harley C.B. Cech T.R. Science. 1997; 277: 955-959Crossref PubMed Scopus (2068) Google Scholar) and an RNA-containing subunit (human telomerase RNA) (2Feng J. Funk W.D. Wang S.S. Weinrich S.L. Avilion A.A. Chiu C.P. Adams R.R. Chang E. Allsopp R.C. Yu J. Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2079) Google Scholar) that is used as a template for extending telomere length. Telomerase activity is repressed in most normal human somatic tissues, whereas the enzyme is active in ∼90% of human cancers (3Shay J.W. Bacchetti S. Eur. J. Cancer. 1997; 33: 787-791Abstract Full Text PDF PubMed Scopus (2398) Google Scholar). However, the mechanism through which telomerase is reactivated in the process of carcinogenesis remains unclear. Telomerase enzymatic activity can be regulated at multiple levels, including hTERT transcription, alternative splicing, chaperone-mediated folding, phosphorylation, and nuclear translocation; however, the major control mechanism of telomerase regulation seems to be at the level of hTERT transcription (for a review of telomerase regulation, see Ref. 4Kyo S. Inoue M. Oncogene. 2002; 21: 688-697Crossref PubMed Google Scholar and references therein). The tumor suppressor gene p53 is a sequence-specific transcription factor that can mediate many downstream effects such as growth arrest and apoptosis through activation or repression of its target genes (5Oren M. Cell Death Differ. 2003; 10: 431-442Crossref PubMed Scopus (910) Google Scholar). p53 is the most frequently altered gene in human cancers. Mutations and deletions of p53 are found in over half of human primary tumors (6Harris C.C. Br. J. Cancer. 1996; 73: 261-269Crossref PubMed Scopus (106) Google Scholar). Absence of functional p53 allows cellular immortalization and predisposes cells to neoplastic transformation (7Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (580) Google Scholar). Several studies have been conducted in an attempt to correlate the status of p53 and telomerase activity during carcinogenesis. p53-null mouse embryonic fibroblasts exhibit increased (>3-fold) basal levels of telomerase activity relative to those present in matched early passage fibroblasts derived from p53 wild-type-expressing embryos (8Drissi R. Zindy F. Roussel M.F. Cleveland J.L. J. Biol. Chem. 2001; 276: 29994-30001Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Moreover, elevation of p53 protein levels, which is usually associated with inactivating p53 mutations, is correlated with telomerase expression in cervical cancer (9Nair P. Jayaprakash P.G. Nair M.K. Pillai M.R. Acta Oncol. 2000; 39: 65-70Crossref PubMed Scopus (28) Google Scholar), breast cancer (10Roos G. Nilsson P. Cajander S. Nielsen N.H. Arnerlov C. Landberg G. Int. J. Cancer. 1998; 79: 343-348Crossref PubMed Scopus (56) Google Scholar), non-small cell lung cancer (11Kiyooka K. Maniwa Y. Okada M. Ann. Thorac. Cardiovasc. Surg. 1999; 5: 293-299PubMed Google Scholar), and ovarian cancers (12Wisman G.B. Hollema H. Helder M.N. Knol A.J. Van der Meer G.T. Krans M. De Jong S. De Vries E.G. Van der Zee A.G. Int. J. Oncol. 2003; 23: 1451-1459PubMed Google Scholar). Overexpression of wild-type p53 was shown to down-regulate telomerase enzymatic activity in a number of cancer cell lines independent of its effects on growth arrest and apoptosis (13Kusumoto M. Ogawa T. Mizumoto K. Ueno H. Niiyama H. Sato N. Nakamura M. Tanaka M. Clin. Cancer Res. 1999; 5: 2140-2147PubMed Google Scholar). This observation was attributed to transcriptional repression of hTERT by wild-type p53 since it was preceded by down-regulation of hTERT mRNA (13Kusumoto M. Ogawa T. Mizumoto K. Ueno H. Niiyama H. Sato N. Nakamura M. Tanaka M. Clin. Cancer Res. 1999; 5: 2140-2147PubMed Google Scholar). This conclusion was supported by promoter activity studies demonstrating the ability of p53 to repress hTERT promoter-reporter constructs. Mapping studies showed that mutation of all five Sp1 transcription factor-binding sites within the core hTERT promoter results in strong down-regulation of the reporter activity, which cannot be further repressed by p53 (14Kanaya T. Kyo S. Hamada K. Takakura M. Kitagawa Y. Harada H. Inoue M. Clin. Cancer Res. 2000; 6: 1239-1247PubMed Google Scholar). In an additional study, activation of exogenous temperature-sensitive p53 in BL41 Burkitt's lymphoma cells triggered rapid down-regulation of hTERT mRNA expression independent of the induction of the p53 target gene p21 (15Xu D. Wang Q. Gruber A. Bjorkholm M. Chen Z. Zaid A. Selivanova G. Peterson C. Wiman K.G. Pisa P. Oncogene. 2000; 19: 5123-5133Crossref PubMed Scopus (221) Google Scholar). Mitomycin C treatment of three breast cancer cell lines results in strong down-regulation of hTERT only in MCF7 cells, which express endogenous wild-type p53, but not in T-47D cells carrying mutant p53 and in p53-null MDA-MB-157 cells. Wild-type p53 inhibits Sp1 binding to the hTERT proximal promoter in a gel shift assay with purified proteins. The ability of p53 and Sp1 to form a complex was shown by co-immunoprecipitation (15Xu D. Wang Q. Gruber A. Bjorkholm M. Chen Z. Zaid A. Selivanova G. Peterson C. Wiman K.G. Pisa P. Oncogene. 2000; 19: 5123-5133Crossref PubMed Scopus (221) Google Scholar). These studies suggest a mechanism of hTERT repression in which p53 binds to Sp1 and renders it inaccessible to hTERT promoter activation (15Xu D. Wang Q. Gruber A. Bjorkholm M. Chen Z. Zaid A. Selivanova G. Peterson C. Wiman K.G. Pisa P. Oncogene. 2000; 19: 5123-5133Crossref PubMed Scopus (221) Google Scholar). However, the physiological relevance of these findings has been questioned because most of these studies were based on non-physiological overexpression of p53 or lacked isogenic controls (16Lin S.Y. Elledge S.J. Cell. 2003; 113: 881-889Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). In this study, we therefore re-examined the role of p53 in telomerase repression using proper isogenic controls and in lines expressing physiological levels of p53. Our results demonstrate that endogenous p53 represses telomerase in a cell type-specific manner. This p53-induced repression occurs through an indirect mechanism and is mediated by the p21/E2F pathway. Cell Lines—Human non-small cell lung cancer cell line H1299 and prostate cancer cell line LNCaP were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics. The human breast cancer cell line MCF7 stably expressing small interfering RNA (siRNA) targeting p53 and its vector control line were a gift from Dr. R. Agami (Netherlands Cancer Institute). The RKO colon carcinoma cell line and its derivative overexpressing E6 were a gift from Dr. C. Hurris (National Institutes of Health). HT1080 fibrosarcoma cells were a gift from Dr. M. Brandeis (Hebrew University, Jerusalem, Israel). Ecotropic Phoenix retrovirus-producing cells were from American Type Culture Collection. MCF7, RKO, HT1080, and Phoenix cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, and antibiotics. The HCT-116 colon carcinoma cell line and its p53-null derivative were a gift from Dr. B. Vogelstein (The John Hopkins University, Baltimore, MD) (17Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2538) Google Scholar). They were maintained in McCoy's medium supplemented with 10% fetal calf serum and antibiotics. All cell lines were grown at 37 °C in a humidified atmosphere of 5% CO2 in air. Plasmids—The reporter construct containing the p21 promoter (pGL3-Waf1), the expression vectors for a dominant-negative p53 peptide (p53-DD) (18Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar), a p53 deletion mutant lacking the proline-rich domain (62–91del), and wild-type E7 and its deletion mutant lacking the pocket protein-binding region (E7del21–35) were a gift from Dr. M. Oren (Weizmann Institute). The pBabe-GSE56-puro retroviral construct was a gift from Dr. A. V. Gudkov (Lerner Research Institute, Cleveland, OH) (19Ossovskaya V.S. Mazo I.A. Chernov M.V. Chernova O.B. Strezoska Z. Kondratov R. Stark G.R. Chumakov P.M. Gudkov A.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10309-10314Crossref PubMed Scopus (160) Google Scholar). pBabe-E1A was a gift from Dr. D. Peeper (Netherlands Cancer Institute). The generation of the reporter constructs containing different lengths of the hTERT promoter and construction of point mutations in the E- and MT-boxes were as described (20Tzukerman M. Shachaf C. Ravel Y. Braunstein I. Cohen-Barak O. Yalon-Hacohen M. Skorecki K.L. Mol. Biol. Cell. 2000; 11: 4381-4391Crossref PubMed Scopus (39) Google Scholar, 21Braunstein I. Cohen-Barak O. Shachaf C. Ravel Y. Yalon-Hacohen M. Mills G.B. Tzukerman M. Skorecki K.L. Cancer Res. 2001; 61: 5529-5536PubMed Google Scholar). The additional point mutations of the hTERT promoter were generated on the template of pGL3-core-phTERT with a QuikChange XL kit (Stratagene) using the following primers (only the sense primer is shown; mutations are in lowercase; and the numbers indicate positions relative to the transcription initiation site (22Takakura M. Kyo S. Kanaya T. Hirano H. Takeda J. Yutsudo M. Inoue M. Cancer Res. 1999; 59: 551-557PubMed Google Scholar)): for mutations of Sp1 sites 1 and 2 (mt2Sp1), –114CCAGCTCaaCCTCCTCCGCGCGGACCC-ataCCCGTCCCG–76; for mutations of Sp1 sites 3–5, –62CCCCGGCCC-AaaCCCCTCCGGGCCCTCCCAaaCCCTCCCCTTCCTTTCCGCGGCC-CataCCTC+1; and for mutation of the E2F site (E2F-I), –3CCTCTCC-TCGCGGCatGAGTTTCAGGCAGC+25. The M6 reporter (point mutation of the most proximal Sp1 site) was constructed by subcloning the SacII and XhoI inserts from the mt5Sp1 plasmid into wild-type pGL3-core-phTERT digested with the same enzymes. Expression plasmids for wild-type human p53 and mutants L22Q/W23S, R273H, R175H, 360del, and 342del were gifts from Dr. C. Hurris and were as described (23Zhou X. Wang X.W. Xu L. Hagiwara K. Nagashima M. Wolkowicz R. Zurer I. Rotter V. Harris C.C. Cancer Res. 1999; 59: 843-848PubMed Google Scholar). E2FdTA expression plasmid pRcCMV-E2F1-(1–363) was as described (24Hofmann F. Martelli F. Livingston D.M. Wang Z. Genes Dev. 1996; 10: 2949-2959Crossref PubMed Scopus (214) Google Scholar). Small hairpin RNA either in the pSuper vector (for luciferase assays) or in the pSuper-Retro-Hygro vector (for stable expression), both of which were gifts from Dr. R. Agami, were prepared as described (25Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar). The following 19-bp sequences were targeted: for p21 (p21i), GACCATGTGGACCTGTCAC; for mouse p63 (mp63i), GGCAGAGCGTGCTGGTCCC; and for the p53 control mutated sequence (p53i-mut), GACTCCCGTTGTAATCTAC. Western Blots—For Western blotting of p53 and p21 proteins, 50 μg of total cell lysate was separated on 12.5% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked overnight in 5% dry skimmed milk in PBS/Tween, and p53 was detected using a polyclonal antibody produced in our laboratory. For detection of p21, a polyclonal antibody (C-19, Santa Cruz Biotechnology) was used. The protein-antibody complexes were detected using a horseradish peroxidase-conjugated secondary antibody (Roche Applied Science) using the enhanced chemiluminescence ECL system (Amersham Biosciences). Equal loading was verified in each experiment by Ponceau red staining. Production of Stable Cell Lines—MCF7, HT1080, and LNCaP cells were stably transfected or infected with an ecotropic receptor. Ecotropic Phoenix packaging cells were transfected with 10 μg of the appropriate retroviral construct by the calcium phosphate method. Culture supernatants were collected 36–48 h post-transfection and filtered. Target cells were infected with the filtered viral supernatants in the presence of 4 μg/ml Polybrene for 12 h, after which the medium was changed. Fresh viral suspensions were added after a 24-h interval for an additional 12 h. For production of stable RNA interference (RNAi) against p21 or mouse p63 in MCF7 cells, pSuper-Retro-Hygro was used. Following infection, cells were selected with 1 mg/ml hygromycin for 1 week. For production of HT1080 and LNCaP cells with inactivated p53, the pBabe-GSE56-puro retroviral construct was used. Following infection, cells were selected with 1 μg/ml puromycin for 1 week. Southwestern Blot Analysis—DNA samples (300 ng of each) were applied to a nitrocellulose membrane and cross-linked to the membrane by UV light at 120 J. The membrane was washed twice with PBS and blocked with 5% milk in PBS for 3 h. Nuclear extracts were prepared as described (26Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) and diluted to a concentration of 0.5 mg/ml protein in binding buffer (12.5 mm Tris-HCl (pH 7.9). 3.1 mm MgCl2, 25 mm KCl, 0.5 mm dithiothreitol, 10% glycerol, 0.25 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/ml aprotinin, 10 μg/ml salmon sperm DNA, and 1 μg/ml pGL3 plasmid DNA for blocking). The diluted nuclear extract was incubated with the membrane overnight at 4 °C. Following washing, the blots were probed with anti-p53 polyclonal antibody, washed with PBS/Tween, probed with anti-rabbit secondary antibody, and developed using the enhanced chemiluminescence ECL kit. Chromatin Immunoprecipitation Analysis—Chromatin immunoprecipitation was performed as described previously (27Zalcenstein A. Stambolsky P. Weisz L. Muller M. Wallach D. Goncharov T.M. Krammer P.H. Rotter V. Oren M. Oncogene. 2003; 22: 5667-5676Crossref PubMed Scopus (104) Google Scholar). The primers used for the detection of hTERT promoter sequences that amplify region –217 to +119 relative to the transcription initiation site were 5′-CAGGCCGGGCTCCCAGTGGA and 3′-CAGCAGGGAGCGCACG-GCTC. The primers used for the detection of p21 promoter sequences that amplify the region near the p53-binding site were 5′-GCACTCTT-GTCCCCCAG and 3′-TCTATGCCAGAGCTCAACAT. Transfections and Reporter Assays—Cells were grown in complete medium and replated at 3 × 104 cells/well in a 24-well plate 16–24 h before transfection. For reporter gene assay, cells were transfected by FuGENE 6 transfection reagent (Roche Applied Science) using 300 ng of promoter-luciferase reporter constructs, the indicated amounts of expression plasmids, and 100 ng of pCMV-β-galactosidase expression vector for normalization of transfection efficiency. After transfection (48 h), cell extracts were prepared, and luciferase and β-galactosidase activities were determined using reagents and procedures from Promega. For experiments with RNAi, 250 ng of Rb RNAi, 250 ng of p107 RNAi, and 700 ng of p130 RNAi were cotransfected with the reporter. The targeted sequences are as follows: for p130, CUCUUGGGCCUGC-CUUGTT; for Rb, CACACUCCAGUUAGGACUGTT; and for p107, CC-AAGAGUCAAGGAAGUUCTT. Reverse Transcription (RT)-PCR and Telomerase Activity Assays— Total RNA was isolated using the RNeasy kit (QIAGEN Inc.), and 2 μg was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) and random hexamer primers (Roche Applied Science). Real-time quantitative RT-PCR for total hTERT mRNA was performed using the Assays-on Demand TaqMan kit (Applied Biosystems) on an ABI 7000 instrument (Applied Biosystems). The values for hTERT were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping control. The primer sequences for SYBR Green real-time PCR of GAPDH were as follows: 5′-ACCCACTC-CTCCACCTTTGA and 3′-CTGTTGCTGTAGCCAAATTCGT. For specific detection of full-length hTERT by semiquantitative PCR, the following primers were used: 5′-TGTCAAGGTGGATGTGACG and 3′-CTGGAGGTCTGTCAAGGTA. The forward primer is directed to the α-splice site so that the α-spliced form is not amplified. Similarly, the design of the reverse primer prevents amplification of the β-spliced form. PCR was performed in the presence of [33P]dCTP, and reaction products were detected by autoradiography. Telomerase enzymatic activity was measured using the real-time telomeric repeat amplification protocol essentially as described (28Wege H. Chui M.S. Le H.T. Tran J.M. Zern M.A. Nucleic Acids Res. 2003; 31: E3-E9Crossref PubMed Google Scholar). p53 Represses Telomerase Activity and hTERT mRNA—To examine the role of endogenous wild-type p53 in telomerase regulation, we used several isogenic cell line pairs specifically differing in their p53 status. To this end, MCF7 breast cancer cells containing wild-type p53 and their derivatives stably expressing p53 RNAi were used (25Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar). As demonstrated in Fig. 1A, induction of the p53 protein following doxorubicin treatment was strongly suppressed in RNAi-containing cells (MCF7-p53i) compared with cells expressing the vector control (MCF7-vector). As shown in Fig. 1B, following 48 h of doxorubicin treatment, a 10-fold down-regulation of telomerase activity was observed in control cells. This inhibition was largely p53-dependent, as only a 2-fold reduction was detected in MCF7-p53i cells. Next, we examined the levels of hTERT mRNA using realtime quantitative RT-PCR. A significant p53-dependent reduction of hTERT mRNA could be detected as early as 18 h post-treatment (Fig. 1C). The long lag between mRNA down-regulation and the decrease in telomerase enzymatic activity is consistent with the reported long half-life of the latter (29Holt S.E. Aisner D.L. Shay J.W. Wright W.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10687-10692Crossref PubMed Scopus (153) Google Scholar). hTERT was shown to have a complex splicing pattern, which gives rise to at least four isoforms (full-length, α, β, and αβ), with only the full-length form catalytically active (30Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman A.R. Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar). Since all alternatively spliced forms of hTERT were detected in our real-time RT-PCR, we tested specifically whether the full-length form that encodes enzymatically active hTERT is down-regulated following doxorubicin treatment. RT-PCR using primers specific for this form indeed showed strong down-regulation of full-length hTERT in MCF7-vector cells (Fig. 1D). To corroborate the ability of endogenous p53 to down-regulate hTERT expression in an additional isogenic system, we used LNCaP prostate cancer cells containing wild-type p53 and their derivatives in which p53 was inactivated using the dominant-negative GSE56 peptide (31Mittelman J.M. Gudkov A.V. Somatic Cell Mol. Genet. 1999; 25: 115-128Crossref PubMed Scopus (10) Google Scholar). Following doxorubicin treatment, we observed strong p53-dependent repression of hTERT mRNA (Fig. 1E). Of note, the phenomenon of p53-dependent repression of hTERT mRNA was not general to all the cell lines examined. In a number of isogenic cell lines, the reduction of hTERT mRNA following drug treatment was in-dependent of the p53 status (Table I).Table IRepression of hTERT by endogenous wild-type p53 is cell type-specificCell lineDescriptionMeans of p53 inactivationp53-dependent hTERT repressionMCF7Breast cancersiRNA for p53YesLNCaPProstate cancerGSE56YesHCT-116Colon cancerGene knockoutNoHT1080FibrosarcomaGSE56NoRKOColon cancerE6No Open table in a new tab p53 Represses the Transcriptional Activity of the hTERT Promoter—To examine whether p53-mediated down-regulation of hTERT mRNA is due to promoter repression, we performed a series of hTERT promoter-luciferase gene reporter assays (Fig. 2A). As demonstrated in Fig. 2B, wild-type p53 repressed the hTERT promoter constructs of different lengths in a dose-dependent manner when cotransfected into p53-null H1299 cells. The core promoter was sufficient to respond to the p53-mediated repression. To avoid the effects of exogenous p53 overexpression and to extend the analysis to another cell type, we assessed the effect of endogenous p53 by inactivating it using the dominant-negative peptide p53-DD (18Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar). The transfection process itself stabilized and activated endogenous p53 (data not shown); and thus, the reporter activity in the absence of p53-DD reflects the effect of activated p53. Inactivation of p53 by cotransfection with p53-DD strongly reduced the promoter activity of a known p53 transactivation target, p21waf1, and resulted in ∼2.5-fold up-regulation of both 5.9-kb and core hTERT promoter activities (Fig. 2C). These results demonstrate that p53-mediated repression maps to the core promoter. Mapping of p53 Domains Required for Telomerase Repression—To map the domains of p53 necessary for telomerase repression, H1299 cells were cotransfected with the core hTERT promoter-luciferase reporter construct together with various p53 mutants. Similar expression levels of the different p53 forms were confirmed by Western blotting (data not shown). Fig. 3 demonstrates that the most frequent naturally occurring point mutations in the DNA-binding core domain (R175H and R273H) as well as the inactivating double mutation in the transactivation domain (L22Q/W23S) eliminated p53-mediated repression. Deletion of 51 amino acids (342del), which disrupts the oligomerization domain of p53, also abolished repression. Thus, the integrity of the DNA-binding core domain, the N-terminal transactivation domain, and the C-terminal oligomerization domains is essential for repression. In contrast, deletion of 33 C-terminal amino acids (360del) or the proline-rich domain (62–91del) did not interfere with repression, meaning that these domains are dispensable for this function. The proline-rich domain is important for the recruitment of the Sin3A corepressor and histone deacetylases (32Zilfou J.T. Hoffman W.H. Sank M. George D.L. Murphy M. Mol. Cell. Biol. 2001; 21: 3974-3985Crossref PubMed Scopus (103) Google Scholar). This mechanism of transcriptional repression by p53 has been demonstrated in the case of stathmin and map4 (33Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar). Thus, these results suggest that recruitment of the Sin3A corepressor by p53 is not involved in hTERT repression. Overall, the pattern of domains essential for hTERT repression is identical to that needed for transactivation by p53. p53 Is Not Associated with the Core hTERT Promoter in Vitro and in Vivo—Since our mapping demonstrated the importance of the intact p53 DNA-binding core domain, we hypothesized that p53 may bind to the hTERT promoter. Examination of the core hTERT promoter sequence revealed a lack of the typical p53 consensus binding sites. However, since this is generally the case for transcriptional repression by p53, we decided to test whether p53 can bind elsewhere to the hTERT promoter. First, we assessed whether p53 can bind the hTERT promoter in vitro using a Southwestern assay. As shown in Fig. 4A, only background binding of wild-type p53 to the pGL3 plasmid containing the core hTERT promoter could be detected. In contrast, strong binding to the same plasmid containing the p21 promoter (serving as a positive control) was observed. Similar results were obtained with endogenous p53 from doxorubicin-treated MCF7 cells (data not shown). To examine whether p53 binds to the hTERT promoter in vivo, we performed a chromatin immunoprecipitation experiment. MCF7 cells and their counterparts expressing p53 siRNA were either treated with 0.4 μm doxorubicin for 16 h or left untreated. Following formaldehyde cross-linking and precipitation of the chromatin with anti-p53 polyclonal antibody, the precipitated DNA was subjected to PCR amplification using primers specific to the hTERT promoter or the p53 binding site in the promoter of a known p53 target gene, p21waf1. As shown in Fig. 4B, no amplification of hTERT promoter sequences could be detected, whereas the p21 promoter sequence was easily detected in the same immunoprecipitation product. The specificity of this immunoprecipitation was demonstrated by the lack of a PCR product of the p21 promoter in the no-antibody control and its lower intensity in MCF7-p53i cells. Thus, these results demonstrate that the p53 protein does not associate with the hTERT promoter. An Atypical E2F Site Is Responsible for Repression of the hTERT Promoter by p53—Since the mutational analysis demonstrated that DNA binding by p53 was required for hTERT repression (Fig. 3), but no direct p53 binding to the core hTERT promoter was apparent, we proposed that p53 must bind elsewhere in the genome to mediate hTERT promoter repression. The complete correlation of the domains necessary for repression with the structural requirements for transactivation by p53 raised the possibility that p53 activates a target gene whose product represses the hTERT promoter. The cell type variability of p53-mediated hTERT repression (Table I) is consistent with this hypothesis and can potentially be explained by the absence of the mediator(s) in some cancer cell lines. We therefore attempted to identify such putative mediator(s) by identification of the cis-regulatory element in the hTERT promoter that is responsible for p53-mediated repression. To this end, we created several deletion and point mutati
Referência(s)