Constitutive Expression of the Cyclin-dependent Kinase Inhibitor p21 Is Transcriptionally Regulated by the Tumor Suppressor Protein p53
1998; Elsevier BV; Volume: 273; Issue: 44 Linguagem: Inglês
10.1074/jbc.273.44.29156
ISSN1083-351X
AutoresHsin-Yi Tang, Kathy Zhao, Joseph F. Pizzolato, Maxim Fonarev, Jessica Langer, James J. Manfredi,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe tumor suppressor protein p53 has been implicated in the response of cells to DNA damage. Studies to date have demonstrated a role for p53 in the transcriptional activation of target genes in the cellular response to DNA damage that results in either growth arrest or apoptosis. In contrast, here is demonstrated a role for p53 in regulating the basal level of expression of the cyclin-dependent kinase inhibitor p21 in the absence of treatment with DNA-damaging agents. Wild-type p53-expressing MCF10F cells had detectable levels of p21 mRNA and protein, whereas the p53-negative Saos-2 cells did not. Saos-2 cells were infected with recombinant retrovirus to establish a proliferating pool of cells with a comparable constitutive level of expression of wild-type p53 protein to that seen in untreated MCF10F cells. Restoration of wild-type but not mutant p53 expression recovered a basal level of expression of p21 in these cells. Constitutive expression of luciferase reporter constructs containing the p21 promoter was inhibited by co-transfection with the human MDM2 protein or a dominant-negative p53 protein and was dependent on the presence of p53 response elements in the reporter constructs. Furthermore, p53 in nuclear extracts of untreated cells was capable of binding to DNA in a sequence-specific manner. These results implicate a role for p53 in regulating constitutive levels of expression of p21 and demonstrate that the p53 protein is capable of sequence-specific DNA binding and transcriptional activation in untreated, proliferating cells. The tumor suppressor protein p53 has been implicated in the response of cells to DNA damage. Studies to date have demonstrated a role for p53 in the transcriptional activation of target genes in the cellular response to DNA damage that results in either growth arrest or apoptosis. In contrast, here is demonstrated a role for p53 in regulating the basal level of expression of the cyclin-dependent kinase inhibitor p21 in the absence of treatment with DNA-damaging agents. Wild-type p53-expressing MCF10F cells had detectable levels of p21 mRNA and protein, whereas the p53-negative Saos-2 cells did not. Saos-2 cells were infected with recombinant retrovirus to establish a proliferating pool of cells with a comparable constitutive level of expression of wild-type p53 protein to that seen in untreated MCF10F cells. Restoration of wild-type but not mutant p53 expression recovered a basal level of expression of p21 in these cells. Constitutive expression of luciferase reporter constructs containing the p21 promoter was inhibited by co-transfection with the human MDM2 protein or a dominant-negative p53 protein and was dependent on the presence of p53 response elements in the reporter constructs. Furthermore, p53 in nuclear extracts of untreated cells was capable of binding to DNA in a sequence-specific manner. These results implicate a role for p53 in regulating constitutive levels of expression of p21 and demonstrate that the p53 protein is capable of sequence-specific DNA binding and transcriptional activation in untreated, proliferating cells. kilobase pair(s) cytomegalovirus Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline N, N′-hexamethyl- ene-bisacetamide. The tumor suppressor protein p53 is a transcription factor that binds to DNA in a sequence-specific manner, has been implicated in the cellular response to DNA damage, and appears to play a role in a variety of cellular responses including growth arrest, apoptosis, differentiation, and senescence (1Gottlieb T.M. Oren M. Biochim. Biophys. Acta. 1996; 1287: 77-102Crossref PubMed Scopus (511) Google Scholar, 2Ko L.J. Prives C. 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Consistent with this, cells that lack p21 expression have an impaired p53-dependent response to DNA damage (6Brugarolas J. Chandrasekaran C. Gordon J.I. Beach D. Jacks T. Hannon G.J. Nature. 1995; 377: 552-557Crossref PubMed Scopus (1159) Google Scholar, 7Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1962) Google Scholar). This transcriptional activation of p21 expression is mediated by the interaction of p53 with two response elements located in the p21 promoter (8el-Deiry W.S. Tokino T. Waldman T. Oliner J.D. Velculescu V.E. Burrell M. Hill D.E. Healy E. Rees J.L. Hamilton S.R. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 2910-2919PubMed Google Scholar). The DNA binding activity of p53 appears to be regulated by the terminal 30 amino acids of the protein. Phosphorylation by either casein kinase II or protein kinase C, acetylation by p300, and binding by a monoclonal antibody 421, or the bacterial dnaK protein all occur within this region of p53 and will activate the ability of p53 to bind to DNA in a sequence-specific manner in vitro (9Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2210) Google Scholar, 10Funk W.D. Pak D.T. Karas R.H. Wright W.E. Shay J.W. Mol. Cell. Biol. 1992; 12: 2866-2871Crossref PubMed Scopus (679) Google Scholar, 11Halazonetis T.D. Davis L.J. Kandil A.N. EMBO J. 1993; 12: 1021-1028Crossref PubMed Scopus (183) Google Scholar, 12Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 13Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 14Mundt M. Hupp T. Fritsche M. Merkle C. Hansen S. Lane D. Groner B. Oncogene. 1997; 15: 237-244Crossref PubMed Scopus (47) Google Scholar, 15Takenaka I. Morin F. Seizinger B.R. Kley N. J. Biol. Chem. 1995; 270: 5405-5411Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). There have been several reports that the ability of p53 in nuclear extracts to bind to DNA requires the presence of antibody 421, leading to the notion that p53 exists in a latent form prior to DNA damage (10Funk W.D. Pak D.T. Karas R.H. Wright W.E. Shay J.W. Mol. Cell. Biol. 1992; 12: 2866-2871Crossref PubMed Scopus (679) Google Scholar, 12Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (887) Google Scholar). Consistent with this idea, microinjection of the antibody 421 into cells activates p53-dependent expression from reporter constructs (13Hupp T.R. Sparks A. Lane D.P. Cell. 1995; 83: 237-245Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 16Abarzua P. LoSardo J.E. Gubler M.L. Neri A. Cancer Res. 1995; 55: 3490-3494PubMed Google Scholar). Thus, it has been proposed that in untreated cells, the p53 protein exists in a latent state that is unable to bind to DNA and that the ability of p53 to activate target gene expression is not merely dependent on the increase in protein level but also requires post-translational modification of p53 to convert this latent form into a form that is active for DNA binding (12Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 17Hupp T.R. Lane D.P. J. Biol. Chem. 1995; 270: 18165-18174Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This notion is supported by studies demonstrating that p53 becomes phosphorylated at particular sites after treatment of cells with DNA-damaging agents (18Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (715) Google Scholar, 19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar). Prior to the cloning of the gene, it was noted that p21 was absent from cyclin/cyclin-dependent kinase complexes in cells lacking functional p53 (20Xiong Y. Zhang H. Beach D. Genes Dev. 1993; 7: 1572-1583Crossref PubMed Scopus (503) Google Scholar). Other studies have noted that the level of p21 mRNA was much lower in fibroblasts and keratinocytes derived from mice containing a homozygous deletion of p53 as compared with the corresponding cells from mice expressing wild-type p53 (21Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 22Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3238) Google Scholar, 23Weinberg W.C. Azzoli C.G. Kadiwar N. Yuspa S.H. Cancer Res. 1994; 54: 5584-5592PubMed Google Scholar, 24Macleod K.F. Sherry N. Hannon G. Beach D. Tokino T. Kinzler K. Vogelstein B. Jacks T. Genes Dev. 1995; 9: 935-944Crossref PubMed Scopus (761) Google Scholar). This suggests that p53 may play a role in the level of p21 expression in untreated, proliferating cells. The experiments presented here tested this idea directly and demonstrate that constitutive expression of the p21 protein in untreated cells is, indeed, dependent on p53 and thus implicate a role for p53 not only in the increased expression of p21 in response to DNA damage leading to either growth arrest or apoptosis but also in the basal level of expression of p21 in normally proliferating cells. The plasmid p21P contains 2.5-kb1 of the humanp21 promoter inserted upstream of a firefly luciferase reporter gene in the vector pGL2 (Promega). The plasmid p21D2.1 has 2.1 kb at the 5′ end of the promoter sequence removed and lacks the two p53 response elements of the p21 promoter (25Datto M.B., Yu, Y. Wang X.-F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). The plasmid pRL-SV40 contains the SV40 enhancer and early promoter upstream of a Renilla luciferase reporter gene (Promega). The plasmid pCMV-hdm2 encodes the human MDM2 protein under control of the cytomegalovirus (CMV) promoter and the plasmid pCMV-p53Ala-143 encodes the tumor-derived mutant human p53 protein containing a missense mutation of valine to alanine at residue 143 (26Friedlander P. Haupt Y. Prives C. Oren M. Mol. Cell. Biol. 1996; 16: 4961-4971Crossref PubMed Scopus (269) Google Scholar). Saos-2 and WI38 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). MCF7 cells were maintained in RPMI medium containing 10% heat-inactivated FBS and 5 μg/ml insulin. MCF10F cells were grown in 50% DMEM and 50% Ham's F12 medium containing 5% horse serum, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 μg/ml insulin, and 500 ng/ml hydrocortisone. PA12-p53BN and PA12-p53EN are cell lines that produce recombinant retrovirus encoding human wild-type p53 or the mutant p53His-273, respectively (27Chen P.L. Chen Y.M. Bookstein R. Lee W.H. Science. 1990; 250: 1576-1580Crossref PubMed Scopus (501) Google Scholar). These cell lines were grown in 10% FBS in DMEM containing high glucose and 400 μg/ml G418 sulfate. The hybridoma cell line producing the mouse monoclonal antibody 1801 was grown in DMEM containing 10% FBS. Hybridoma cell lines expressing the mouse monoclonal antibodies 421 and 419 were grown in 50% DMEM and 50% Fischer's medium containing 10% FBS. Monoclonal antibody 1801 specifically reacts with human p53 (28Banks L. Matlewshki G. Crawford L. Eur. J. Biochem. 1986; 159: 529-534Crossref PubMed Scopus (540) Google Scholar), 421 recognizes p53 from a variety of species, and 419 recognizes an epitope on the SV40 large T antigen (29Harlow E. Crawford L.V. Pim D.C. Williamson N.M. J. Virol. 1981; 39: 861-869Crossref PubMed Google Scholar). All cell lines were grown at 37 °C in a humid incubator containing 5% CO2. Antibody against p21WAF1, CIP1 was obtained commercially (Ab-1/clone EA10, Calbiochem). For treatment with ultraviolet light, the medium was removed, and the cells were exposed to ultraviolet light using a UV Stratalinker (Stratagene). Total RNA was extracted from 5 × 106 cells using RNAzol (Tel-test), and Northern blot analysis was performed following conventional procedures, using a 2.1-kb full-length human p21 cDNA or human glyceraldehyde-3-phosphate dehydrogenase cDNA (Ambion) as probes. Cells were lysed in a buffer containing 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate (SDS), 50 mm NaCl, 25 mm Tris-HCl, pH 7.5, and the protease inhibitors, phenylmethylsulfonyl fluoride (1 mm), aprotinin (50 μg/ml), and leupeptin (50 μg/ml) for 10 min on ice. Lysates were spun at 15,000 rpm for 10 min, and the supernatant was saved. Protein levels were determined by the bicinchoninic acid protein assay (Pierce). Appropriate amounts of total cellular protein were loaded on 10% SDS-polyacrylamide gels and electrophoresed at 150 V constant voltage for 3 h. Samples were transferred to nitrocellulose paper and probed with the appropriate antibody. Second antibody was a horseradish peroxidase-conjugated goat anti-mouse IgG, and the signal was detected by the enhanced chemiluminescence method (Amersham Pharmacia Biotech). The retrovirus-producing cell lines PA12-BN and PA12-EN were grown to 75% confluence and fed with fresh DMEM containing 10% FBS. After incubation at 37 °C for 16 h, the supernatant was harvested and filtered through a 0.2-μm filter. Old medium was removed from a subconfluent 60-mm dish of Saos-2 cells and replaced with 1 ml of filtered supernatant containing 8 μg/ml Polybrene. Dishes were rocked for 2 h at 37 °C in a humid incubator containing 5% CO2 and then 3 ml of DMEM containing 10% FBS was added to the dish, and it was further incubated for 48 h. The cells were then trypsinized and replated in a 100-mm dish using DMEM containing 10% FBS and 400 μg/ml G418 sulfate. Cells were fed every 3 days with this same medium. After 2 weeks, the resulting drug-resistant colonies were pooled and passaged. For detecting replicative DNA synthesis, cells were incubated with 10 μm bromodeoxyuridine for 30 min prior to fixation. The proportion of cells actively synthesizing DNA was quantitated by anti-bromodeoxyuridine immunoanalysis, and the total DNA content was analyzed by staining with propidium iodide as described previously (30Tang H. Davis M.A. Strickfaden S.M. Maybaum J. Lawrence T.S. Radiat. Res. 1994; 138: S109-S112Crossref PubMed Scopus (19) Google Scholar). Cells were fixed with 70% ethanol for at least 2 h, resuspended in the following solutions in order: 0.25% paraformaldehyde in phosphate-buffered saline (PBS), 0.5 mg/ml ribonuclease A in PBS, 0.5% Triton X-100 in 0.1 n HCl, and finally distilled water. Samples were then heated at 97 °C for 10 min, immediately placed on ice for additional 10 min, and washed with 0.5% Tween 20 in PBS. The incorporation of bromodeoxyuridine was detected by monoclonal anti-bromodeoxyuridine antibody conjugated to fluorescein isothiocyanate (Becton Dickinson). Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson). MCF7, MCF10F, or Saos-2 cells were transfected using theN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts liposomal transfection reagent (DOTAP, Boehringer Mannheim). One confluent 100-mm dish of cells was split into three 6-well dishes and incubated for 24 h. Cells were fed with complete medium containing serum and incubated for an additional 3 h.N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium salts/DNA mixtures containing 2 μg of the relevant reporter plasmid plus 50 ng of the p53 expression plasmid or an equal amount of an empty vector plasmid were prepared according to the manufacturer's instructions and incubated at room temperature for 15 min. Serum-free medium was then added to the mixtures and used to replace the media in the wells. The dishes were incubated at 37 °C for 3 h, after which the transfection mix was removed and replaced with complete medium containing serum. After 48 h, the 6-well plates were placed on ice and washed once with PBS. The cells were then lysed by scraping into 120 μl of Reporter Buffer (Promega Luciferase Assay System), and samples were spun for 1 min at 14,000 rpm at 4 °C. Total protein concentration was determined using a commercially available assay (Bio-Rad). 40 μl of each sample was warmed to room temperature and mixed with luciferase assay substrate that was reconstituted with Luciferase Assay Buffer (Promega). Light emission was determined in a TD-20e luminometer (Turner). Nuclear and cytosolic extracts were performed as described by Graeber et al. (31Graeber T.G. Peterson J.F. Tsai M. Monica K. Fornace A.J. Giaccia A.J. Mol. Cell. Biol. 1994; 14: 6264-6277Crossref PubMed Google Scholar). Cells were homogenized in 10 mm Tris-HCl, pH 7.4, containing 10 mm NaCl, 6 mmMgCl2, 1 mm dithiothreitol, 0.4 mmphenylmethylsulfonyl fluoride, and 100 μmNa3VO4 and spun at 10,000 rpm for 30 s. The supernatant was saved as the cytosolic extract. The pellet was repacked by spinning at 14,000 rpm for 1 min and then nuclei were suspended in a nuclear extraction buffer (20 mm Hepes, pH 7.5, containing 20% glycerol, 500 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 1 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 50 μm leupeptin, and 50 μg/ml aprotinin), incubated at 4 °C for 1 h, and spun at 14,000 rpm for 10 min. This supernatant was saved as the nuclear extract. Lactate dehydrogenase activity was assayed according to Ramirez et al. (32Ramirez V.D. Gautron J.P. Epelbaum J. Pattou E. Zamora A. Kordon C. Mol. Cell. Endocrinol. 1975; 3: 339-350Crossref PubMed Scopus (26) Google Scholar), and histone levels were determined by immunoblotting using an anti-histone antibody that reacts with an epitope that is present on all five histone proteins (H11-4, Boehringer Mannheim). Such assays showed less than 10% cross-contamination between cytosolic and nuclear extracts. The specific probe that was used for binding, TCGAGCCGGGCATGTCCGGGCATGTCCGGGCATGTC, contains the high affinity binding sequence identified by Halazonetiset al. (11Halazonetis T.D. Davis L.J. Kandil A.N. EMBO J. 1993; 12: 1021-1028Crossref PubMed Scopus (183) Google Scholar) named by them BC or BB.9. In the competition experiments, the nonspecific oligonucleotide (referred to as Sens-1), TCGAAGAAGACGTGCAGGGACCC, was used. Complementary single-stranded oligonucleotides were annealed by incubation at 95 °C for 4 min, 65 °C for 10 min, and then gradually brought to room temperature. Ends were filled using the Klenow fragment of DNA polymerase to produce a labeled double-stranded oligonucleotide. Appropriate amounts of extracts (1–7 μl) were mixed with 1 ng of labeled double-stranded oligonucleotide in a total reaction mixture of 30 μl containing 6 μl of 5× electrophoretic mobility shift assay buffer (100 mm Hepes, pH 7.9, 0.5 mm EDTA, 50% glycerol, 10 mm MgCl2), 1.5 μl of 40 mmspermidine, 1.5 μl of 10 mm dithiothreitol, 1 μl of 500 μg/ml double-stranded poly(dI/dC), and 5–13 μl of water with a final salt concentration of 85 mm. The amount of total protein per reaction was normalized, and the reactions were carried out at room temperature for 30 min. For antibody supershift analysis, 2 μl of the appropriate undiluted hybridoma supernatant was added. His-tagged human p53 was produced by infection of insect cells with a recombinant baculovirus and purified by nickel-agarose chromatography and used as a positive control (52Resnick-Silverman L. St. Clair S. Maurer M. Zhao K. Manfredi J.J. Genes Dev. 1998; 12: 2102-2107Crossref PubMed Scopus (105) Google Scholar). Samples were electrophoretically separated on a native 4% polyacrylamide gel at 4 °C at 200 V for 2 h. After drying, gels were exposed to Kodak XAR film at −70 °C with an intensifying screen. Previous studies have noted that either fibroblasts or keratinocytes from mice that were homozygously deleted for p53 expressed lower basal levels of p21 mRNA as compared with fibroblasts or keratinocytes from mice expressing both alleles of the wild-type p53 gene (21Michieli P. Chedid M. Lin D. Pierce J.H. Mercer W.E. Givol D. Cancer Res. 1994; 54: 3391-3395PubMed Google Scholar, 22Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3238) Google Scholar, 23Weinberg W.C. Azzoli C.G. Kadiwar N. Yuspa S.H. Cancer Res. 1994; 54: 5584-5592PubMed Google Scholar, 24Macleod K.F. Sherry N. Hannon G. Beach D. Tokino T. Kinzler K. Vogelstein B. Jacks T. Genes Dev. 1995; 9: 935-944Crossref PubMed Scopus (761) Google Scholar). To characterize further a role for p53 in the basal level of expression of p21, the p53-negative cell line Saos-2 was compared with the wild-type p53-expressing cell line MCF10F. Total RNA was extracted from each cell line, and Northern analysis was performed. The p53-negative Saos-2 cell line expressed low levels of p21 mRNA as compared with the wild-type p53-expressing MCF10F cells (Fig. 1 A). Total cellular extracts of each cell line were subjected to SDS-polyacrylamide gel electrophoresis and subsequent immunoblotting with an anti-p21-specific antibody (Fig. 1 B). MCF10F cells expressed a detectable level of p21, whereas the level of p21 expression in Saos-2 cells was undetectable. To confirm that Saos-2 cells retained the ability to synthesize p21, both MCF10F and Saos-2 cells were treated with 10 mm N, N′-hexamethylene-bisacetamide (HMBA). HMBA is a non-retinoid, differentiating agent that has previously been shown to induce p21 expression in a p53-independent manner (24Macleod K.F. Sherry N. Hannon G. Beach D. Tokino T. Kinzler K. Vogelstein B. Jacks T. Genes Dev. 1995; 9: 935-944Crossref PubMed Scopus (761) Google Scholar). Treatment of Saos-2 cells with HMBA induced expression of p21 demonstrating that Saos-2 cells retained the ability to synthesize p21. Thus, both the level of protein and messenger RNA for p21 were much higher in the p53-expressing MCF10F cells than in the p53-negative Saos-2 cells. Previous studies have shown that restoration of wild-type p53 expression through transfection of a suitable expression plasmid did not allow for establishment of stable cell lines expressing wild-type p53 (33Johnson P. Gray D. Mowat M. Benchimol S. Mol. Cell. Biol. 1991; 11: 1-11Crossref PubMed Scopus (106) Google Scholar, 34Diller L. Kassel J. Nelson C.E. Gryka M.A. Litwak G. Gebhardt M. Bressac B. Ozturk M. Baker S.J. 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Science. 1990; 250: 1576-1580Crossref PubMed Scopus (501) Google Scholar) utilized recombinant retroviral infection to restore a level of wild-type p53 expression in Saos-2 cells that was comparable to that seen in normal cells and that was compatible with continued proliferation of these cells. To that end, Saos-2 cells were infected with recombinant retroviruses expressing either wild-type human p53 or the mutant human p53His-273, and pools of G418 sulfate-resistant cells were established. Immunoblotting of whole cell extracts from these drug-resistant pools demonstrated that both wild-type (Fig. 2 A, lane 4) and mutant (Fig. 2 A, lane 3) p53 expression could be detected in comparison to the parent cells which are p53-negative (Fig. 2 A, lane 2). Furthermore, the pool of Saos-2 cells expressing wild-type p53 expressed a level that is comparable to the endogenous p53 level in MCF10F cells (Fig. 2 A, lane 5). Consistent with previous observations, this level of expression of wild-type p53 that was obtained using recombinant retroviral infection was sufficiently low to allow the cells to continue to grow (Table I). These drug-resistant pools were labeled with bromodeoxyuridine and subjected to flow cytometric analysis to demonstrate that they were actively incorporating DNA. Indeed, the pools expressing wild-type p53 had a similar percentage of bromodeoxyuridine-positive cells as the parent cell line, the pool expressing mutant p53, or the wild-type p53 expressing cell lines WI38, MCF10F, or MCF7 (Table I). These pools were then examined for the level of p21 expression. Immunoblotting of whole cell extracts demonstrated that Saos-2 cells expressing wild-type but not mutant p53 expressed a level of p21 that was comparable to that of WI38 or MCF7 cells and, in fact, was greater than that seen with MCF10F cells (Fig. 2 B). Thus, restoration of expression of wild-type p53 in a p53-negative cell line also restored a basal level of expression of p21.Table IIncorporation of bromodeoxyuridine into the DNA of various cell linesCell lineBromodeoxyuridine positive cells1-aCells were labeled with 1 μmbromodeoxyuridine for 30 min, fixed, and processed for flow cytometric analysis as described under “Experimental Procedures.”%WI3812MCF10F22MCF727Saos-224Saos-2 (wt)1-bPools of G418 sulfate-resistant Saos-2 cells that had been infected with recombinant retrovirus expressing either wild-type (wt) or mutant (His273) p53 proteins.23Saos-2 (His273)1-bPools of G418 sulfate-resistant Saos-2 cells that had been infected with recombinant retrovirus expressing either wild-type (wt) or mutant (His273) p53 proteins.241-a Cells were labeled with 1 μmbromodeoxyuridine for 30 min, fixed, and processed for flow cytometric analysis as described under “Experimental Procedures.”1-b Pools of G418 sulfate-resistant Saos-2 cells that had been infected with recombinant retrovirus expressing either wild-type (wt) or mutant (His273) p53 proteins. Open table in a new tab The observation that reintroduction of p53 expression in Saos-2 cells restored a basal level of p21 expression (Fig. 2) suggests that in the absence of DNA damage, p53 regulates expression of p21. To test directly this notion and to confirm that such regulation is at the level of transcription, wild-type p53-expressing MCF7 cells were transfected with a luciferase reporter construct containing 2.4 kb of the human p21 promoter. To determine whether the basal level of expression that is observed was p53-dependent, an expression plasmid for the human MDM2 protein was co-transfected with the reporter. Mdm2 binds to p53 and inhibits its transcriptional activity, apparently by targeting the p53 protein for degradation (19Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar,38Oliner J.D. Pietenpol J.A. Thiagalingam S. Gyuris J. Kinzler K.W. Vogelstein B. 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In contrast, co-transfection of the plasmid encoding Mdm2 into the p53-negative Saos-2 cells had no effect on the low level of luciferase activity seen in these cells from the same reporter construct (Figs. 3 and 4 C). Treatment of MCF7 cells with ultraviolet light induced expression of the full-length p21 promoter construct but not the construct that lacks the p53-binding sites (Fig. 3). Furthermore, treatment of Saos-2 cells with ultraviolet light had no effect on the expression of luciferase from the full-length p21 promoter reporter construct (Fig. 3). These latter results are consistent with the fact that MCF7 cells express a functional wild-type p53 protein (42Guillot C. Falette N. Paperin M.P. Courtois S. Gentil-Perret A. Treilleux I. Ozturk M. Puisieux A. Oncogene. 1997; 14: 45-52Crossref PubMed Scopus (28) Google Scholar, 43Fan S. Smith M.L. Rivet D.J.I. Duba D. Zhan Q. Kohn K.W. Fornace A.J.J. O'Connor P.M. Cancer Res. 1995; 55: 1649-1654PubMed Google Scholar, 44Casey G. Lo H.M. Lopez M.E. Vogelstein B. Stanbridge E.J. Oncogene. 1991; 6: 1791-1797PubMed Google Scholar). 2H.-Y. Tang, K. Zhao, J. Langer, S. Waxman, and J. J. Manfredi, submitted for publication.Figure 4Ectopic expression of the human MDM2
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