Activation of p53-p21 Pathway in Response to Disruption of Cell-Matrix Interactions
1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês
10.1074/jbc.272.46.29091
ISSN1083-351X
AutoresRay‐Chang Wu, Axel H. Schönthal,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoThe proliferation of most cells is strictly dependent on cell-matrix interactions, a phenomenon called anchorage dependence. Because tumor cells often are independent of this regulation, it is important to characterize the molecular pathways that control cellular proliferation after detachment of cells from their matrix. In this report, we investigated a possible role of p53 and one of its target genes, p21 waf1/cip1 , as components of anchorage-dependent cell growth control. We found that p53 protein is rapidly activated upon the disruption of cellular attachment. This led to p21 transcriptional activation via two p53-binding sites in its promoter. Elevated p21 protein levels blocked transcription and activity of the cell cycle-regulator cyclin A, and cells became arrested in G1 of the cell cycle. Under the same conditions, fibroblasts from p53 knock-out mice did not activate p21 and did not down-regulate cyclin A expression but rather induced another cell cycle inhibitor, p27. Thus, our results characterize a chain of events, starting from the activation of p53 and proceeding via p21 to cyclin A, that is activated in response to the loss of cellular adherence. This p53-regulated pathway may constitute one of a few redundant systems to ensure proper cell control in multicellular organisms. The proliferation of most cells is strictly dependent on cell-matrix interactions, a phenomenon called anchorage dependence. Because tumor cells often are independent of this regulation, it is important to characterize the molecular pathways that control cellular proliferation after detachment of cells from their matrix. In this report, we investigated a possible role of p53 and one of its target genes, p21 waf1/cip1 , as components of anchorage-dependent cell growth control. We found that p53 protein is rapidly activated upon the disruption of cellular attachment. This led to p21 transcriptional activation via two p53-binding sites in its promoter. Elevated p21 protein levels blocked transcription and activity of the cell cycle-regulator cyclin A, and cells became arrested in G1 of the cell cycle. Under the same conditions, fibroblasts from p53 knock-out mice did not activate p21 and did not down-regulate cyclin A expression but rather induced another cell cycle inhibitor, p27. Thus, our results characterize a chain of events, starting from the activation of p53 and proceeding via p21 to cyclin A, that is activated in response to the loss of cellular adherence. This p53-regulated pathway may constitute one of a few redundant systems to ensure proper cell control in multicellular organisms. The proliferation of normal cells, with the exception of some hematopoietic cells, is strictly dependent on cell-matrix interactions. Nonadherent mesenchymal cells fail to proliferate despite the presence of growth factors, a characteristic called anchorage dependence. In contrast, many transformed cells have lost their anchorage dependence and grow independently of cell-matrix interactions. This anchorage-independent phenotype in cell culture has been found to closely correlate with the ability of cells to form tumors in animals (1Shin S.-I. Freedman V.H. Risser R. Pollack R. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4435-4439Crossref PubMed Scopus (662) Google Scholar). The proliferation of all cells, and their progression through the cell cycle, is regulated by the sequential activity of various cyclin-dependent kinases (cdks). 1The abbreviations used are: cdks, cyclin-dependent kinases; CKIs, cdk inhibitors; HEMA, poly(2-hydroxyethyl methacrylate); EMSA, electrophoretic mobility shift assay; MEFs, mouse embryo fibroblasts; tk, thymidine kinase. The enzymatic activity of cdks is dependent on posttranslational modifications, as well as on physical interactions with one of the cyclin proteins that are the regulatory subunits of cdks (2Hunter T. Pines J. Cell. 1994; 79: 573-582Abstract Full Text PDF PubMed Scopus (2181) Google Scholar, 3Graña X. Reddy E.P. Oncogene. 1995; 11: 211-219PubMed Google Scholar). The expression of cyclins is stimulated in response to growth factor stimulation of resting (G0) cells and is required for cell cycle progression. In addition, there are two families of cdk inhibitors (CKIs) as follows: p21 waf1/cip1 , p27 kip1 , and p57 kip2 , which bind to and inactivate most cyclin-cdk complexes, and the INK4s, which only inhibit complexes containing cdk4 and cdk6 (4Harper J.W. Elledge S.J. Curr. Opin. Genet. & Dev. 1996; 6: 56-64Crossref PubMed Scopus (362) Google Scholar, 5Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3227) Google Scholar). An important regulator of p21 transcription is the tumor suppressor protein p53 (6Harris C.C. Carcinogenesis. 1996; 17: 1187-1198Crossref PubMed Scopus (242) Google Scholar, 7El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (8032) Google Scholar). It is a transcription factor that binds to a specific DNA response element in the promoter of regulated genes, such as the muscle creatine kinase gene, the GADD45 gene, and theMDM-2 oncogene (8Bates S. Vousden K.H. Curr. Opin. Genet. & Dev. 1996; 6: 12-19Crossref PubMed Scopus (342) Google Scholar, 9Donehower L.A. Bradley A. Biochim. Biophys. Acta. 1993; 1155: 181-205PubMed Google Scholar, 10Milner J. Semin. Cancer Biol. 1994; 5: 211-219PubMed Google Scholar). In the promoter of the p21 gene, two such binding sites have been identified and shown to confer transcriptional activation by p53 (11El-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). In addition, the p21 gene has binding sites for other transcription factors that may play a role in the regulation of p21 expression by p53-independent pathways (12Macleod K.F. Sherry N. Hannon G. Beach D. Tokino T. Kinzler K. Vogelstein B. Jacks T. Genes Dev. 1995; 9: 935-944Crossref PubMed Scopus (759) Google Scholar, 13Liu Y. Martindale J.L. Gorospe M. Holbrook N.J. Cancer Res. 1996; 56: 31-35PubMed Google Scholar, 14Alpan R.S. Pardee A.B. Cell Growth Differ. 1996; 7: 893-901PubMed Google Scholar, 15Prowse D.M. Bolgan L. Molnár Á. Dotto G.P. J. Biol. Chem. 1997; 272: 1308-1314Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 16Qiu X. Forman H.J. Schönthal A.H. Cadenas E. J. Biol. Chem. 1996; 271: 31915-31921Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Zeng Y.-X. El-Deiry W.S. Oncogene. 1996; 12: 1557-1564PubMed Google Scholar). A link between cyclin expression and anchorage-dependent cell cycle progression has recently been established. In mouse and human fibroblasts, the serum-stimulated induction of cyclin A and cyclin D1 expression was dependent on cell adhesion (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar, 19Guadagno T.M. Ohtsubo M. Roberts J.M. Assoian R.K. Science. 1993; 262: 1572-1575Crossref PubMed Scopus (368) Google Scholar, 20Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (355) Google Scholar). In non-adherent cells, the expression of either mRNA and protein was blocked, and the cells were arrested in G1 of the cell cycle. However, the forced ectopic expression of either cyclin A or cyclin D1 cDNA resulted in anchorage-independent cell division, suggesting that both cyclins might be targets of the adhesion-dependent signals that control cell proliferation. At least part of this control might be exerted at the transcriptional level. Although one report suggested the involvement of the E2F-binding site in the cyclin A promoter (21Schulze A. Zerfass-Thome K. Berges J. Middendorp S. Jansen-Dürr P. Henglein B. Mol. Cell. Biol. 1996; 16: 4632-4638Crossref PubMed Scopus (122) Google Scholar), another report indicated the presence of a novel CCAAT-binding protein that may mediate the adhesion-dependent transcriptional activation of cyclin A (22Krämer A. Carstens C.-P. Fahl W.E. J. Biol. Chem. 1996; 271: 6579-6582Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In addition, another part of this control was exerted posttranscriptionally. It was shown that the kinase activity of cdk-cyclin E complexes was repressed after detachment of cells (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar,20Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (355) Google Scholar). However, there appear to be cell type-specific differences in the control of anchorage-dependent growth, not only between normal and tumor cells but also between cell lines that are strictly anchorage-dependent. For example, in anchorage-dependent NIH3T3 cells there is no cyclin D synthesis after serum stimulation of G0-synchronized cells when the cells are detached (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar). Consequently, the retinoblastoma protein, a substrate of cdk4-cyclin D complexes, cannot become phosphorylated. Hence, retinoblastoma protein stays in its hypophosphorylated, active form and prevents cells from entering S phase. In contrast, in NRK cells, which are also anchorage-dependent, cyclin D synthesis is stimulated under the same conditions, and retinoblastoma protein becomes phosphorylated (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar). However, the cells are still arrested in the cell cycle because they are unable to induce cyclin A expression to a level sufficient for proliferation. Together, these data also emphasize a certain redundancy in adhesion-dependent cell cycle control (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar, 19Guadagno T.M. Ohtsubo M. Roberts J.M. Assoian R.K. Science. 1993; 262: 1572-1575Crossref PubMed Scopus (368) Google Scholar, 20Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (355) Google Scholar). In our report, we sought to investigate molecular events that took place early after the disruption of cell-matrix interactions, and we analyzed their possible connection to later processes, such as the regulation of cyclin expression. In our studies, we found that p53 protein is rapidly activated upon the disruption of cellular attachment of logarithmically growing cells. This led to the transcriptional activation of the p21 gene via the two p53-binding sites in its promoter region. Elevated p21 protein levels blocked cyclin A transcription and activity and subsequently led to proliferation arrest in G1 of the cell cycle. In contrast, in cells lacking p53, p21 expression was not elevated, and cyclin A expression was not down-regulated; rather, in these cells another inhibitor of cyclin-dependent kinases, p27, was induced after the disruption of cell-matrix interactions. Thus, our results characterize a cell type-specific chain of events that starts from the activation of p53, proceeds via p21, and targets cyclin A. This p53-regulated pathway may constitute one of several partially redundant systems to ensure proper cell control in multicellular organisms. HEMA (poly-HEME; poly(2-hydroxyethyl methacrylate)) was obtained from Sigma and dissolved in ethanol at 10 mg/ml. Synthetic oligonucleotides for EMSAs were provided by the Core Facility of the K. Norris Jr. Comprehensive Cancer Center. C3 10T 1/2 mouse fibroblasts were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in a 5% CO2 atmosphere. Mouse embryo fibroblasts (MEFs) from p53 knock-out mice were kindly provided by Lawrence A. Donehower (Baylor College of Medicine, Houston, TX) and cultured as described above. For the disruption of cell-matrix interactions, monolayered cells were scraped off the culture dish and dispersed by pipetting. Then one-half was seeded back into a culture dish for re-attachment, and the other half was cultured in HEMA-coated plates which prevented the attachment of cells (23Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2828) Google Scholar). Most experiments were repeated with agar-coated plates (1.4% agar in complete medium) in which attachment of cells was prevented as well. Transient transfections were performed using the calcium-phosphate-DNA precipitation technique (24Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994Google Scholar). The transfection mixture was added into the medium, and the cells were exposed to the precipitate for 12 h. Then the monolayer was washed twice with phosphate-buffered saline, and the cells were scraped off the plate. One-half was put into a fresh plate, the other half was put into a HEMA-coated plate. After another 24 h the cells were harvested, and cellular lysates were prepared. After determining the protein concentration by use of the bicinchoninic assay reagent (Pierce), the luciferase activity of each lysate was determined in a luminometer. In all transfections a plasmid encoding β-galactosidase (CMV-β-gal) was included to monitor transfection efficiencies. All transfections were repeated at least three times. Luciferase reporter plasmids containing the human p21 promoter (11El-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) as well as plasmid PG13 (containing 13 p53-response elements upstream of a polyoma basal promoter) (7El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (8032) Google Scholar) were obtained from Wafik El-Deiry (University of Pennsylvania, Philadelphia, PA). To generate plasmid p21-tk, a 1.1-kilobase pair fragment containing both p53-binding sites, was excised from the p21-wt plasmid (called WWP-luc in Ref. 17Zeng Y.-X. El-Deiry W.S. Oncogene. 1996; 12: 1557-1564PubMed Google Scholar) with HindIII andAccI. This fragment was inserted upstream of a minimal thymidine kinase (tk) promoter-luciferase reporter. The expression vector for p21 was generated by excising a 0.85-kilobase pair mouse p21 cDNA fragment from plasmid p21-9c (25Huppi K. Siwarski D. Dosik J. Michieli P. Chedid M. Reed S. Mock B. Givol D. Mushinski J.F. Oncogene. 1994; 9: 3017-3020PubMed Google Scholar) with EcoRI and inserting it downstream of a cytomegalovirus promoter in plasmid pCMV-blue (PharMingen, San Diego, CA). Construct cyclin A-luciferase (26Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar) was obtained from Toshio Nikaido (Shinshu University, Matsumoto, Japan). PP2A-luciferase was generated by subcloning a 1200-base pairAccI/HindIII fragment of the PP2A catalytic subunit α promoter (27Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Biochemistry. 1991; 30: 89-97Crossref PubMed Scopus (84) Google Scholar) into pGL3-luc basic (Promega, Madison, WI). Total RNA was isolated using the guanidinium thiocyanate method (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (64498) Google Scholar), followed by poly(A) extraction using oligo(dT) beads (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Equal amounts of each RNA sample were separated on formaldehyde/agarose gels and transferred onto nitrocellulose membranes. For hybridization, specific riboprobes were generated using T7 RNA polymerase according to manufacturer's instructions. The hybridization was carried out essentially as described (30Schönthal A.H. Feramisco J.R. Oncogene. 1993; 8: 433-441PubMed Google Scholar). After hybridization, the membranes were washed twice at 80 °C in 0.2 × SSPE and 0.5% SDS for 30 min and subsequently exposed to Kodak X-AR autoradiographic film. After exposure, the filters were stripped and rehybridized to a probe for β-actin to control for equal amounts of RNA loaded in each lane. Cells were lysed in RIPA buffer as described (31Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). 20 μg of each sample was separated by polyacrylamide gel electrophoresis and blotted onto nitrocellulose. After blocking with blotto (5% milk, 0.1% Tween 20, 10 mm Tris/HCl, pH 7.5, 150 mm NaCl) for 1 h, the membrane was exposed to the primary antibody diluted in blotto at 4 °C overnight. All antibodies against cell cycle-regulatory proteins were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and diluted according to the manufacturer's instructions. The secondary antibodies were coupled to horseradish peroxidase and were detected by chemiluminescence using the SuperSignalTM Substrate (Pierce). Nuclear extracts from 107 cells per point were prepared exactly as described previously (32Pfeffer K. Matsuyama T. Kündig T.M. Wakeham A. Kishihara K. Shahinian A. Wiegmann K. Ohashi P.S. Krönke M. Mak T.W. Cell. 1993; 73: 457-467Abstract Full Text PDF PubMed Scopus (1546) Google Scholar). The protein concentration was measured using the bicinchoninic acid assay (Pierce) with bovine serum albumin as the standard. The binding reaction was performed by incubating 12 μg of nuclear extract with 0.5 μg of poly(dI-dC) and32P-labeled oligonucleotides in binding buffer (5 mm HEPES, pH 7.8, 5 mm MgCl2, 50 mm KCl, 0.5 mm dithiothreitol, 10% glycerol) in a final volume of 20 μl. The sequences for the double-stranded,in vitro synthesized oligonucleotides were as follows: p53-binding site at position −2800 of the mouse p21 promoter, 5′-GGAACATGTCTTGACATGTTC-3′; inactivated binding site, 5′-GGAATATATCTTGAATTCTTC-3′ (nucleotides that deviate from the wild type binding site are underlined). These double-stranded oligonucleotides were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase, purified over a column, and used at 4 × 104 cpm per reaction. To produce supershifts, the nuclear extracts were incubated with 100 ng of mouse monoclonal antibody against p53 (PAb 421, Calbiochem) for 30 min prior to the addition of oligonucleotides (33Milner J. Trends Biochem. Sci. 1995; 20: 49-51Abstract Full Text PDF PubMed Scopus (92) Google Scholar). For competition purposes, 100 ng (160-fold molar excess) of unlabeled wild type or mutant p53 oligonucleotides were included in the binding reaction. All binding reactions were incubated for 30 min at room temperature and separated on a 5% acrylamide gel. Several previous studies that analyzed the molecular mechanisms of anchorage dependence were performed by using G0-synchronized cells (18Zhu X. Ohtsubo M. Bohmer R.M. Roberts J.M. Assoian R.K. J. Cell Biol. 1996; 133: 391-403Crossref PubMed Scopus (408) Google Scholar, 19Guadagno T.M. Ohtsubo M. Roberts J.M. Assoian R.K. Science. 1993; 262: 1572-1575Crossref PubMed Scopus (368) Google Scholar, 20Fang F. Orend G. Watanabe N. Hunter T. Ruoslahti E. Science. 1996; 271: 499-502Crossref PubMed Scopus (355) Google Scholar). The studies showed that these cells, after detachment, were unable to induce expression of cyclin A and cyclin D1 in response to stimulation with serum growth factors and were not able to progress through the cell cycle. In our study presented here, we used cells that were growing logarithmically,i.e. that were distributed throughout the cell cycle at the onset of detachment. In contrast to G0-synchronized cells where the expression of cyclins is shut down and cdk activity is marginal, logarithmically growing cell cultures exhibit high levels of cyclin expression and cdk activity. Our goal was to determine how this highly active cell cycle machinery was controlled after the loss of cellular adherence. 10T 1/2 murine fibroblasts were either cultured in plastic tissue culture dishes (adherent) or on top of agar or HEMA-coated dishes (non-adherent) in the continuous presence of serum growth factors. Under non-adherent conditions, the cells became growth-arrested and accumulated in G1 of the cell cycle (not shown). At the same time, the kinase activity associated with cyclin A-containing cdks was down-regulated rapidly (Fig.1). To determine the mechanisms of inhibition of cdk activity, we next analyzed the protein levels of various cdks and cyclins (Fig.2). Cyclin A protein was down-regulated and became undetectable at 36 h after the onset of detachment. Cyclin D1 was down-regulated as well but was still detectable after 48 h. There was also some decrease in cdc2 protein levels, and no decrease in cdk4 protein. Importantly, however, none of these proteins exhibited a decrease within the first 4 h (Fig. 2 B), which is the period when cyclin A-containing complexes lost most of their kinase activity. This finding indicated the effects of two regulatory mechanisms: the first one at the post-translational level which controls the enzymatic activity of the kinase (within the first few hours) and the second at the level of expression which controls the synthesis of the various kinase subunits (a later event). Because p21 waf1/cip1 and p27 kip1 are known posttranslational regulators of cdk activity, we analyzed their expression levels during the non-attached culture of cells (Fig.2 A). Whereas the amount of p27 protein did not increase during the course of the experiment, p21 protein levels increased 3-fold after detachment. Because this increase was already maximal at 4 h, we analyzed its kinetics of induction at earlier time points as well. We found that p21 expression was induced very early after detachment. As shown in Fig. 2 B, the increase in p21 protein could be detected already after 1 h and reached its maximum at 3–4 h. At these time points, there was no change in the levels of cyclin A, cyclin D1, cdc2, and cdk4 proteins (Fig. 2 B). Because cdks containing cyclin A are known targets for p21, our results suggest that the posttranslational inhibition of cdk activity after detachment of cells may be due to the increased levels of the cdk inhibitor p21. To test this directly, we investigated the amount of p21 protein in cyclin A-containing protein complexes. For this purpose, cyclin A was immunoprecipitated, and the collected complexes were analyzed for their p21 content by Western blotting. As shown in Fig.3, the amount of p21 protein associated with these complexes was greatly increased after the loss of cellular adherence. This finding directly implicated p21 as a negative posttranslational regulator of cdk activity in response to cell-matrix disruption. To determine further whether elevated levels of p21 protein may also be involved in the observed down-regulation of cyclin A expression, we investigated the mechanisms of cyclin A down-regulation. As shown in Fig. 4 A, the mRNA levels of cyclin A decline after cellular detachment and are undetectable at 24 h thereafter. This down-regulation appeared to start around 6 h, which is well after the induction of p21 protein (Fig. 2) and p21 mRNA (Fig. 4 B). This finding raised the possibility that elevated levels of p21 may result in decreased expression of cyclin A. To test this more directly, we co-transfected an expression vector containing p21 cDNA together with a luciferase reporter construct under the control of the cyclin A promoter. This co-transfection was performed in adherent cells to avoid the induction of the endogenous p21 gene (which occurs after detachment of cells). As shown in Fig. 5, elevated levels of p21 caused a decrease in cyclin A promoter activity, indicating that p21 protein is able to inhibit, directly or indirectly, transcription from the cyclin A promoter. Similarly, after detachment of cells,i.e. under conditions where the amount of endogenous p21 protein is increased, the activity of a transfected cyclin A promoter is reduced as well (not shown). As a control, we also transfected the promoter of a different gene, protein phosphatase type 2A. In this case, no down-regulation was observed neither during detachment (not shown) nor with co-transfected p21 cDNA (Fig. 5), indicating that the observed inhibitory effects on the cyclin A promoter were specific. Rather, there was a weak (1.6-fold) induction of the PP2A promoter. As PP2A has been characterized as a negative regulator of cell growth (34Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (635) Google Scholar), this effect may warrant some further investigation in the future.Figure 5Cyclin A promoter activity in the presence of transfected p21 cDNA. Cells were grown attached to cell culture dishes. They were transiently co-transfected with 4 μg of cyclin A-luciferase reporter construct (cyclin A) together with 2 μg of either an expression vector for p21 (+p21, black bars) or the same expression vector without the p21 cDNA insert (−p21,white bars). As a control, p21 cDNA was also co-transfected with a luciferase reporter plasmid under the regulation of the promoter for serine/threonine protein phosphatase type 2A (PP2A). 24 h after transfection, the cells were harvested, and luciferase activity was determined. Shown is the average of 3–5 independent experiments. The luciferase activity from cells without co-transfected p21 cDNA was arbitrarily set at 100%.Error bars reflect standard deviation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results above indicated the involvement of p21 protein in the transcriptional and post-translational inhibition of cyclin A expression and activity during cellular detachment. Because this control seemed to be exerted through elevated levels of p21, and because the tumor suppressor protein p53 had been shown to be a major regulator of p21 expression, we investigated whether the increased expression of p21 during detachment was controlled by p53. Two p53-binding sites have been identified in the p21 promoter and shown to bind p53 protein (11El-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). One is located at −2225 and the other at −1330 with respect to the start of transcription of the gene. We used p21 promoter deletion mutants containing either both, only one, or none of the p53-binding sites, fused to the luciferase reporter gene (Fig. 6). After transfection of these constructs, the cells were split in half and cultured either under adherent or non-adherent conditions. As shown in Fig. 6, the p21 promoter containing both p53-binding sites (construct p21-wt) was induced nearly 5-fold after detachment of cells, as compared with adherent cells. When only one p53-binding site was present (construct p21-sm) this induction was reduced to 2-fold. The construct where both p53-binding sites had been deleted (p21-dm) was not induced at all. To confirm the importance of the p53-binding sites further, we transferred the fragment of the p21 promoter, which contained the two binding sites, to a heterologous promoter, the thymidine kinase (tk) promoter. The tk promoter alone, fused to the luciferase gene (ptk), was not induced after detachment of cells. However, after insertion of the two p53-binding sites (p21-tk), this construct was induced nearly 5-fold (Fig. 6). In addition, a construct containing 13 repeats of a consensus p53-binding site (pG13) was induced 3-fold after cellular detachment. Thus, these results demonstrate that the p53-binding site is necessary and sufficient for the induction of p21 during non-adherent culture conditions. The activation of p53 was confirmed by electrophoretic mobility shift assays (EMSAs). Extracts from adherent or non-adherent cells were incubated with an oligonucleotide representing the distal p53-binding site of the p21 promoter. This sequence has been shown before to bind p53 protein (11El-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). As can be seen in Fig.7, there was four times more binding activity in cells that were detached, indicating elevated p53 activity. A mutated oligonucleotide with several point mutations did not compete for specific binding and did not exhibit any increased binding in detached cells. Together with the observed transcriptional activation via the p53-binding sites, these experiments demonstrate activation of p53 after detachment of cells. Our data so far indicated that disruption of cell-matrix interactions activated a chain of events that involve p53, followed by the transcriptional activation of p21, which is followed by the transcriptional and posttranslational inhibition of cyclin A. We therefore investigated next whether or not these events would take place in cells that lack functional p53 protein. We used mouse embryo f
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