Title: The Calcium-binding Protein S100A2 Interacts with p53 and Modulates Its Transcriptional Activity
Abstract: Head and neck squamous cell carcinoma express high levels of the EF-hand calcium-binding protein S100A2 in contrast to other tumorigenic tissues and cell lines where the expression of this protein is reduced. Subtractive hybridization of tumorigenic versus normal tumor-derived mammary epithelial cells has previously identified the S100A2 protein as potential tumor suppressor. The biological function of S100A2 in carcinogenesis, however, has not been elucidated to date. Here, we report for the first time that during recovery from hydroxyurea treatment, the S100A2 protein translocated from the cytoplasm to the nucleus and co-localized with the tumor suppressor p53 in two different oral carcinoma cells (FADU and SCC-25). Co-immunoprecipitation experiments and electrophoretic mobility shift assay showed that the interaction between S100A2 and p53 is Ca2+-dependent. Preliminary characterization of this interaction indicated that the region in p53 involved with binding to S100A2 is located at the C terminus of p53. Finally, luciferase-coupled transactivation assays, where a p53-reporter construct was used, indicated that interaction with S100A2 increased p53 transcriptional activity. Our data suggest that in oral cancer cells the Ca2+- and cell cycle-dependent p53-S100A2 interaction might modulate proliferation. Head and neck squamous cell carcinoma express high levels of the EF-hand calcium-binding protein S100A2 in contrast to other tumorigenic tissues and cell lines where the expression of this protein is reduced. Subtractive hybridization of tumorigenic versus normal tumor-derived mammary epithelial cells has previously identified the S100A2 protein as potential tumor suppressor. The biological function of S100A2 in carcinogenesis, however, has not been elucidated to date. Here, we report for the first time that during recovery from hydroxyurea treatment, the S100A2 protein translocated from the cytoplasm to the nucleus and co-localized with the tumor suppressor p53 in two different oral carcinoma cells (FADU and SCC-25). Co-immunoprecipitation experiments and electrophoretic mobility shift assay showed that the interaction between S100A2 and p53 is Ca2+-dependent. Preliminary characterization of this interaction indicated that the region in p53 involved with binding to S100A2 is located at the C terminus of p53. Finally, luciferase-coupled transactivation assays, where a p53-reporter construct was used, indicated that interaction with S100A2 increased p53 transcriptional activity. Our data suggest that in oral cancer cells the Ca2+- and cell cycle-dependent p53-S100A2 interaction might modulate proliferation. S100A2 is a member of the subfamily of S100 Ca2+-binding proteins, characterized by two distinct EF-hand structural motifs. It is a homodimeric protein that upon binding of calcium undergoes a conformational change (1Rustandi R.R. Baldisseri D.M. Weber D.J. Nat. Struct. Biol. 2000; 7: 570-574Crossref PubMed Scopus (292) Google Scholar, 2Bhattacharya S. Bunick C.G. Chazin W.J. Biochim. Biophys. Acta. 2004; 1742: 69-79Crossref PubMed Scopus (215) Google Scholar). The transduction of calcium signals in that form regulates many cellular functions such as the control of cell growth and proliferation (3Taylor D.A. Bowman B.F. Stull J.T. J. Biol. Chem. 1989; 264: 6207-6213Abstract Full Text PDF PubMed Google Scholar), transcription (4Kao J.P. Alderton J.M. Tsien R.Y. Steinhardt R.A. J. Cell Biol. 1990; 111: 183-196Crossref PubMed Scopus (147) Google Scholar), and p53-dependent growth arrest and apoptosis (5Lam M. Dubyak G. Chen L. Nunez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (611) Google Scholar, 6Heizmann C. Fritz G. Schafer B. Front. Biosci. 2002; 7: d1356-d1368Crossref PubMed Google Scholar). The S100A2 protein has been first detected in lung and kidney and is mainly expressed in a subset of tissues and cells such as breast epithelia and liver (6Heizmann C. Fritz G. Schafer B. Front. Biosci. 2002; 7: d1356-d1368Crossref PubMed Google Scholar, 7Fritz G. Heizmann C.W. Messerschmidt A.W. Bode A.M. Cygler M. Handbook of Metalloproteins. 3. Wiley, New York2004: 529-540Google Scholar, 8Franz C. Durussel I. Cox J. Schafer B. Heizmann C. J. Biol. Chem. 1998; 273: 18826-18834Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Glenney Jr., J.R. Kindy M.S. Zokas L. J. Cell Biol. 1989; 108: 569-578Crossref PubMed Scopus (88) Google Scholar, 10Zhang T. Woods T.L. Elder J.T. J. Invest. Dermatol. 2002; 119: 1196-1201Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). S100A2 and twenty other S100 genes are located on a cluster on human chromosome 1q21, a region frequently rearranged in human cancer (6Heizmann C. Fritz G. Schafer B. Front. Biosci. 2002; 7: d1356-d1368Crossref PubMed Google Scholar, 11Marenholz I. Heizmann C.W. Fritz G. Biochem. Biophys. Res. Commun. 2004; 322: 1111-1122Crossref PubMed Scopus (677) Google Scholar). Interestingly the cDNA coding for the S100A2 protein was identified as a novel tumor suppressor gene by subtractive hybridization between normal and tumor-derived human mammary epithelial cells (12Lee S. Tomasetto C. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2825-2829Crossref PubMed Scopus (316) Google Scholar). Expression studies showed that the S100A2 gene is markedly down-regulated in several tumor tissues of various origins like melanomas (13Maelandsmo G. Florenes V. Mellingsaeter T. Hovig E. Kerbel R. Fodstad O. Int. J. 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Recently, much attention has been paid to the expression of the S100A2 gene and gene product in head and neck squamous cell carcinoma (HNSCC). 1The abbreviations used are: HNSCC, head and neck squamous cell carcinoma; FADU/SCC-25, oral epithelial carcinoma cells; HBL100, human breast epithelia cell line; HU, hydroxyurea; IP, immunoprecipitation; GST, glutathione S-transferase; IVT, in vitro translated; FACS, fluorescence-activated cell sorting. In contrast to breast (14Pedrocchi M. Schafer B. Mueller H. Eppenberger U. Heizmann C. Int. J. Cancer. 1994; 57: 684-690Crossref PubMed Scopus (132) Google Scholar, 16Wicki R. Franz C. Scholl F. Heizmann C. Schafer B. Cell Calcium. 1997; 22: 243-254Crossref PubMed Scopus (120) Google Scholar) and colon carcinoma (17Bronckart Y. Decaestecker C. Nagy N. Harper L. Schafer B.W. Salmon I. Pochet R. Kiss R. Heizman C.W. Histol. Histopathol. 2001; 16: 707-712PubMed Google Scholar), the S100A2 protein is overexpressed in a subset of HNSCC (18Nagy N. Brenner C. Markadieu N. Chaboteaux C. Camby I. Schafer B.W. Pochet R. Heizmann C.W. Salmon I. Kiss R. Decaestecker C. Lab. Invest. 2001; 81: 599-612Crossref PubMed Scopus (89) Google Scholar, 19Lauriola L. Michetti F. Maggiano N. Galli J. Cadoni G. Schafer B.W. Heizmann C.W. Ranelletti F.O. Int. J. Cancer. 2000; 89: 345-349Crossref PubMed Scopus (59) Google Scholar). Furthermore, S100A2 expression in HNSCC has been positively associated with squamous cell differentiation and negatively with tumor grading (20Shrestha P. Muramatsu Y. Kudeken W. Mori M. Takai Y. Ilg E.C. Schafer B.W. Heizmann C.W. Virchows Arch. 1998; 432: 53-59Crossref PubMed Scopus (68) Google Scholar). Immunolocalization studies revealed that the protein, preferably located in the nucleus in normal tissue (21Mandinova A. Atar D. Schafer B. Spiess M. Aebi U. Heizmann C. J. Cell Sci. 1998; 111: 2043-2054Crossref PubMed Google Scholar, 22Mueller A. Bachi T. Hochli M. Schafer B. Heizmann C. Histochem. 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Anticancer Res. 1996; 16: 2385-2388PubMed Google Scholar) and tumor progression (28Nylander K. Stenling R. Gustafsson H. Zackrisson B. Roos G. Cancer. 1995; 75: 87-93Crossref PubMed Scopus (83) Google Scholar). Induction of p53 transactivation activity by DNA damage results in increased S100A2 transcription (29Tan M. Heizmann C.W. Guan K. Schafer B.W. Sun Y. FEBS Lett. 1999; 445: 265-268Crossref PubMed Scopus (67) Google Scholar). Furthermore, S100B, which is present in neuronal tissues and is associated with brain tumors (8Franz C. Durussel I. Cox J. Schafer B. Heizmann C. J. Biol. Chem. 1998; 273: 18826-18834Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 30Baudier J. Delphin C. Grunwald D. Khochbin S. Lawrence J.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11627-11631Crossref PubMed Scopus (303) Google Scholar, 31Delphin C. Ronjat M. Deloulme J. Garin G. Debussche L. Higashimoto Y. Sakaguchi K. Baudier J. J. Biol. Chem. 1999; 274: 10539-10544Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Scotto C. Delphin C. Deloulme J. Baudier J. Mol. Cell. Biol. 1999; 19: 7168-7180Crossref PubMed Scopus (57) Google Scholar), and S100A4, overexpressed in metastatic breast cancer cell lines (33Grigorian M. Andresen S. Tulchinsky E. Kriajevska M. Carlberg C. Kruse C. Cohn M. Ambartsumian N. Christensen A. Selivanova G. Lukanidin E. J. Biol. Chem. 2001; 276: 22699-22708Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), were recently reported to interact with p53, and this in turn was shown to cause decreased p53 transcriptional activity. However, although interactions between p53 and S100 proteins are of particular interest, the mechanism of S100-p53-regulated growth arrest, and in particular the role of S100A2 in carcinogenesis in HNSCC, has not been elucidated to date. In an attempt to study the biological role of S100A2, we investigated the subcellular localization of S100A2 and p53 proteins in HNSCC cell lines synchronized with the DNA-replication inhibitor hydroxyurea (34Timson J. Mutat. Res. 1975; 32: 115-132Crossref PubMed Scopus (216) Google Scholar). Next, we examined the calcium dependence of S100A2-p53 association in vivo using two distinct HNSCC lines as well as cells derived from breast cancer epithelia. The interaction between the two proteins was confirmed through in vitro pull-down assay using full-length and truncated p53 proteins. Moreover, the effect of this interaction on p53 transactivation was examined in a luciferase-coupled reporter assay. Our data provide the first insights into the regulation of p53 activity by S100A2. DNA Constructs—Full-length cDNA of human p531-393 and the deletion construct p5373-393, both containing a Kozak consensus start, were cloned into the mammalian expression vector pcDNA3 (Invitrogen) by PCR employing a 5′ BamHI site and a 3′ NotI site. p21-luc (in pGL2), p53Asp281→Gly, and p531-362 (lacking the S100B binding site) were gifts from Patrick Chène, Novartis, Basel and have been described previously (35Atema A. Chene P. Cancer Lett. 2002; 185: 103-109Crossref PubMed Scopus (11) Google Scholar, 36Chene P. Ory K. Ruedi D. Soussi T. Hegi M.E. Int. J. Cancer. 1999; 82: 17-22Crossref PubMed Scopus (6) Google Scholar). The full-length S100A2 cDNA was cloned as a fusion into pGEX-3X vector (Amersham Biosciences) by PCR using a 5′ BamHI site and 3′ EcoRI site. All constructs were controlled by sequencing. Human Squamous Cell Carcinoma Tissues Biopsies—Human HNSCC tissue sections originating from patients suffering from hypopharynx and tongue carcinoma were fixed in formalin and embedded in paraffin. The sections were stained using the following antibodies: monoclonal mouse anti-human p53-DO-1 (p53-DO-1, Santa Cruz, Biotechnology, Santa Cruz, CA), monoclonal mouse anti-human p53-1801 (p53-1801; Santa Cruz, Biotechnology), polyclonal human anti-rabbit S100A2 (anti-S100A2), polyclonal human anti-rabbit S100A4 (anti-S100A4), and polyclonal human anti-rabbit S100A6 (anti-S100A6, all from Dako, Glostrup, Denmark), at a dilution of 1:25. Human tissue samples were analyzed using a wide field microscope (Leica, Switzerland), at a resolution of ×20. Human HNSCC-Cell Lines—FADU (originating from the hypopharynx, HTB-43, ATCC, Manassas, VA) and SCC-25 cells (from a tongue, ATCC CCL-1628, kindly provided by Dr. C. Decaestecker, Institute de Pharmacy, Université Libre de Bruxelles, Bruxelles), normal breast epithelial cell line HBL-100 (HCC1187, ATCC), and H1299 lung adenocarcinoma cells (CRL-5803, ATCC) were all grown at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin (complete medium). Transfections were performed using the calcium phosphate method. Cell Synchronization Assay and Cell Cycle Analysis—Exponentially growing FADU, SCC-25, and HBL100 cells were treated with 2 mm HU (Fluka, Buchs, Switzerland) for 24, 28, and 20 h, respectively, to obtain G1/S-phase arrest. Synchronized cells were released from the HU block and subjected to cell cycle analysis. For FACS analysis, samples were collected at the indicated time points, trypsinized, washed, and stained with propidium iodide (25 μg/ml) according to the manufacturer's guidelines (Cycle Test™Plus DNA Reagent Kit, BD Biosciences). Stained samples were analyzed in a fluorescence-activated cell sorter (FACSCalibur, BD Biosciences), and cell cycle distribution was analyzed with WinMDI software. Immunofluorescence—FADU and SCC-25 cells were grown on glass coverslips and synchronized with HU. Cells were fixed in 2% paraformaldehyde for 15 min at room temperature, washed four times with phosphate-buffered saline, permeabilized using 0.1% Triton for 1 min, and blocked in Dulbecco's modified Eagle's medium-horse serum (1%) for 1 h at room temperature after extensive washes with phosphate-buffered saline. Slides were incubated with the following antibodies: monoclonal anti-p53-DO1 at a dilution of 1:100 and polyclonal anti-S100A2 at a dilution of 1:500 for 1 h at 37 °C. Samples were washed with phosphate-buffered saline and incubated with the secondary CY2- and CY5-conjugated anti-mouse and the CY3-conjugated anti-rabbit antibody (Dianova, Hamburg, Germany) both at a dilution of 1:200 as described previously (37Ilg E.C. Troxler H. Burgisser D.M. Kuster T. Markert M. Guignard F. Hunziker P. Birchler N. Heizmann C.W. Biochem. Biophys. Res. Commun. 1996; 225: 146-150Crossref PubMed Scopus (42) Google Scholar). Nuclear stainings were performed using 4′,6-diamidino-2-phenylindole. Control stainings were performed on untreated cells and with the secondary antibody alone. Localization of the proteins was obtained with a Leica confocal microscope (DMIRE, Wetzlar, Germany). Western Blot—For whole cell extracts, cells were lysed in ice-cold lysis buffer (50 mm Tris buffer (pH 7.5), 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1% Triton, 5 mm β-mercaptoethanol, 100 mm NaCl), and samples were clarified by centrifugation for 15 min at 14,000 rpm in an Eppendorf centrifuge. 100 μg of protein extracts were resuspended in loading buffer, heated for 5 min at 95 °C and loaded onto a 4-12%-gradient SDS-PAGE gel (Invitrogen). Upon separation of proteins under denaturing conditions, proteins were transferred to Nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) and probed with the following antibodies: polyclonal anti-S100A2 at 1:2000 dilution, monoclonal anti-p53-DO1 antibodies at 1:5000 dilution for 1 h at room temperature in TBST/3% milk powder. Membranes were washed in TBST, incubated with the secondary anti-rabbit- or the anti-mouse-horseradish peroxidase-conjugated antibody (1:10000), respectively, for 1 h at room temperature, and exposed to ECL detection reagent (ECL, Amersham Biosciences). Protein bands were visualized on Kodak films. Co-immunoprecipitation—FADU and HBL100 cell extracts were prepared as described above and precleared with Protein G- and Protein A-Sepharose beads (Amersham Biosciences). Monoclonal anti-p53-DO-1 and polyclonal anti-S100A2 antibodies were coupled to Protein-G- and Protein-A-Sepharose beads, respectively, and washed with NET-80 buffer (20 mm Tris, pH 7.5, 80 mm NaCl, 1 mm ETDA) for 1 h at room temperature. The beads were again washed with NET-80 buffer and precleared cell extracts were incubated for 6 h at 4°C in the presence of 0.1-2 mm CaCl2. Pellets were centrifuged at 11,000 rpm, rinsed once with NET-80 buffer, and three times with buffer A (50 mm Tris, pH 8.0, 0.2% Triton, 500 mm NaCl), buffer B (50 mm Tris, pH 8.0, 0.1% Triton, 150 mm NaCl, 0.1% SDS), and buffer C (50 mm Tris, pH 8.0, 0.1% Triton). Western blotting was carried out as described above. Electrophoretic Mobility Shift Assay—An oligonucleotide derived from the p21/WAF promoter (33Grigorian M. Andresen S. Tulchinsky E. Kriajevska M. Carlberg C. Kruse C. Cohn M. Ambartsumian N. Christensen A. Selivanova G. Lukanidin E. J. Biol. Chem. 2001; 276: 22699-22708Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar) was labeled with [γ-32P]ATP (Amersham Biosciences). Human p53 proteins in nuclear extracts were obtained from H1299 cells after transient transfection using the calcium phosphate method. Human recombinant S100A2 protein was purified as previously described (8Franz C. Durussel I. Cox J. Schafer B. Heizmann C. J. Biol. Chem. 1998; 273: 18826-18834Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Nuclear extracts were incubated in the presence of 5× gel shift binding buffer (20% glycerol, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, 250 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.25 mg/ml poly(dI-dC)) and 0.5 and 1 μg of recombinant S100A2 protein. For the S100A2 interaction with p53, the reaction mix was incubated in the presence of 2 mm calcium or 5 mm EDTA for 1-2 h at 4 °C. Radiolabeled oligonucleotide p21/WAF was added, and the reaction was allowed to proceed for an additional 20 min at room temperature. The assay was terminated with 1 μl of gel loading 10× buffer (250 mm Tris-HCl, pH 7.5, 0.2% bromphenol blue, 40% glycerol). Samples were run on 5% non-denaturing polyacrylamide gel (CleanGel™ System 25S, Amersham Biosciences) according to the manufacturer's guidelines. The gel was dried and exposed to x-ray film at -80 °C overnight. GST-Pull-down Assay—The GST-S100A2 fusion construct was expressed from pGEX-3X and purified as previously described (38Fikrig E. Barthold S.W. Kantor F.S. Flavell R.A. Science. 1990; 250: 553-556Crossref PubMed Scopus (358) Google Scholar). p53 full-length and deletion mutants were cloned in pcDNA3 plasmid (Invitrogen) and used to produce 35S-labeled proteins (Amersham Biosciences) with the TnT-coupled transcription-translation system (IVT, Promega, Madison, WI): 2 μg of GST-S100A2 coupled to glutathione-Sepharose beads washed with 2 mm CaCl2 were incubated with 4 μl of the IVT reaction mix and NET-80 buffer (80 mm NaCl, 20 mm Tris) for 3 h at 4 °C. Radiolabeled proteins were pulled down, washed extensively with NET-80 buffer, and separated on a 4-12% SDS-Gel NuPAGE (Invitrogen). The gel was dried and exposed to x-ray film at -80 °C to visualize radiolabeled proteins. Transcriptional Activation Assay—A luciferase construct driven under the control of the p21 promoter (p21-luc) was used to study the transcriptional activity of p53. H1299 cells (6 × 105) were plated in 60-mm dishes in the presence of complete medium and transfected after 24 h using the calcium phosphate method. The following constructs were used: p531-393, p5373-393, and p531-362 (lacking the S100B-p53 binding site) and S100A2. After 48 h of transfection, luciferase activity was measured according to the manufacturer's guidelines (Promega). Transfection efficiency was normalized using β-galactosidase activity. Results are the mean of three independent experiments. Statistical significance was evaluated using the Student t test. p53 and S100 Proteins Are Differentially Localized in Human HNSCC Tissue Biopsies—S100 and p53 display a characteristic nuclear and cytoplasmic staining in tumor biopsy sections (Fig. 1) originating from the tongue (upper panel) and the hypopharynx (lower panel). Wild-type p53 predominantly accumulated in the nucleus in both tissue sections but was found in the cytoplasm of sections of the tongue (upper panel, a). S100A2 was expressed in the cytoplasm and diffusely in the nucleus in sections of the tongue (upper panel, c) as well as the hypopharynx (lower panel, c). Compared with S100A2, S100A4 (upper and lower panels, d) and S100A6 (upper and lower panels, e) were both exclusively expressed in the cytoplasm of HNSCC tissue biopsies. The staining for the S100B protein was negative (upper and lower panels, f). To further investigate the evidence on the cytosolic localization of S100A2 and p53, which are normally nuclear proteins (14Pedrocchi M. Schafer B. Mueller H. Eppenberger U. Heizmann C. Int. J. Cancer. 1994; 57: 684-690Crossref PubMed Scopus (132) Google Scholar, 22Mueller A. Bachi T. Hochli M. Schafer B. Heizmann C. Histochem. Cell Biol. 1999; 111: 453-459Crossref PubMed Scopus (63) Google Scholar), we examined cell lines derived from HNSCC tissues (FADU and SCC-25 cells). Cell Cycle-dependent Localization of S100A2 in Human FADU and SCC-25 Cells—To assess whether transition through the cell cycle affects S100A2 subcellular localization, FADU and SCC-25 cells expressing both endogenous S100A2 and p53 proteins were treated with HU. HU blocks ribonucleoside diphosphate reductase and leads to rapid depletion of deoxyribonucleotide pool, thereby arresting the cells at the G1/S boundary. Immunofluorescence staining of untreated FADU cells showed that p53 and S100A2 were predominantly localized in the nucleus, whereas cytoplasmic staining for both proteins was evident in SCC-25 cells (Fig. 2, A and B). The merged images indicated partial co-localization of p53 and S100A2 in the nucleus in FADU but mainly cytoplasmic co-localization in SCC-25 cells. Treatment of FADU or SCC-25 with 2 mm HU for 24 or 28 h, respectively, resulted in synchronization at G1/S as shown by flow cytometric analysis (Fig. 3, A and B). Under these conditions, p53 and S100A2 co-localized in the nucleus and the cytoplasm in both cell lines (Fig. 2, t = 0, panels C and D). Upon HU removal (t = 1), cells synchronously moved into S-phase (Fig. 3, A and B), and this was characterized by translocation of p53 to the nucleus in FADU and SCC-25 cells. At this time point, S100A2 translocated to the nucleus in both cell lines (Fig. 2, C and D). At 8 and 10 h, respectively, after release from the HU block (t = 2), cells were mostly in G2 phase (Fig. 3, A and B), and both p53 and S100A2 proteins were exclusively present in the nucleus. During transition through the next cell cycle, at 12 and 16 h after release from the HU block (t = 3), p53 and S100A2 were redistributed to the cytoplasm where they co-localized. Finally, in FADU cells, 24 h after release from the HU block (t = 4), p53 and S100A2 staining returned to the pattern displayed in untreated cells.Fig. 3Cell cycle analysis of FADU and SCC-25 cells synchronized with HU. FADU (A), SCC-25 (B), and HBL100 cells (C) synchronized with HU (2 mm) for 24, 28, and 20 h, respectively, were released at the indicated times in complete medium and stained with propidium iodide prior to flow cytometric analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) FACS analysis of FADU and SCC-25 cells indicated that the less differentiated SSC-25 cells had a longer cell cycle as compared with the former (Fig. 3, A and B). Taken together these data show cell cycle-dependent shuttling of p53 and S100A2 in FADU and SSC-25 cells with co-localization in the nucleus at late S/G2-phase. Endogenous S100A2 and p53 Interact in FADU and HBL100 Cells in a Calcium-dependent Manner—Co-localization of p53 suggested that the two proteins may physically interact. To substantiate this finding, we performed co-immunoprecipitation experiments using total extracts of FADU cells. Precleared FADU cell lysates were immunoprecipitated with p53-DO-1 antibody in the presence of increasing calcium concentrations or 5 mm EDTA. Proteins were resolved by SDS-PAGE and detected by Western blot analysis with anti-S100A2 (Fig. 4A). The results showed that S100A2 co-immunoprecipitated with p53 in the presence of calcium concentrations higher than 0.1 mm (lanes 3-5). In contrast, no interaction between p53 and S100A2 could be detected in the presence of a calcium chelator (lane 7) or at low calcium concentration (lane 2). Identical results were obtained when immunoprecipitations (IP) were performed with S100A2 antibody and p53 was detected with p53-DO-1 antibody (Fig. 4B). To rule out the possibility that the signal given by the IgG-heavy chain of the antibody used in IP could be erroneously interpreted as p53, we performed control-IP using anti-S100A2 antibody in the absence of cell extract. The results indicated that no protein band at the level of ∼50 kDa could be detected when using antibody p53-DO-1 for Western blotting (Fig. 4, B and D, lane 1). Western blotting of S100A2 confirmed the efficiency of the antibody used for IP (Fig. 4, C and E). To support the evidence obtained from FADU cells, we employed HBL100 cells that originate from breast cancer tissue and, like FADU cells, express wild-type p53 and S100A2 (Fig. 4D). As shown in Fig. 3C, HBL100 cells could also be synchronized with HU. Precleared HBL100 cell lysates were incubated with anti-S100A2 antibody under the same conditions used for FADU cells. Also in the case of HBL100 cells, the interaction between p53 and S100A2 was readily observable though at slightly higher calcium concentrations (lane 5) than in FADU cells. As observed in the latter, addition of 5 mm EDTA during IP completely abrogated this interaction (lane 7). Taken together, these findings suggest that the interaction between p53 and S100A2 is Ca2+-dependent and occurs in cell lines expressing the two proteins, independent of the tumor type from which the cells originate. S100A2 Binding to p53 Affects Its DNA Binding Activity—To characterize the physiological consequences of S100A2 binding to p53, we examined p53 DNA binding activity (Fig. 5). To this end, we performed electrophoretic mobility shift using nuclear extracts from H1299 cells and a labeled oligonucleotide containing the p53-consensus sequence of the p21 promoter. Because H1299 cells lack both p53 and S100A2, they were transiently transfected with the p531-393 construct (lane 1). Addition of 0.5 μg of recombinant S100A2 protein to H1299 nuclear extracts resulted in supershift of the oligonucleotide-p53 complex in the presence (lane 4) but not in the absence of 2 mm calcium (lane 3). Addition of 5 mm EDTA to the p53-S100A2 complex completely reversed the effect of calcium (lane 5). Furthermore, we tested DNA binding in the presence of increasing amounts of S100A2. Previous reports have shown that increasing amounts of S100B reduce the intensity of p53 complex binding to its responsive element (30Baudier J. Delphin C. Grunwald D. Khochbin S. Lawrence J.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11627-11631Crossref PubMed Scopus (303) Google Scholar, 31Delphin C. Ronjat M. Deloulme J. Garin G. Debussche L. Higashimoto Y. Sakaguchi K. Baudier J. J. Biol. Chem. 1999; 274: 10539-10544Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 39Lin J. Blake M. Tang C. Zimmer D. Rustandi R. Weber D. Carrier F. J. Biol. Chem. 2001; 276: 35037-35041Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 40Ko L. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar, 41Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1177) Google Scholar). Similarly, in the presence of 1 μg of S100A2 and 2 mm calcium, the intensity of the S100A2-p53 complex was clearly reduced (lane 7). Again, calcium was essential for the induction of the supershift, and the latter could be reversed by 5 mm EDTA (lane 8). Ectopic expression of p53 proteins in transiently transfected H1299 cells was controlled by Western blot analysis (Fig. 5B). These results show that the Ca