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 Ca(2+)-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 Ca(2+)- and cell cycle-dependent p53-S100A2 interaction might modulate proliferation.
Highlights
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
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)
The S100A2 protein belongs to the calcium binding, EF-hand protein family of S100 proteins and has attracted interest due to the fact that it is localized to the nucleus [22], it is downregulated in various tumor tissues [24, 42, 43], and it is presumably a potential tumor suppressor gene [12, 16]
Summary
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 5Ј BamHI site and a 3Ј NotI site. p21-luc (in pGL2), p53Asp2813Gly, and p531–362 (lacking the S100B binding site) were gifts from Patrick Chene, Novartis, Basel and have been described previously [35, 36]. Human p53 proteins in nuclear extracts were obtained from H1299 cells after transient transfection using the calcium phosphate method. Nuclear extracts were incubated in the presence of 5ϫ 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 g of recombinant S100A2 protein. 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 g of GST-S100A2 coupled to glutathioneSepharose beads washed with 2 mM CaCl2 were incubated with 4 l of the IVT reaction mix and NET-80 buffer (80 mM NaCl, 20 mM Tris) for 3 h at 4 °C.
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