Cooperation between ARID3A and p53 in the transcriptional activation of p21WAF1 in response to DNA damage.

ARID3A/DRIL1/Bright is a family member of the AT rich interaction domain (ARID) DNA-binding proteins that are involved in diverse biological processes. We have reported that p53 activates ARID3A transcription, and ARID3A overexpression induces G1 arrest. However, the role of ARID3A in the p53 pathway remains unclear. Here, we show that ARID3A cooperates with p53 to transcriptionally activate p21(WAF1), a p53-target gene important for cell-cycle arrest. ARID3A bound to its binding sites in the p21(WAF1) promoter in vivo and in vitro, and induced p21(WAF1) transcription in U2OS cells expressing wild-type p53 but not Saos-2 cells lacking p53. The co-expression of ARID3A with p53 cooperates to activate p21(WAF1) transcription and the stably transfected p21(WAF1) promoter. Mutation of the ARID3A binding sites reduced the p21(WAF1) promoter activity, and siRNA-based ARID3A knockdown suppressed the transcription of p21(WAF1), but not the proapoptotic NOXA and PUMA in response to DNA damage. Furthermore, p53 knockdown decreased ARID3A transcription, and, conversely, ARID3A overexpression and knockdown resulted in an increase or decrease in p53 stability, respectively. These results indicate both cooperative and interdependent roles for ARID3A and p53 in the transcriptional activation of p21(WAF1) in response to DNA damage.

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation.

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.

Interaction of E2F-Rb family members with corepressors binding to the adjacent E2F site.

Cell cycle-dependent transcriptional repression of the E2F1 and B-myb promoters is mediated through E2F-binding sites and adjacent corepressor site (cell cycle gene homology region (CHR)/downstream repression site (DRS)). Here, we show that a factor binding to the B-myb CHR is co-purified with E2F DNA-binding activity, and coimmunoprecipitated with components of E2F/Rb-family repressor complexes, E2F4 and retinoblastoma (Rb) family proteins. In spite of structural and functional similarities, however, the E2F1 and B-myb CHRs exhibited distinct factor-binding specificities. Furthermore, substitution of E2F1 CHR with the B-myb CHR in the E2F1 promoter revealed that the B-myb CHR was unable to repress the E2F1 promoter completely in the G0 phase. These results suggest that transcriptional repression of the E2F1 and B-myb promoters is mediated by physical interaction of E2F/Rb-family repressor complexes with promoter-specific corepressors.

Loss of cyclin E requirement in cell growth of an oral squamous cell carcinoma cell line implies deregulation of its downstream pathway.

Cyclin E and Cdk2 have been shown to play an important role in G1/S transition of the cell cycle. Two E-type cyclins (E1 and E2) have been identified to date and share functionally similarities. Upregulation of these cyclins has been observed frequently in human cancers. We examined the expression profile of cyclin E1 and E2 in cell lines derived from human oral squamous cell carcinoma (SCC), and found that the expression of cyclin E1 protein was hardly detected in HSC-2 cells. Although cyclin E2 was abundantly expressed, histone H1 kinase activities of both E-type cyclins were virtually undetectable in this cell line. Inhibition of cyclin E1, but not that of E2, by using vectors expressing antisense-oriented their cDNAs induced drastic growth suppression on HOC313 cells that express both E-type cyclins. Inhibition of neither cyclin E1 nor E2 suppressed the growth of HSC-2 cells, and compensatory elevation of cyclin E1 was not evident in cyclin E2-inhibited HSC-2 cells. In contrast, HSC-2 cells expressed cyclin D1 and hyperphosphorylated forms of Rb family proteins, and were arrested in G1 by overexpression of p16(INK4), a specific inhibitor against D-type cyclin activity. These results indicate that HSC-2 cells lost proper growth control specifically mediated by cyclin E and suggest that deregulation of its downstream pathway may contribute to tumorigenesis of oral SCC.

Differential Expression of c-myc and N-myc during Oral Organogenesis of the Mouse Embryo.

The expressions of the c- and N-myc proto-oncogenes during oral development of midgestational mouse embryos were examined by in situ hybridization in order to analyze their roles. In the mandibular rudiment, c-myc RNA was strongly expressed in the mesenchymal condensation around the ossification center in which high-level expression of 2 ar (osteopontin) was detected. In tooth germs, c-myc was strongly expressed in the epithelia at the bud stage, and its expression gradually became restricted to the inner enamel epithelia from the cap to bell stages. In contrast, N-myc expression was detected in the undifferentiated mesenchymal cells of the dental papilla. Incorporation of BrdU was examined immunohistochemically to study the relationship between the expressions of c- and N-myc and cell proliferation. Unexpectedly, the distribution of BrdU labelled regions was not coincident with the expressions of c- and N-myc. These results suggest that the level of myc expression is not necessarily related to cell proliferation.