A novel single-base deletion of the RUNX Family Transcription Factor 2 gene associated with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare, autosomal dominant hereditary disorder characterized by skeletal malformations and dental abnormalities. The purpose of this study was to explore the functional role of a novel mutation in the pathogenesis of CCD. Genomic DNA was extracted from peripheral blood mononuclear cells collected from family members of a Chinese patient with CCD. An analysis of their RUNX Family Transcription Factor 2 (RUNX2) gene sequences was performed by PCR amplification and Sanger sequencing. The function of the mutant RUNX2 was studied by bioinformatics, real-time PCR, western blotting, and subcellular localization analysis. Sanger sequencing identified a novel single-base deletion (NM_001024630.4:c.132delG;NP_001019801.3: Val45Trpfs* 99) in the RUNX2 gene present in the Chinese patient with CCD. In vitro, functional studies showed altered protein localization and increased expression of mutant RUNX2 mRNA and mutant Runt-related transcription factor 2 (RUNX2). Luciferase reporter assay demonstrated that the novel RUNX2 mutations significantly increased the transactivation activity of RUNX2 on the osteocalcin gene promoter. In conclusion, we identified a patient with sporadic CCD carrying a novel deletion/frameshift mutation of the RUNX2 gene and performed screening and functional analyses to determine the cause of the CCD phenotype. This study provides new insights into the pathogenesis of CCD.3.

[Celecoxib enhances chemosensitivity of oral cancer cells by blocking cell cycle progression in vitro].

OBJECTIVE: To investigate the effect of celecoxib in enhancing the chemosensitivity of oral cancer cells and the correlation of this effect with cell cycle arrest. METHODS: KB/VCR cell line was treated with celecoxib (10, 20, 40, and 80 µmol/L) and/or VCR (0.375, 0.75, 1.5, and 3 µmol/L), and the growth inhibition rates of KB/VCR cells were assessed with MTT assay. Flow cytometry was employed to analyze the distribution of cell cycle. Western blotting was performed to detect the expression of P-glycoprotein (P-gp) and the cell cycle related proteins Cyclin D1 and p21(WAF1/CIP1). RESULTS: Low concentrations of celecoxib (<20 µmol/L) produced no obvious effect on the proliferation of the cells. But at 10 µmol/L, celecoxib significantly enhanced the toxicity of VCR in a time-dependent manner, and the combined treatments for 24, 48, and 72 h caused growth inhibition rates of (37.53∓2.05)%, (46.67∓3.17)% and (54.02∓1.53)%, respectively, significantly higher than those following treatments with celecoxib or VCR alone (P<0.01). Compared with the cells treated with VCR alone , the cells with combined treatments showed a significantly increased cell percentage in G0/G1 phase [(56.08∓0.46)%] with decrease percentages in S phase [(22.83∓0.20)%] and G2/M phase [(21.09%∓0.66)%]. The combined treatment also significantly down-regulated cyclin D1, up-regulated p21(WAF1/CIP1), and reduced P-gp expressions in the cells. CONCLUSIONS: Celecoxib enhances the chemosensitivity of KB/VCR cells by down-regulating P-gp expression, which is partially mediated by modification of cyclin D1 and p21(WAF1/CIP1) to result in cell cycle arrest.