Vanadate-induced cell growth arrest is p53-dependent through activation of p21 in C141 cells

Vanadium is widely used in industry. It is a potent toxic agent and carcinogen. The mechanisms involved in its toxicity and carcinogenesis are still unclear. Improper cell growth is believed to be involved in cancer development. The present study investigated the regulation of p53 on vanadate-induced cell growth arrest using both p53 wild type C141 cells and p53 deficient embryo fibroblasts (p53 -/-). On vanadate stimulation, C141 cells exhibited a dose- and time-dependent S phase arrest as determined by DNA content analysis. In contrast, vanadate was unable to increase the percentage of S phase in p53 -/- cells. Luciferase assay showed that vanadate induced p53 activation in a dose- and time-dependent manner in p53 wild type C141 cells. Addition of pifithrin-alpha (PFT), a specific inhibitor of p53, reduced the activation of p53 with a concomitant decrease in growth arrest at S phase. Western blotting analysis demonstrated that vanadate caused a dose- and time-dependent increase of p21 level in C141 cells. Pretreatment of C141 cells with PFT decreased p21 expression induced by vanadate while the p21 expression did not vary in vanadate stimulated p53 -/- cells. The results obtained from the present study suggest that vanadate is able to induce S phase arrest through p53- and p21-dependent pathway.     

p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.