WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells

Abstract

Purpose : There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as gamma-rays and X-rays is well known to induce WAF1 (p21/CIP1/sdi1) gene expression in a p53 -dependent manner. In the present study, it was examined whether WAF1 accumulation was induced after carbon-ion (C-) beam or alpha-particle irradiation in four glioblastoma cell lines. Materials and methods : A colony assay for radiosensitivity and Western blot analysis of WAF1 were applied to two human glioblastoma cell lines, A-172 bearing wild-type p53 (wt p53) and T98G bearing mutated p53 (m p53) . A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a m p53 expression vector respectively. Results : The amount of WAF1 increased markedly after X-ray irradiation in A-172 and A-172/neo cells but not in T98G and A-172/mp53 cells. The level of WAF1 reached a plateau at 3-10 h after X-ray irradiation at 5 Gy in A-172 and A-172/neo cells. Likewise, the levels of WAF1 in A-172 and A-172/neo cells reached a plateau at 3-10 h and 6-24 h after C-beam (3.0Gy) and alpha-particle (4.5 Gy) irradiation respectively. The amount of WAF1 increased markedly in a dose-dependent manner 10 h after X-ray, C-beam or alpha-particle irradiation in A-172 and A172/neo cells but not in T98G or A-172/mp53 cells. In addition, cell survival assay showed that these cell lines were most sensitive to C-beams, less sensitive to alpha-particles and least sensitive to X-rays at 10% survival. There was no difference in sensitivity among these cell lines against C-beam and alpha-particle irradiation whereas wt p53 cells (A-172 and A-172/neo) were more sensitive to X-rays than m p53 cells (A-172/mp53 and T98G). Conclusions : These results indicate that C-beams and alpha-particles induce p53 -dependent WAF1 accumulation as well as is the case with X-rays, suggesting that WAF1 protein accumulation may not contribute to cell killing.