WAF1 accumulation by carbon-ion beam and alpha-particle irradiation in human glioblastoma cultured cells.
OBJECTIVE
There have been no reports about the effects of heavy-ion beams on the expression of the WAF1 gene, although ionizing radiation such as y-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.


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 (wtp53) and T98G bearing mutated p53 (mp53). A-172/neo and A-172/mp53 were transfected with a control vector (containing only a neo selection marker) and a mp53 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 (3.0 Gy) 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 A-172/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 wtp53 cells (A-172 and A-172/neo) were more sensitive to X-rays than mp53 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.
Takahashi
A
A
Department of Biology, Nara Medical University, Kashihara, Japan.
Ohnishi
K
K
Tsuji
K
K
Matsumoto
H
H
Aoki
H
H
Wang
X
X
Tamamoto
T
T
Yukawa
O
O
Furusawa
Y
Y
Ejima
Y
Y
Tachibana
A
A
Ohnishi
T
T
D000512:Alpha Particles / Q000627:therapeutic use*; D015153:Blotting, Western; D002244:Carbon; D002470:Cell Survival / Q000528:radiation effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis*; D004307:Dose-Response Relationship, Radiation; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000532:radiotherapy*; D063193:Heavy Ion Radiotherapy; D006801:Humans; D015398:Signal Transduction / Q000235:genetics / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D014965:X-Rays
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016159:Tumor Suppressor Protein p53; D002244:Carbon