1996 Fiscal Year Final Research Report Summary
Analysis of radiation-induced apoptosis using radioresistant mutants
| Project/Area Number |
07680576
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
環境影響評価(含放射線生物学)
|
|---|
| Research Institution | HIROSHIMA UNIVERSITY (1996)
Kanazawa University (1995) |
|---|
Principal Investigator |
SUZUKI Fumio Res.Inst.Radiat.Biol.Med.HIROSHIMA UNIVERSITY Professor, 原爆放射能医学研究所, 教授 (10019672)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
ISHIGAKI Yasuhito Kanazawa Univ., Facul.Pharm.Sci.Associate Researcher, 薬学部, 助手 (20232275)
|
|---|
| Project Period (FY) |
1995 – 1996
|
| Keywords | Mouse / Thymic lymphoma / X-rays / Mutant / Apoptosis / Radioresistant / PCR-SSCP analysis / p53 gene |
|---|
| Research Abstract |
Apoptosis is an important physiological process that can be activated by various physical and chemical stimuli such as ionizing radiation and various chemicals. In order to analyze molecular mechanism of radiation-induced apoptosis, we isolated apoptosis-resistant mutants from a radiosensitive mouse thymic lymphoma 3SB cell line following treatment with ethyl methanesulfonate (EMS). After enrichment by repeated X-irradiation, EMS treated cells were grown in a semi-solid medium containing 0.25% agar, and 12 independent colonies were isolated. These clonal cell lines were cultured in a liquid medium for a few weeks and then inoculated into the semi-solid medium following irradiation with 5 Gy X-rays, to isolate more stable radioresistant mutants. We finally obtained 5 stable cell lines and found that one clone, 1B1C4, is more resistant to X-ray-induced apoptotic cell death than other clones. When 3SB cells were exposed to 5 Gy of X-rays, the fraction of cells stained with erythrosin B increased quickly within 8 h of incubation following irradiation. However, no such apoptosis occurred in 1B1C4 cells, even after incubation for 24 h. Similar radioresistance was observed using agarose gel electrophoresis of DNA from X-irradiated 1B1C4 cells. PCR-SSCP analysis of p53 cDNA fragments containing exons 5 to 9 suggested that two clones, 1B1C4 and 1D5-8 cells, contain a mutant p53 gene. PCR direct sequence analysis revealed that 1B1C4 cells have a transition from C to T in the second position of p53 codon 238. This mutation was found to cause a functional defect in the p53 protein as revealed by the sequence-specific DNA binding assay. These results indicate that the process of radiation-induced apoptosis in the mouse thymic lymphoma cell line may take at least two routs, p53-dependent or -independent pathways.
|
