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Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line
H Kawai1, Y Kitamura, O Nikaido
1Department of Regulatory Radiobiology, Research Institute for Radiation, Biology and Medicine, Hiroshima University, Japan.
Abstract:
To analyze specific genes related to radiation-induced apoptosis, 12 apoptosis-resistant clones were isolated from cells of the radiosensitive mouse thymic lymphoma 3SB line after treatment with ethyl methanesulfonate. Five of 12 clonal cell lines were recloned and were examined for their susceptibility to X-ray-induced apoptosis. A cell survival assay showed that all five secondary cell lines were two to three times more resistant to X rays than 3SB cells. 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 after irradiation. However, no apoptosis occurred in these secondary mutant cells. In particular, the percentage of cells undergoing apoptosis in one clone, 1B1C4, was low even after incubation for 48 h. In contrast to X rays, after exposure to 20 J/m2 UV radiation, the proportion of apoptotic cells in these mutant cells increased and reached about 60 to 100% at 24 h, indicating a difference in the ability of X rays and UV radiation to induce apoptosis. A similar radioresistance was observed using agarose gel electrophoresis of DNA from cells of all X-irradiated secondary lines. Western blot analysis and a sequence-specific DNA-binding assay demonstrated that 1B1C4 cells had a functional defect in p53 protein, but the other four cell lines displayed wild-type p53 after X irradiation. Our results suggest the existence of separate radiation-specific p53-dependent and independent apoptosis in thymic lymphoma cells. Thus these apoptosis-resistant cell lines provide a useful tool to identify the genes involved in the signaling pathways leading to X-ray-specific apoptosis.
Insights
Researchers developed apoptosis-resistant cell lines from mouse thymic lymphoma cells to study radiation-induced cell death. These cell lines revealed distinct pathways for X-ray and UV radiation-induced apoptosis, highlighting p53-dependent and independent mechanisms.
Area of Science:
- Molecular Biology
- Radiobiology
- Cell Biology
Background:
- Apoptosis, or programmed cell death, is crucial for development and tissue homeostasis.
- Radiation exposure can induce apoptosis, a process vital for eliminating damaged cells.
- Understanding the genetic basis of radiation-induced apoptosis is key to cancer therapy and radiation protection.
Purpose of the Study:
- To isolate and characterize apoptosis-resistant cell lines from radiosensitive mouse thymic lymphoma cells.
- To investigate the differential response of these cell lines to X-ray and UV radiation-induced apoptosis.
- To identify genetic factors involved in radiation-specific apoptosis signaling pathways.
Main Methods:
- Isolation of ethyl methanesulfonate-induced apoptosis-resistant clones from 3SB mouse thymic lymphoma cells.
- Assessment of radiosensitivity using cell survival assays and apoptosis assays (erythrosin B staining).
- Analysis of p53 protein function using Western blot and DNA-binding assays, and DNA damage using agarose gel electrophoresis.
Main Results:
- Five apoptosis-resistant cell lines were generated, exhibiting 2-3 times greater resistance to X-rays than parental cells.
- These resistant cell lines showed significantly reduced X-ray-induced apoptosis, with one clone (1B1C4) exhibiting a defect in p53 protein function.
- In contrast, the resistant cell lines remained sensitive to UV radiation-induced apoptosis, indicating distinct response mechanisms.
- DNA damage analysis revealed similar radioresistance patterns across X-irradiated secondary lines.
Conclusions:
- The study suggests the existence of separate radiation-specific apoptosis pathways: one dependent on p53 and another independent of it.
- The generated apoptosis-resistant cell lines serve as a valuable tool for dissecting the molecular mechanisms of X-ray-specific apoptosis.
- These findings contribute to understanding cellular responses to radiation and may inform strategies for cancer treatment and radiation safety.