Isolation and Characterization of Apoptosis-Resistant Mutants from a Radiosensitive Mouse Lymphoma Cell Line

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.

Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis

“…1 c-e ). (52,53), APP-mediated antiapoptotic effects and APP-dependent inhibition of p53 activation might be parallel events. To determine whether hAPP695 protects against p53-mediated apoptosis, we used a p53-encoding recombinant adenovirus (p53-rAd) that elicits apoptosis in primary neuronal cultures (24,25).…”

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

“…wild-type p53) murine cells. It has been observed that inducibility of the mutant form of p53 by DNA damage may be relatively low [12] or high [25], depending on the cell line. We have observed a difference between the sublines in the response to x-irradiation: in LY-R cells the p53 increase is very weak and dose-independent at 2 h and 24 h after irradiation (Fig.…”

Suppression of Postmitochondrial Signaling and Delayed Response to UV‐induced Nuclear Apoptosis in HeLa Cells

“…UV causes rapid or delayed apoptosis depending on the cell type In a previous study, 27) we demonstrated 3SB, a mouse thymic lymphoma cell line that expresses wild-type p53 protein, to exhibit apoptosis shortly after exposure to X-rays or UV radiation. In this study, we stained the cells with DAPI and examined changes in nuclear morphology under a fluorescence microscope.…”

“…Contamination of mitochondria in cytosolic extracts was monitored with COX IV. Western blot analysis Cell lysates were prepared as described previously, 27) electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). Western blotting was performed with the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL).…”

“…After centrifugation at 16 000 rpm, the supernatant was used for the precipitation of DNA, as described previously. 27) DNA samples were loaded onto 2% agarose gels for electrophoresis and the gels were stained with 0.5 µg/ ml ethidium bromide solution. Preparation of cytosolic and mitochondrial fractions Cells were harvested, washed twice with ice-cold PBS, resuspended in ice-cold transport buffer (20 mM HEPES pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM glycoletherdiaminetetraacetic acid (EGTA), 2 mM dithiothreitol (DTT), 1 µg/ml each of aprotinin, leupeptin, and pepstatin ) containing 100 µg/ml (for 3SB and Jurkat cells) or 300 µg/ml (for HeLa and MCF-7 cells) digitonin and allowed to permeabilize at 37°C for 5 min.…”