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Bax protein is not involved in hypoxia-induced apoptosis
I. J. Radiation O n c o l o g y • Biology • Physics
136
V o l u m e 42, N u m b e r 1 S u p p l e m e n t , 1998
23 OVEREXPRESSION OF THE HUMAN MNSOD TRANSGENE I N V I T R O PROTECTS 32D CL 3 MURINE I-IEMATOPOIETIC P R O G E N I T O R CELLS F R O M IRR:A-DIATION-INDUCED APOPTOSIS. Epperly Michael W; Bray Jenifer A; Escobar Patricia; Bigbee William L; Watldns Simon C; and Greenberger Joel S. Departments of Radiation Oncology, Environmental/Occupational Health, and Cell Biology & Physiology; University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. Purpose/Objective: •ncreased expressi•n •f MnS•D in vitr• and in viv• has been dem•nstrated t• pr•tect ce••s fr•m irradiati•n• chemotherapy, TNF-cL and cytokine withdrawal. The mechanism of MnSOD protection is unknown. Using the 32D cl 3 murine hematopoietic progenitor cell line and clones overexpressing the human MnSOD transgene, the mechanism for cell protection was been investigated. Materials & Methods: The 32D cl 3 cell line was co-electroporated with pRK5-MnSOD and pSV2-neo plasmids, selected for G418 resistance, and cloned in 4% methylcellulose. The clones were selected for overexpression of MnSOD by nested RT-PCR for the human MnSOD transgene, northern analysis, western analysis, and biochemical assay. Radiation survival curves were performed by irradiating the ceils to doses ranging from 0 to 800 cGy, plating the cells in methylcellulosecultures, and counting colonies of >50 cells 7 days later. Cell cycle analysis was performed by irradiating the cells to 500 cGy, and fixing the cells in 70% ethanol at 0, 1, 3, 6, 24, and 48 hrs after irradiation. The cells were stained with propidium iodide and analyzed by flow cytometry. The percent of apoptotic cells was determined by irradiating the cells to 1000 cGy, and analyzing for apoptosis at 0, 6, 24, and 48 hrs later using Promega's Apoptosis Detection System. DNA strand breaks were determined by Comet assay where the cells were irradiated on ice at 0, 200, 400, and 600 cGy. The cells were plated in a 1% low melt agarose solution, lysed, with proteins and RNA digested. The DNA was electrophoresed and the length of the Comet tails analyzed. Expression of proteins involved in apoptosis such as bcl-2, bcl/xl, caspaces 1 and 3, and PARP was determined by Western analysis. Results: Two clones, IF2 and 2C6, were obtained which demonstrated increased human MnSOD expression by nested RT-PCR, Northern and Western analyses, and by biochemical assay. Increased radioresistance was detected in 1F2 and 2C6, as demonstrated by an increased fi of 4.94_+0.48 (p=0.042) and 4.954_+0.13 (p=0.011), respectively, compared to 2.767_+0.20 for 32D cl 3. There was no significant difference in the Do. All cell lines had a similar cell cycle distribution following 500 cGy with both a GI/S and G2/M phase blocks 6 hrs after irradiation. The GI/S phase arrest continued for at least 48 hrs. Apoptosis was detected in 20.3+4.5% of32D cl 3 cells, compared to 4.6-,L-1.3 (p=0.028) and 5.9+2.1% ( # . 0 4 3 ) for 1F2 and 2C6 cells, respectively, 48 hrs after irradiation. Comet assay results demonstrated a significant decrease in comet tail length at 400 and 600 cGy for IF2 (p<0.02) and 2C6 (p<0.009), compared to 32D cl 3. Western analysis demonstrated lower levels of caspace 3 and PARP activity in 1F2 and 2C6, compared to 32I) cl 3. Protein levels forp53, bcl-2, bcl/xl and caspace 1 (ICE) were comparable for all three cell lines. Conclusion: MnSOD overexpressing clonal cell lines 1F2 and 2C6 are more resistant to irradiation than the parental 32D cl 3 cell line. Overexpression of MuSOD results in a decrease in DNA strand breaks following irradiation and decrease in apoptosis as demonstrated by decreased PARP and caspace 3 activity. The underlying mechanism of irradiation resistance by overexpressiou of MnSOD is not yet known but may result from the stabilization of mitochondria.
24 BAX PROTEIN IS NOT INVOLVED IN HYPOXIA-INDUCED APOPTOSIS
Sheri D. Marquez ~, Jennifer Den"2, Constantinos Koumenis2, Amato J. Giaccia~" Department of Radiation Oncology ~, and Division of Radiobiology2, Stanford University School of Medicine, Stanford, CA 94305 Purpose/Objective: Tumors with high hypoxic fractions have a poor prognosis and have been shown to be more resistant to irradiation and chemotherapy. An involvement of p53 in hypoxia-induced apoptosis has been previously established. Hypoxia acts as a selective agent in eliminating wild type p53 tumor ceils, thereby allowing selective clonal expansion of tumor cells without wild type p53 function, creating a subpopulation resistant to treatment-induced apoptosis in certain cell lines. The mechanism by which p53 induces apoptosis under hypoxia remains unknown. There is some evidence that suggests bax protein is involved in p53-dependent radiation induced apoptosis. Bax disruption has been linked to tumor progression and treatment resistance. In this study, we investigated the potential role of bax protein in hypoxia induced apoptosis. Materials & Methods: Embryonic fibroblasts expressing E1A and Ha-ras from p53 and bax wild type and deficient mice were grown under aerobic (20%02). Hypoxia treatments were performed by exposing cells to 0.02% O2 for various time periods. A separate, aerobic irradiated control group was also included in our study. Western (and Northern) blot analyses of bax protein levels (mRNA transcripts) in cells with wild type p53 versus cells deficient in wild type p53 were performed to identify induction of protein expression in response to radiation and hypoxia. Apoptotic morphology was by staining cell subpopulations with Hoechst dye 33342 and propidium iodide. Cells with apoptotic morphology were identified with a fluorescent microscope and scored as a percentage of the total number of plated tumor cells. Results: Western (and Northern) blot analysis demonstrated that neither hypoxia nor irradiation induces bax protein (or mRNA) levels in comparison to oxygenated controls. However, basal levels of bax are elevated in the presence of wild type p53. Similar rates of hypoxia-induced apoptosis were observed between cell subpopulations, independent of the presence or the absence of functional bax protein. Conclusion: Hypoxia-induced apoptosis in oncologically transformed mouse fibroblasts did not correlate with bax protein expression. Although there is evidence to suggest a role for bax protein in -radiation-induced apoptosis in a p53-dependent fashion, our data suggests that bax does not appear to be involved in hypoxia-induced apoptosis. This implies that the mechanism by which p53 induces apoptosis in response to hypoxia may be different than the role p53 plays in radiation- or chemotherapy-induced apoptosis. This work was supported by NIH Grant POI CA67166
136
V o l u m e 42, N u m b e r 1 S u p p l e m e n t , 1998
23 OVEREXPRESSION OF THE HUMAN MNSOD TRANSGENE I N V I T R O PROTECTS 32D CL 3 MURINE I-IEMATOPOIETIC P R O G E N I T O R CELLS F R O M IRR:A-DIATION-INDUCED APOPTOSIS. Epperly Michael W; Bray Jenifer A; Escobar Patricia; Bigbee William L; Watldns Simon C; and Greenberger Joel S. Departments of Radiation Oncology, Environmental/Occupational Health, and Cell Biology & Physiology; University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213. Purpose/Objective: •ncreased expressi•n •f MnS•D in vitr• and in viv• has been dem•nstrated t• pr•tect ce••s fr•m irradiati•n• chemotherapy, TNF-cL and cytokine withdrawal. The mechanism of MnSOD protection is unknown. Using the 32D cl 3 murine hematopoietic progenitor cell line and clones overexpressing the human MnSOD transgene, the mechanism for cell protection was been investigated. Materials & Methods: The 32D cl 3 cell line was co-electroporated with pRK5-MnSOD and pSV2-neo plasmids, selected for G418 resistance, and cloned in 4% methylcellulose. The clones were selected for overexpression of MnSOD by nested RT-PCR for the human MnSOD transgene, northern analysis, western analysis, and biochemical assay. Radiation survival curves were performed by irradiating the ceils to doses ranging from 0 to 800 cGy, plating the cells in methylcellulosecultures, and counting colonies of >50 cells 7 days later. Cell cycle analysis was performed by irradiating the cells to 500 cGy, and fixing the cells in 70% ethanol at 0, 1, 3, 6, 24, and 48 hrs after irradiation. The cells were stained with propidium iodide and analyzed by flow cytometry. The percent of apoptotic cells was determined by irradiating the cells to 1000 cGy, and analyzing for apoptosis at 0, 6, 24, and 48 hrs later using Promega's Apoptosis Detection System. DNA strand breaks were determined by Comet assay where the cells were irradiated on ice at 0, 200, 400, and 600 cGy. The cells were plated in a 1% low melt agarose solution, lysed, with proteins and RNA digested. The DNA was electrophoresed and the length of the Comet tails analyzed. Expression of proteins involved in apoptosis such as bcl-2, bcl/xl, caspaces 1 and 3, and PARP was determined by Western analysis. Results: Two clones, IF2 and 2C6, were obtained which demonstrated increased human MnSOD expression by nested RT-PCR, Northern and Western analyses, and by biochemical assay. Increased radioresistance was detected in 1F2 and 2C6, as demonstrated by an increased fi of 4.94_+0.48 (p=0.042) and 4.954_+0.13 (p=0.011), respectively, compared to 2.767_+0.20 for 32D cl 3. There was no significant difference in the Do. All cell lines had a similar cell cycle distribution following 500 cGy with both a GI/S and G2/M phase blocks 6 hrs after irradiation. The GI/S phase arrest continued for at least 48 hrs. Apoptosis was detected in 20.3+4.5% of32D cl 3 cells, compared to 4.6-,L-1.3 (p=0.028) and 5.9+2.1% ( # . 0 4 3 ) for 1F2 and 2C6 cells, respectively, 48 hrs after irradiation. Comet assay results demonstrated a significant decrease in comet tail length at 400 and 600 cGy for IF2 (p<0.02) and 2C6 (p<0.009), compared to 32D cl 3. Western analysis demonstrated lower levels of caspace 3 and PARP activity in 1F2 and 2C6, compared to 32I) cl 3. Protein levels forp53, bcl-2, bcl/xl and caspace 1 (ICE) were comparable for all three cell lines. Conclusion: MnSOD overexpressing clonal cell lines 1F2 and 2C6 are more resistant to irradiation than the parental 32D cl 3 cell line. Overexpression of MuSOD results in a decrease in DNA strand breaks following irradiation and decrease in apoptosis as demonstrated by decreased PARP and caspace 3 activity. The underlying mechanism of irradiation resistance by overexpressiou of MnSOD is not yet known but may result from the stabilization of mitochondria.
24 BAX PROTEIN IS NOT INVOLVED IN HYPOXIA-INDUCED APOPTOSIS
Sheri D. Marquez ~, Jennifer Den"2, Constantinos Koumenis2, Amato J. Giaccia~" Department of Radiation Oncology ~, and Division of Radiobiology2, Stanford University School of Medicine, Stanford, CA 94305 Purpose/Objective: Tumors with high hypoxic fractions have a poor prognosis and have been shown to be more resistant to irradiation and chemotherapy. An involvement of p53 in hypoxia-induced apoptosis has been previously established. Hypoxia acts as a selective agent in eliminating wild type p53 tumor ceils, thereby allowing selective clonal expansion of tumor cells without wild type p53 function, creating a subpopulation resistant to treatment-induced apoptosis in certain cell lines. The mechanism by which p53 induces apoptosis under hypoxia remains unknown. There is some evidence that suggests bax protein is involved in p53-dependent radiation induced apoptosis. Bax disruption has been linked to tumor progression and treatment resistance. In this study, we investigated the potential role of bax protein in hypoxia induced apoptosis. Materials & Methods: Embryonic fibroblasts expressing E1A and Ha-ras from p53 and bax wild type and deficient mice were grown under aerobic (20%02). Hypoxia treatments were performed by exposing cells to 0.02% O2 for various time periods. A separate, aerobic irradiated control group was also included in our study. Western (and Northern) blot analyses of bax protein levels (mRNA transcripts) in cells with wild type p53 versus cells deficient in wild type p53 were performed to identify induction of protein expression in response to radiation and hypoxia. Apoptotic morphology was by staining cell subpopulations with Hoechst dye 33342 and propidium iodide. Cells with apoptotic morphology were identified with a fluorescent microscope and scored as a percentage of the total number of plated tumor cells. Results: Western (and Northern) blot analysis demonstrated that neither hypoxia nor irradiation induces bax protein (or mRNA) levels in comparison to oxygenated controls. However, basal levels of bax are elevated in the presence of wild type p53. Similar rates of hypoxia-induced apoptosis were observed between cell subpopulations, independent of the presence or the absence of functional bax protein. Conclusion: Hypoxia-induced apoptosis in oncologically transformed mouse fibroblasts did not correlate with bax protein expression. Although there is evidence to suggest a role for bax protein in -radiation-induced apoptosis in a p53-dependent fashion, our data suggests that bax does not appear to be involved in hypoxia-induced apoptosis. This implies that the mechanism by which p53 induces apoptosis in response to hypoxia may be different than the role p53 plays in radiation- or chemotherapy-induced apoptosis. This work was supported by NIH Grant POI CA67166