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160 Role of P53 status in radiation sensitivity and cell cycle progression
Proceedings of the 37th Annual ASTRO Meeting
160 ROLE OF P53 STATUS IN RADIATION SENSITIVITY AND CELL CYCLE PROGRESSION Richard C. ZeUars, Tania Loney, Ann F. Schott, Mary A. Davis, Jonathan Maybaum, Michael F. Clarke, and Theodore S. Lawrence University of Michigan Medical Center Puroose: Although p53 function plays a major role in G1 arrest after radiation, the influence of p53 status on progress through other phases of the cell cycle and on radiation sensitivity of human tumors is tess clear. We investigated these issues using cells with a conditional expression system for wild type p53. Methods: A temperature sensitive murine wild type p53 plasmid was used (Ginsberg D, et el: Mol. Cell. Biol. 11:582, 1991). At the permissive temperature (32oc), this plasmid produces a protein which assumes a conformation that exhibits wild type p53 function. However, when cells are cultured at 38oc, this protein assumes an inactive conformation. HT29 human colon cancer cells (which are p53 mutant) were transduced with this plasmid (designated PEP A and PEP G cells) or a control vector (designated CCH1 cells) using electroporation and Geneticin selection. The presence of murine p53 transcript in the PEP cells was confirmed by Northern analysis. Results: Cells were cultured under 3 conditions: 1) 38oc at all times; 2) 32oc for 24 hours prior to irradiation and 3) 32oc for 24 hours after irradiation. We found that culturing under permissive temperatures produced a small decrease in surviving fraction in the PEP clones (0.61 ± 0.10 and 0.64 + 0.07, for PEP A and G, respectively) but not the CCH1 controls (1.14 ± 0.15). PEP cells tended to be more radiosensitive than CCH1 cells (even under non-permissive conditions) and demonstrated a trend towards increased radiosensitivity under both Conditions 2 and 3. In addition, flow cytometry revealed that a 24 hour exposure 1o permissive conditions increased the fraction of cells in G1 slightly and in G2/M substantially. S phase was almost absent. Conclusion: Restoration of p53 function in HT29 human colon cancer cells using this temperature sensitive system produced increased cytotoxicity and radiation sensitivity as well as cell cycle redistribulion. It will be important to assess the role of programmed cell death in these phenomena as well as to elucidate the mechanism of G2 arrest. Supported by NIH grants CA 53440 and CA42761 and an ASTRO fellowship (Richard C. Zellars)
161 PROLONGED G2 DELAY IN MURINE LYMPHOMA CELLS SUSCEPTIBLE TO RADIATION-INDUCED APOPTOSIS CORRELATES WITH CYCLIN BI EXPRESSION. Endlich, Brian P, Almasan, Alexandru and Macklis, Roger M Deparlment of Radiation Oncology, Cleveland Clinic Foundation, Cleveland OH, 44195 Purpose/Objective: Apoptosis is an important component of radiation-induced ceil killing in lymphoma cells. Proteins implicated in cell cycle control, such as p53, Rb, myc and cyclins are likely to play an important role in the apoptotic response. Materials and Methods: We have investigated the apoptotic response in two exponentially growing isogenic murine T-ceil lymphoma cell lines, L5178Y-R (LY-R)and L5178Y-L (LY-S), trradiated with ~~TCs ganuna-rays. Apuptosis was assayed by gel electrophoresis of total genomic DNA and estimated by the appearance of a typical nucleosome size DNA ladder
The position of the cells in the cell c3)cle was
determined by flow cytometry Fluctuations in the levels of several proteins, known to be involved in cell cycle control or apoptosis, such as bax, cyclin BI, c-myc, p53 and Rb were monitored by Western immunoblotting or immunostaining. Results: Our previous work (Macklis, el. al 1993, lnt J Radiation Oncology Bio[ Phys 27, 643-650.) has identified a mitotic delay in
lymphoma cells treated with gamma-irradiation We confirmed previous observations that LY-S cells are radiosensitive, undergoing apoptosis at low doses of radiation. Flow cytomet~ demonstrated that LY-R cells undergo a brief G2 delay in response to radiation exposure that is detectable at 4 hrs post irradiation and reaches a peak 4-8 hrs later, after which cells will resume normal cycling and their distribution in different phases of the cell cycle resembles that of untreated cells In contrast, at isotoxic doses, LY-S cells wxJxibita prolonged, if not permanent, cell cycle block within 2 hrs post-irradiation which reaches a maximum within g hrs and persists for at least 48 hrs Correlating with the G2 arrest in response to gamma-irradiation, cyc[in BI levels in LY-R cells are elevated 4-12 hrs after irradiation (6-7 fold) and decrease by 24 hrs In contrast, LY-S cell have a constitutively high level of cyc[in B l, similar to the highest levels reached in LY-R cells after induction The high cyclin BI level in LY-S cells is persistent throughout the 48 hr period post irradiation The levels of other proteins investigated did not sigmficantly change after gamma-irradiation Conclusions: These observations suggest that gamma-irradiation mediated DNA damage promotes a prolonged, if not permanent G2/M-
phase block, which correlates with apoptosis in LY-S cells Constitutively high levels ofcyclin B1, likely to correspond to high cyclin B l/cdc2 kinase activity in LY-S cells may play an important role in this process An attenuation of G2 checkpoint function in LY-R cells is associated with a fluctuation in cyclin B l levels, which are transiently elevated after irradiation but rapidly decrease permiting exit from mitosis
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160 ROLE OF P53 STATUS IN RADIATION SENSITIVITY AND CELL CYCLE PROGRESSION Richard C. ZeUars, Tania Loney, Ann F. Schott, Mary A. Davis, Jonathan Maybaum, Michael F. Clarke, and Theodore S. Lawrence University of Michigan Medical Center Puroose: Although p53 function plays a major role in G1 arrest after radiation, the influence of p53 status on progress through other phases of the cell cycle and on radiation sensitivity of human tumors is tess clear. We investigated these issues using cells with a conditional expression system for wild type p53. Methods: A temperature sensitive murine wild type p53 plasmid was used (Ginsberg D, et el: Mol. Cell. Biol. 11:582, 1991). At the permissive temperature (32oc), this plasmid produces a protein which assumes a conformation that exhibits wild type p53 function. However, when cells are cultured at 38oc, this protein assumes an inactive conformation. HT29 human colon cancer cells (which are p53 mutant) were transduced with this plasmid (designated PEP A and PEP G cells) or a control vector (designated CCH1 cells) using electroporation and Geneticin selection. The presence of murine p53 transcript in the PEP cells was confirmed by Northern analysis. Results: Cells were cultured under 3 conditions: 1) 38oc at all times; 2) 32oc for 24 hours prior to irradiation and 3) 32oc for 24 hours after irradiation. We found that culturing under permissive temperatures produced a small decrease in surviving fraction in the PEP clones (0.61 ± 0.10 and 0.64 + 0.07, for PEP A and G, respectively) but not the CCH1 controls (1.14 ± 0.15). PEP cells tended to be more radiosensitive than CCH1 cells (even under non-permissive conditions) and demonstrated a trend towards increased radiosensitivity under both Conditions 2 and 3. In addition, flow cytometry revealed that a 24 hour exposure 1o permissive conditions increased the fraction of cells in G1 slightly and in G2/M substantially. S phase was almost absent. Conclusion: Restoration of p53 function in HT29 human colon cancer cells using this temperature sensitive system produced increased cytotoxicity and radiation sensitivity as well as cell cycle redistribulion. It will be important to assess the role of programmed cell death in these phenomena as well as to elucidate the mechanism of G2 arrest. Supported by NIH grants CA 53440 and CA42761 and an ASTRO fellowship (Richard C. Zellars)
161 PROLONGED G2 DELAY IN MURINE LYMPHOMA CELLS SUSCEPTIBLE TO RADIATION-INDUCED APOPTOSIS CORRELATES WITH CYCLIN BI EXPRESSION. Endlich, Brian P, Almasan, Alexandru and Macklis, Roger M Deparlment of Radiation Oncology, Cleveland Clinic Foundation, Cleveland OH, 44195 Purpose/Objective: Apoptosis is an important component of radiation-induced ceil killing in lymphoma cells. Proteins implicated in cell cycle control, such as p53, Rb, myc and cyclins are likely to play an important role in the apoptotic response. Materials and Methods: We have investigated the apoptotic response in two exponentially growing isogenic murine T-ceil lymphoma cell lines, L5178Y-R (LY-R)and L5178Y-L (LY-S), trradiated with ~~TCs ganuna-rays. Apuptosis was assayed by gel electrophoresis of total genomic DNA and estimated by the appearance of a typical nucleosome size DNA ladder
The position of the cells in the cell c3)cle was
determined by flow cytometry Fluctuations in the levels of several proteins, known to be involved in cell cycle control or apoptosis, such as bax, cyclin BI, c-myc, p53 and Rb were monitored by Western immunoblotting or immunostaining. Results: Our previous work (Macklis, el. al 1993, lnt J Radiation Oncology Bio[ Phys 27, 643-650.) has identified a mitotic delay in
lymphoma cells treated with gamma-irradiation We confirmed previous observations that LY-S cells are radiosensitive, undergoing apoptosis at low doses of radiation. Flow cytomet~ demonstrated that LY-R cells undergo a brief G2 delay in response to radiation exposure that is detectable at 4 hrs post irradiation and reaches a peak 4-8 hrs later, after which cells will resume normal cycling and their distribution in different phases of the cell cycle resembles that of untreated cells In contrast, at isotoxic doses, LY-S cells wxJxibita prolonged, if not permanent, cell cycle block within 2 hrs post-irradiation which reaches a maximum within g hrs and persists for at least 48 hrs Correlating with the G2 arrest in response to gamma-irradiation, cyc[in BI levels in LY-R cells are elevated 4-12 hrs after irradiation (6-7 fold) and decrease by 24 hrs In contrast, LY-S cell have a constitutively high level of cyc[in B l, similar to the highest levels reached in LY-R cells after induction The high cyclin BI level in LY-S cells is persistent throughout the 48 hr period post irradiation The levels of other proteins investigated did not sigmficantly change after gamma-irradiation Conclusions: These observations suggest that gamma-irradiation mediated DNA damage promotes a prolonged, if not permanent G2/M-
phase block, which correlates with apoptosis in LY-S cells Constitutively high levels ofcyclin B1, likely to correspond to high cyclin B l/cdc2 kinase activity in LY-S cells may play an important role in this process An attenuation of G2 checkpoint function in LY-R cells is associated with a fluctuation in cyclin B l levels, which are transiently elevated after irradiation but rapidly decrease permiting exit from mitosis
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