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158 Acute dose and low dose-rate irradiation of carcinoma cells expressing human papillomavirus E6 and E7 oncoproteins — The significance of p53, Rb and G1 arrest status
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Radiation Oncology, Biology, Physics Volume 32, Supplement 1
158 ACUTE DOSE AND L O W DOSE-RATE IRRADIATION OF CARCINOMA CELLS EXPRESSING HUMAN PAPILLOMAVIRUS E6 AND E 7 0 N C O P R O T E I N S - THE S I G N I F I C A N C E OF p53, Rb and GI ARREST STATUS DeWeese, Theodore L.I, Walsh, Jonathan C. 2, Dillehay, Larry E. 1, Shad, y l, Kessis, Theodore D. 3, Cho, Kathleen R. 1,3 and Nelson, William G.I, 2 The Johns Hopkins University School of Medicine, Baltimore, MD, Departments of Oncology 1, Urology 2 and Pathology 3 Purpose: The development of carcinomas in a number of sites including the cervix, vulva and anus have been associated with cellular infection by human papillomaviruses (HPV), including HPV 16 and HPV 18. The mechanism by which these viruses contribute to tumor development or progression seems in part to be related to the integration of the viral genome into the host cells DNA, and the binding of p53 protein by the HPV E6 oncoprotein as well as the binding of the retinoblastoma (Rb) protein and Rb-like proteins by the HPV E7 oncoprotein. These interactions lead to loss of p53 and Rb function including loss of the G 1 cell cycle checkpoint. Although it is believed that both p53 and Rb play a role in the radiosensitivity of the cell, whether alteration in either protein enhances or diminishes cellular radiation response is not clear from the literature. Because HPV-associated tumors such as cervical cancer are often treated with acute dose and/or low dose-rate radiation, we set out to evaluate the radiation response of several carcinoma cell sublines expressing either oncogenic E6 or E7 to both types of radiation, and to determine if p53/Rb dependent G1 arrest is an important determinant of cell fate after irradiation. Materials and Methods: We have previously developed a series of RKO colorectal carcinoma ceil sublines expressing both low-risk (HPV 11) and high-risk (HPV 16) E6 and E7 genes, p53-dependeot G1 arrest is intact in RKO parental cells and cells expressing low-risk E6 proteins, while the G 1 arrest is abrogated in cells expressing high-risk E6 or E7. Clonogenic survival was assessed after exposure to acute dose (1 Gy/min) and low dose-ram (0.25 Gy/hour) radiation. The radiobiologic parameters co, 13and the surviving fraction at 2 Gy (SF2) were determined. SDS-PAGE/immunoblotting was carried out to assess both p53 and p21WAFI/CIPI levels after exposure to radiation. Flow cytometry was performed before and after exposure to 1o~ dose-rate radiation to confirm high-risk E6 and E7 disruption of the GI cell cycle checkpoint. Results: Cell survival curves showed no difference in radiosensitivity among any of the cell lines after acute dose or low dose-rate irradiation. The tested cells include RKO parental cells and cells transfecmd with low-risk E6, high-risk E6, high-risk E7 and vector alone. The radiobiologic parameters a, 13and SF2 were likewise similar between the cell lines. SDS-PAGE/immunoblotting revealed that there was no increase in p53 or p21WAFI/CIPI protein levels after DNA damage induced by radiation in cells containing high-risk E6, confirming the action of E6 and the subsequent disruption of the p53-mediated pathway, p53 and p21W,~Ft/CIPI remain intact in the parent, neoresistant, low-risk E6 and high-risk E7 expressing cells. FIow cytometry revealed an intact G1 arrest after low dose-rate exposure in the parent, neoresistant and low-risk E6 lines and a disrupted 01 block in the high-risk E6 and E7 lines, as previously described. The G2 checkpoint was not affected.
Conclusions: These data suggest that in this series of cell lines, where the only difference between cells is the presence or absence of HPV oncogenic E6 or E7, that inactivation of p53 and Rb, two critical proteins important in DNA damage response, does not alter the radiosensitivity of the cell to either acute dose or to low dose-rate radiation. In addition, the presence or absence of the GI cell cycle checkpoint does not seem to be a determinant of cell death after either acute dose or low dose-rate radiation exposure.
159 HYPOXIA ACTS AS A SELECTIVE PRESSURE FOR THE LOSS OF WILD-TYPE P53 IN SOLID TUMOR DEVELOPMENT. Amato J. Gi.accia 1, Mitchell Tsai 1, Scott W. Lowe 2, and Thomas G. Graeber I 1Dept. of Radiation Oncofogy, Stanford University, Stanford, CA 94305, 2 Dept. of Biology, MIT, Cambridge, MA 02139 Purpose/Objective: We have previously shown that hypoxia causes the induction of wild-type p53 activity, but that the Gl-phase block caused by hypoxia is not mediated by p53. Since p53 mutation is an intermediate to late event in solid tumor development, usually occurring before the transition from benign to malignant growth, we investigated whether a tumor specific stress such as hypoxia may provide a selective pressure for loss of functional p53 through apoptotic cell death. In such a model, as a developing neoplasm grows to exceed its blood supply and develops hypoxic regions, wild-type p53 expressing cells would initiate apoptosis, thereby providing mutant p53 ceils with a growth advantage. Such a model would help explain the clonal expansion of mutant p53 tumor cells in the later stages of solid tumor development
Materials & Methods: To demonstrate that hypoxia acts as a tumor specific selective stress, we compared cell killing in vitro and in vivo by hypoxia in minimally transformed mouse embryonic fibmblasts (MEFs) that are homozygons for wild-type p53 to mouse embryonic fibroblasts that are homozygous null for p53. Nuclear fragmentation and DNA degradation was assayed by staining 100-200 cells with Hoechst 33342, and calculating the fraction of cells with fragmented nuclei. DNA degradation was quantitated in vivo using terminal transferase frUNEL) to label fragmented ends. Loss of membrane integrity was assessed by incubating cells with pmpidium iodide and calculating the numbex of cells that excluded the dye. In vitro sel¢cCon experiments were performed by mixing 100 to 1000 times more wild-type p53 ceils with null p53 cells expressing a 13-galactosidase exln'essinn vector and exposing them to hypoxia for 72 hours before aerobic growth and staining for ~galaetosidas¢ activity. The same ratio of cells were maintained in aerobic conditions as conm31s. For tumor formation, (2 X 106) cells were injected into each flank of an athymic nude mice 4 to 8 weeks old. Results: Since another activity that is strongly associated with wild-type p53 is apoptosis, we examined whether hypoxia induced cell killing depended on wild-type p53 activity. Using a combination of cellula,r and biochemical assays, we will show that minimally lransformed cells possessing wild-type p53 am 5 times more sensitive to hypoxic exposure than transformed ceils lacking wild-type p53 in vitro. We will morphologically demonstrate that hypoxia induces nuclear fragmentation and loss of membrane integrity, two hallmarks of apoptosis, To show the oxygen dependency of hypoxic cell killing on wild-type p53 activity, we compared the kinetics of cell killing of two independently derived clones of ME.Fs that are homozygous wild-type for p53 with two clones that axe homozygous null for p53. In this paradigm of oncogenieally transformed embryonic cells, those cells that possess wild-type p53 were extremely sensitive to killing by hypoxia compared to ceils lacking p53 activity. In addition, we will also show that wild-type p53 ceils transfected with a dominant mutant p53 geoe or the bcl-2 oncogene exhibit the same sensitivity to hypoxia as cells devoid of p53 activity. To more directly demonstrate the selectivity of cell ldlling by hypoxia, we mixed different ratios of mutant p53 cells with wild-type p53 cells and compared the growth of cells exposed to hypoxia for 72 hours with the same mixtures of aerobic cells. Using this approach, we could demonstrate that a single exposm'e to hypoxia emiehed for the proliferation of mutant p53 cells 500-1000 fold within one week's time. We will also show that hypoxia causes the selective killing of wild-type p53 cells in tumors using a combination of end-labeling to identify the apoptotic cells and biochemical markers to identify hypoxic cells. Conclusion: Hypoxia is a strong tumor specific stress that could select for tumor cells possessing a mutant p53 phenotype. These experiments also suggest that tumors with high hypoxic fractions may have a worse prognosis independent of treatment due to the loss of their p53 dependent apoptotic pathway.
Radiation Oncology, Biology, Physics Volume 32, Supplement 1
158 ACUTE DOSE AND L O W DOSE-RATE IRRADIATION OF CARCINOMA CELLS EXPRESSING HUMAN PAPILLOMAVIRUS E6 AND E 7 0 N C O P R O T E I N S - THE S I G N I F I C A N C E OF p53, Rb and GI ARREST STATUS DeWeese, Theodore L.I, Walsh, Jonathan C. 2, Dillehay, Larry E. 1, Shad, y l, Kessis, Theodore D. 3, Cho, Kathleen R. 1,3 and Nelson, William G.I, 2 The Johns Hopkins University School of Medicine, Baltimore, MD, Departments of Oncology 1, Urology 2 and Pathology 3 Purpose: The development of carcinomas in a number of sites including the cervix, vulva and anus have been associated with cellular infection by human papillomaviruses (HPV), including HPV 16 and HPV 18. The mechanism by which these viruses contribute to tumor development or progression seems in part to be related to the integration of the viral genome into the host cells DNA, and the binding of p53 protein by the HPV E6 oncoprotein as well as the binding of the retinoblastoma (Rb) protein and Rb-like proteins by the HPV E7 oncoprotein. These interactions lead to loss of p53 and Rb function including loss of the G 1 cell cycle checkpoint. Although it is believed that both p53 and Rb play a role in the radiosensitivity of the cell, whether alteration in either protein enhances or diminishes cellular radiation response is not clear from the literature. Because HPV-associated tumors such as cervical cancer are often treated with acute dose and/or low dose-rate radiation, we set out to evaluate the radiation response of several carcinoma cell sublines expressing either oncogenic E6 or E7 to both types of radiation, and to determine if p53/Rb dependent G1 arrest is an important determinant of cell fate after irradiation. Materials and Methods: We have previously developed a series of RKO colorectal carcinoma ceil sublines expressing both low-risk (HPV 11) and high-risk (HPV 16) E6 and E7 genes, p53-dependeot G1 arrest is intact in RKO parental cells and cells expressing low-risk E6 proteins, while the G 1 arrest is abrogated in cells expressing high-risk E6 or E7. Clonogenic survival was assessed after exposure to acute dose (1 Gy/min) and low dose-ram (0.25 Gy/hour) radiation. The radiobiologic parameters co, 13and the surviving fraction at 2 Gy (SF2) were determined. SDS-PAGE/immunoblotting was carried out to assess both p53 and p21WAFI/CIPI levels after exposure to radiation. Flow cytometry was performed before and after exposure to 1o~ dose-rate radiation to confirm high-risk E6 and E7 disruption of the GI cell cycle checkpoint. Results: Cell survival curves showed no difference in radiosensitivity among any of the cell lines after acute dose or low dose-rate irradiation. The tested cells include RKO parental cells and cells transfecmd with low-risk E6, high-risk E6, high-risk E7 and vector alone. The radiobiologic parameters a, 13and SF2 were likewise similar between the cell lines. SDS-PAGE/immunoblotting revealed that there was no increase in p53 or p21WAFI/CIPI protein levels after DNA damage induced by radiation in cells containing high-risk E6, confirming the action of E6 and the subsequent disruption of the p53-mediated pathway, p53 and p21W,~Ft/CIPI remain intact in the parent, neoresistant, low-risk E6 and high-risk E7 expressing cells. FIow cytometry revealed an intact G1 arrest after low dose-rate exposure in the parent, neoresistant and low-risk E6 lines and a disrupted 01 block in the high-risk E6 and E7 lines, as previously described. The G2 checkpoint was not affected.
Conclusions: These data suggest that in this series of cell lines, where the only difference between cells is the presence or absence of HPV oncogenic E6 or E7, that inactivation of p53 and Rb, two critical proteins important in DNA damage response, does not alter the radiosensitivity of the cell to either acute dose or to low dose-rate radiation. In addition, the presence or absence of the GI cell cycle checkpoint does not seem to be a determinant of cell death after either acute dose or low dose-rate radiation exposure.
159 HYPOXIA ACTS AS A SELECTIVE PRESSURE FOR THE LOSS OF WILD-TYPE P53 IN SOLID TUMOR DEVELOPMENT. Amato J. Gi.accia 1, Mitchell Tsai 1, Scott W. Lowe 2, and Thomas G. Graeber I 1Dept. of Radiation Oncofogy, Stanford University, Stanford, CA 94305, 2 Dept. of Biology, MIT, Cambridge, MA 02139 Purpose/Objective: We have previously shown that hypoxia causes the induction of wild-type p53 activity, but that the Gl-phase block caused by hypoxia is not mediated by p53. Since p53 mutation is an intermediate to late event in solid tumor development, usually occurring before the transition from benign to malignant growth, we investigated whether a tumor specific stress such as hypoxia may provide a selective pressure for loss of functional p53 through apoptotic cell death. In such a model, as a developing neoplasm grows to exceed its blood supply and develops hypoxic regions, wild-type p53 expressing cells would initiate apoptosis, thereby providing mutant p53 ceils with a growth advantage. Such a model would help explain the clonal expansion of mutant p53 tumor cells in the later stages of solid tumor development
Materials & Methods: To demonstrate that hypoxia acts as a tumor specific selective stress, we compared cell killing in vitro and in vivo by hypoxia in minimally transformed mouse embryonic fibmblasts (MEFs) that are homozygons for wild-type p53 to mouse embryonic fibroblasts that are homozygous null for p53. Nuclear fragmentation and DNA degradation was assayed by staining 100-200 cells with Hoechst 33342, and calculating the fraction of cells with fragmented nuclei. DNA degradation was quantitated in vivo using terminal transferase frUNEL) to label fragmented ends. Loss of membrane integrity was assessed by incubating cells with pmpidium iodide and calculating the numbex of cells that excluded the dye. In vitro sel¢cCon experiments were performed by mixing 100 to 1000 times more wild-type p53 ceils with null p53 cells expressing a 13-galactosidase exln'essinn vector and exposing them to hypoxia for 72 hours before aerobic growth and staining for ~galaetosidas¢ activity. The same ratio of cells were maintained in aerobic conditions as conm31s. For tumor formation, (2 X 106) cells were injected into each flank of an athymic nude mice 4 to 8 weeks old. Results: Since another activity that is strongly associated with wild-type p53 is apoptosis, we examined whether hypoxia induced cell killing depended on wild-type p53 activity. Using a combination of cellula,r and biochemical assays, we will show that minimally lransformed cells possessing wild-type p53 am 5 times more sensitive to hypoxic exposure than transformed ceils lacking wild-type p53 in vitro. We will morphologically demonstrate that hypoxia induces nuclear fragmentation and loss of membrane integrity, two hallmarks of apoptosis, To show the oxygen dependency of hypoxic cell killing on wild-type p53 activity, we compared the kinetics of cell killing of two independently derived clones of ME.Fs that are homozygous wild-type for p53 with two clones that axe homozygous null for p53. In this paradigm of oncogenieally transformed embryonic cells, those cells that possess wild-type p53 were extremely sensitive to killing by hypoxia compared to ceils lacking p53 activity. In addition, we will also show that wild-type p53 ceils transfected with a dominant mutant p53 geoe or the bcl-2 oncogene exhibit the same sensitivity to hypoxia as cells devoid of p53 activity. To more directly demonstrate the selectivity of cell ldlling by hypoxia, we mixed different ratios of mutant p53 cells with wild-type p53 cells and compared the growth of cells exposed to hypoxia for 72 hours with the same mixtures of aerobic cells. Using this approach, we could demonstrate that a single exposm'e to hypoxia emiehed for the proliferation of mutant p53 cells 500-1000 fold within one week's time. We will also show that hypoxia causes the selective killing of wild-type p53 cells in tumors using a combination of end-labeling to identify the apoptotic cells and biochemical markers to identify hypoxic cells. Conclusion: Hypoxia is a strong tumor specific stress that could select for tumor cells possessing a mutant p53 phenotype. These experiments also suggest that tumors with high hypoxic fractions may have a worse prognosis independent of treatment due to the loss of their p53 dependent apoptotic pathway.