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31 Apoptosis and radiation clonogenic survival in a solid tumor model system expressing BCL-2
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Radiahon Oncology, Biology, Physics Volume 32, Supplement 1
3O Radiation-Induced Apoptosis and the Intrinsic Radiation Sensitivity of Glial Cells G. Proulx, S. Mei, K. Kanady, Michael Duffy and Francisco S Pardo Dept of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School Boston, Mass. Introduction:Following exposure to ionizing radiation in vitro, cells of wild type p53 phenotype undergo a G 1 arrest, or may be comrmtted to a programmed cell death or apoptotic pathway. Apoptotic cell death may be an mlportant component of glial cell survival following exposure to ionizing radiation in vttro, and m clinical glial radiotherapeunc response tn vivo. Methods: Apoptosis was assayed in early-passage human glioma cell lines using a technique which rehes on the detec'ti~m o~ changes in ce[hilar morphology via an immunohistochemically-derivedenzyme substratc reaction. The ff,ethod was employed on two early-passage cell lines derived from high grade astrocytic cell lines, and two derived ti'om low grade astrocytic cell lines. Restriction fragment length polymorphtsms and karyotypes substantiated that the cell lines were of human origin. Cellular populations were examined up to 48-72 hrs after irradiation for evidence of apoptosis after doses vary,ing from 2 20 Gy. Agarose gel electrophoresis was used to assess DNA fragmentation following exposure to similar levels of ionizing radialitm Both asynchronous and synchronous cell aggregates were analyzed at 40X magnification following fixation. Radiation sensitivity was assayed using standard clonogenic survival assays under oxic conditions. Results: Asynchronous high grade glioma cell lines revealed no radiation-induced apoptosis following exposures of 2-2(l Gy of ionizing radiation in vitro. The low grade glioma lines, however, revealed a significant amount of radiation-induced alx)ptosis following single fraction sizes in the 2-20 Gy range. Clonogenic radiation survival assays indicate significant differences in radiation sensitivity in glial cellular populations. Cell lines derived from low or intermediate grade tumors renderred radiobiologic indices that were relatively more sensitive to ionizing radiation in vitro (SF2.33-.45"~ than the two corresponding cell lines of high grade glial origin. (p<.l)5, two-tailed test SF2 and D). Conclusions: Apoptosis, as assayed immunohistoc'hemically, may be one mechanism COnlFibUling to the increased sensitivity of low grade glioma cells to ionizing radiation in vuro lmmunohJstochemical evidence of :lpoptosi,, may be a more sensitwe indicatorof apoptotic cell death than analyses of DNA fragmentation following radiation exposure. The mechanisms responsible for apoptosis and the relative clinicaI radiation sensitwity of low grade gliomas ~n vitro and the relevance to the clinical radiation sensitivity of low grade astrocytic tumors~n ~ivo warrants further sludy
31 A P O P T O S I S AND R A D I A T I O N C L O N O G E N I C SURVIVAL IN A SOLID T U M O R M O D E L SYSTEM E X P R E S S I N G BCL-2 Marc S. Rudoltz, M.D. ~, Eric J. Bernhard, PhD. ~, Gary' D Kao, M.D. ~. Vincent Baukanauskas, M.S. ~, Ruth J. Muschel, M.D.. Ph.D. 2, W. Gillies McKenna, M.D.. Ph.D) Department of Radiation Oncology ~, Deparhnent of Pathology and Laboratory_ Medicine ~, Hospital of the University of Pennsylvania, Philadelphia, PA 19104 Purpose/Background: The bcl-2 proto-oncogene encodes a protein which prevents apoptosis following a variety of insults, including irradiation. Bcl-2 has been most extensively studied in hematopoetic malignancies, and data now suggests that its expression increases radioresistance in these cells, presumably by interfering with radiation induced apoptosis. Recently, Bcl-2 has been noted to be expressed by many solid tumors commonly treated with radiation therapy, such as non small cell lung cancer and breast cancer. Supnsingly, Bcl-2 expression in these tumors has been correlated with a favorable prognosis. To the authors knowledge there are no studies, either in malignant cell lines or model tumor systems, which have evaluated the effect of Bcl-2 expression on radiation sensitivity. This study evaluates the effect of Bcl-2 expression on radiation induced apoptosis and clonogenic survival in a previously characterized solid tumor model system, 3.7, a rat embryo fibroblast line (REF) transformed by cotransfection of v-myc and H-ras. This cell line has been shown to be radioresistant when compared to the same REF line transfected with v-myc "alone (MR4). This resistance appears to be related to a prolongation of the G2 delay following radiation. Materials and Methods: Bcl-2 was transfected by calcium phosphate precipitation of plasmid pZipneo-bcl-2 into the 3.7 cells. In this vector, the cDNA for human bcl-2 is placed under the control of a MoMLV constitutive promoter+ Twenty four hours after transfection the ceils were washed with fresh media. Selection was begun at 48 hours with G4t8. Independent clones were identified by western and southern blotting and expanded. Apoptosis was studied by plating 5 x 104 cells in 100 mm. tissue culture dishes 24 hours prior to irradiation. Medium was changed just prior to irradiation. Apoptosis was confirmed by visual inspection following propidium iodide staining, assessing for brightly staining nuclear f~'agmentation. Apoptotic laddering of DNA degradation into nucleosomal fragments was evaluated by I% agarose gel electrophoresis of DNA samples isolated by phenol/chloroform extraction. Clonogenic survival was performed by plating known numbers of cells into 100 mm. plates and irradiating them with doses ranging from 2 Gy through 10 Gy using 225 kVp photons. Results: Preliminary data comparing REFs expressing v-myc (MR4) with 3.7 (v-mye + H-ras) cells revealed that H-ras provides partial protection from radiation induced apoptosis, reducing the rate of apoptosis following IO Gy from 30-40% to 15-20%. Expression of Bcl-2 in 3.7 cells reduced the level of apoptosis to < 3%, even at doses up to 20 Gy. However, there does not appear to be a change in radiosensitivity in these cells expressing Bcl-2. Do'S ranging from 1.6-1.7 were tound in clones transfected with either pZipneo-bcl-2 or with the vector alone. The effects of bcl-2 on the shoulder of the survival curve and the cell cycle specific effects of Bcl-2 are currently under investigation. Conclusion: In our model tumor system Bcl-2 expression enhances protection from radiation induced apoptosis but does not further increase clonogenic survival. We have previously shown that H-ras provides partial protection from apoptosis while increasing clonogenic survival following radiation, implying that a prolongation of the G2 delay may be the cause of these phenomena. This suggests that apoptosis is but one mechanism of radiation induced cell death. Protection from apoptosis in cells which undergo abundant apoptosis, such as hematopoetic cells, will increase clonogenic survival. However, in cells already partially protected from apoptosis, such as our 3.7 model solid tumor cells, a further decrease in apoptosis does not alter clonogenic survival. The relationship between cell cycle perturbations, Bcl-2 expression, and apoptosis will also be discussed.
Radiahon Oncology, Biology, Physics Volume 32, Supplement 1
3O Radiation-Induced Apoptosis and the Intrinsic Radiation Sensitivity of Glial Cells G. Proulx, S. Mei, K. Kanady, Michael Duffy and Francisco S Pardo Dept of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School Boston, Mass. Introduction:Following exposure to ionizing radiation in vitro, cells of wild type p53 phenotype undergo a G 1 arrest, or may be comrmtted to a programmed cell death or apoptotic pathway. Apoptotic cell death may be an mlportant component of glial cell survival following exposure to ionizing radiation in vttro, and m clinical glial radiotherapeunc response tn vivo. Methods: Apoptosis was assayed in early-passage human glioma cell lines using a technique which rehes on the detec'ti~m o~ changes in ce[hilar morphology via an immunohistochemically-derivedenzyme substratc reaction. The ff,ethod was employed on two early-passage cell lines derived from high grade astrocytic cell lines, and two derived ti'om low grade astrocytic cell lines. Restriction fragment length polymorphtsms and karyotypes substantiated that the cell lines were of human origin. Cellular populations were examined up to 48-72 hrs after irradiation for evidence of apoptosis after doses vary,ing from 2 20 Gy. Agarose gel electrophoresis was used to assess DNA fragmentation following exposure to similar levels of ionizing radialitm Both asynchronous and synchronous cell aggregates were analyzed at 40X magnification following fixation. Radiation sensitivity was assayed using standard clonogenic survival assays under oxic conditions. Results: Asynchronous high grade glioma cell lines revealed no radiation-induced apoptosis following exposures of 2-2(l Gy of ionizing radiation in vitro. The low grade glioma lines, however, revealed a significant amount of radiation-induced alx)ptosis following single fraction sizes in the 2-20 Gy range. Clonogenic radiation survival assays indicate significant differences in radiation sensitivity in glial cellular populations. Cell lines derived from low or intermediate grade tumors renderred radiobiologic indices that were relatively more sensitive to ionizing radiation in vitro (SF2.33-.45"~ than the two corresponding cell lines of high grade glial origin. (p<.l)5, two-tailed test SF2 and D). Conclusions: Apoptosis, as assayed immunohistoc'hemically, may be one mechanism COnlFibUling to the increased sensitivity of low grade glioma cells to ionizing radiation in vuro lmmunohJstochemical evidence of :lpoptosi,, may be a more sensitwe indicatorof apoptotic cell death than analyses of DNA fragmentation following radiation exposure. The mechanisms responsible for apoptosis and the relative clinicaI radiation sensitwity of low grade gliomas ~n vitro and the relevance to the clinical radiation sensitivity of low grade astrocytic tumors~n ~ivo warrants further sludy
31 A P O P T O S I S AND R A D I A T I O N C L O N O G E N I C SURVIVAL IN A SOLID T U M O R M O D E L SYSTEM E X P R E S S I N G BCL-2 Marc S. Rudoltz, M.D. ~, Eric J. Bernhard, PhD. ~, Gary' D Kao, M.D. ~. Vincent Baukanauskas, M.S. ~, Ruth J. Muschel, M.D.. Ph.D. 2, W. Gillies McKenna, M.D.. Ph.D) Department of Radiation Oncology ~, Deparhnent of Pathology and Laboratory_ Medicine ~, Hospital of the University of Pennsylvania, Philadelphia, PA 19104 Purpose/Background: The bcl-2 proto-oncogene encodes a protein which prevents apoptosis following a variety of insults, including irradiation. Bcl-2 has been most extensively studied in hematopoetic malignancies, and data now suggests that its expression increases radioresistance in these cells, presumably by interfering with radiation induced apoptosis. Recently, Bcl-2 has been noted to be expressed by many solid tumors commonly treated with radiation therapy, such as non small cell lung cancer and breast cancer. Supnsingly, Bcl-2 expression in these tumors has been correlated with a favorable prognosis. To the authors knowledge there are no studies, either in malignant cell lines or model tumor systems, which have evaluated the effect of Bcl-2 expression on radiation sensitivity. This study evaluates the effect of Bcl-2 expression on radiation induced apoptosis and clonogenic survival in a previously characterized solid tumor model system, 3.7, a rat embryo fibroblast line (REF) transformed by cotransfection of v-myc and H-ras. This cell line has been shown to be radioresistant when compared to the same REF line transfected with v-myc "alone (MR4). This resistance appears to be related to a prolongation of the G2 delay following radiation. Materials and Methods: Bcl-2 was transfected by calcium phosphate precipitation of plasmid pZipneo-bcl-2 into the 3.7 cells. In this vector, the cDNA for human bcl-2 is placed under the control of a MoMLV constitutive promoter+ Twenty four hours after transfection the ceils were washed with fresh media. Selection was begun at 48 hours with G4t8. Independent clones were identified by western and southern blotting and expanded. Apoptosis was studied by plating 5 x 104 cells in 100 mm. tissue culture dishes 24 hours prior to irradiation. Medium was changed just prior to irradiation. Apoptosis was confirmed by visual inspection following propidium iodide staining, assessing for brightly staining nuclear f~'agmentation. Apoptotic laddering of DNA degradation into nucleosomal fragments was evaluated by I% agarose gel electrophoresis of DNA samples isolated by phenol/chloroform extraction. Clonogenic survival was performed by plating known numbers of cells into 100 mm. plates and irradiating them with doses ranging from 2 Gy through 10 Gy using 225 kVp photons. Results: Preliminary data comparing REFs expressing v-myc (MR4) with 3.7 (v-mye + H-ras) cells revealed that H-ras provides partial protection from radiation induced apoptosis, reducing the rate of apoptosis following IO Gy from 30-40% to 15-20%. Expression of Bcl-2 in 3.7 cells reduced the level of apoptosis to < 3%, even at doses up to 20 Gy. However, there does not appear to be a change in radiosensitivity in these cells expressing Bcl-2. Do'S ranging from 1.6-1.7 were tound in clones transfected with either pZipneo-bcl-2 or with the vector alone. The effects of bcl-2 on the shoulder of the survival curve and the cell cycle specific effects of Bcl-2 are currently under investigation. Conclusion: In our model tumor system Bcl-2 expression enhances protection from radiation induced apoptosis but does not further increase clonogenic survival. We have previously shown that H-ras provides partial protection from apoptosis while increasing clonogenic survival following radiation, implying that a prolongation of the G2 delay may be the cause of these phenomena. This suggests that apoptosis is but one mechanism of radiation induced cell death. Protection from apoptosis in cells which undergo abundant apoptosis, such as hematopoetic cells, will increase clonogenic survival. However, in cells already partially protected from apoptosis, such as our 3.7 model solid tumor cells, a further decrease in apoptosis does not alter clonogenic survival. The relationship between cell cycle perturbations, Bcl-2 expression, and apoptosis will also be discussed.