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Beta Ratio for Radiation-induced CT Density Changes in Patients Receiving Thoracic Radiation: Comparison of Data from SRS and Conventional Fractionation
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Volume 84 Number 3S Supplement 2012 Materials/Methods: SAMP6 and SAMR1 mice (4 months old) were irradiated to 0 or 20 Gy to the right hind leg with a Linear Accelerator at 300 monitor units per minute using 6 MV photons. The nonirradiated left hind leg was used as a 0 Gy control. Twenty-four hrs later a 2-mm diameter circular unicortical wound was made in each proximal tibia. At day 21, the mice were sacrificed with the tibiae removed, fixed, and radiographed. The x-rays were used to measure the wound diameter as an index of wound healing. Results: Radiographic analysis at day 21 showed healing, smaller wounds in control non-irradiated SAMR1 mice (0.23 mm 0.06, n Z 8) compared with non-irradiated SAMP6 mice (0.48 mm 0.01, p Z 0.0318, n Z 6); this denotes a relative defect in bone formation in the SAMP6 strain. Both SAMR1 and SAMP6 showed delayed bone healing after 20 Gy to one limb. Limb irradiation in SAMR1 mice impaired healing and resulted in 280 % larger wounds (0.64 0.09, p Z 0.0015, n Z 5), compared with non-irradiated mice. Hind limb irradiation of SAMP6 mice further delayed healing and resulted in persistent, 170% large wounds than in non-irradiated SAMP6 mice (0.81 0.07, p Z 0.0240, n Z 5). Conclusion: SAMP6 mice demonstrated delayed bone wound repair compared with control SAMR1 mice and irradiation inhibited wound healing in both strains of mice. Acknowledgement: This project was supported by NIAID U191A168021 and NIH R21AG034254. Author Disclosure: M. Epperly: None. S. Cao: None. T.M. Dixon: None. J. Goff: None. J. Glowacki: None. P. Wipf: None. J.S. Greenberger: None.
3215 Estimating the Alpha/Beta Ratio for Radiation-induced CT Density Changes in Patients Receiving Thoracic Radiation: Comparison of Data from SRS and Conventional Fractionation D. Fried, S.M. Miller, M. Fayda, D.E. Morris, T.M. Zagar, M. Lawrence, E. Schreiber, M. Ivanovic, W. McCartney, and L.B. Marks; University of North Carolina at Chapel Hill, Chapel Hill, NC Purpose: To estimate the Alpha/Beta (a/b) ratio for radiation(RT)-induced lung injury by comparing the density changes observed in patients treated with hypofractionated stereotactic body radiation therapy (SBRT) versus those treated with conventionally-fractionated (i.e., 2 Gy/fraction) radiation. Methods: Images from patients treated with hypofractionated SBRT (48 Gy in 12 fractions through multiple non-axial beams) were compared with the corresponding images from patients treated with conventionally-fractionated radiation treatment (usually with large fields- anteroposterior/posteroanterior and off-cord-obliques, either sequentially or concurrently). For all patients, the pre- and post-computed tomography (CT) images were compared to each other and to the three-dimensional dose distribution, using image fusion and treatment planning software. Therefore, we were able to define changes in regional lung density within different regions of the lung that received different doses of radiation (and hence different radiation fraction sizes). Data from both treatment cohorts was pooled for analysis. Linear regression was used to estimate the a/b ratio by comparing the observed dose response curve (DRC) data in the hypofractionated vs. conventionally-fractionated patients. Results: Considering the data <6 months post-RT, the degree of density change was greater in the hypofractionated patients vs. conventionally-fractionated patients across the entire dose spectrum. Those treated with conventional fractionation, the slope of the dose response curve for radiation-associated density changes was z 1.0, vs. a slope of z2.2 those treated with hypofractionated SBRT. Different values for a/b were estimated for different regions of the lung exposed to different total doses of radiation. For lungs exposed to total doses 12, 24, 36, and 48 Gy, the estimated values for a/ b were 1.3, 2.5, 3.7, and 5.0, respectively. Conclusion: The estimated a/b ratio for acute RT-induced changes in lung density (assessed by CT) is in the range of 1.3-5.0, and appears to
vary with total dose. The lower a/b ratio in the low dose region might reflect the lesser degree of injury observed with these lung doses. Additional work is needed to better define the fractionation sensitivityassociated lung injury, and also the impact of total dose on the fractionation sensitivity. This information might be useful in computing isoeffective dose distributions for competing treatment plans with variable fraction sizes, and for predicting subsequent degree of RT-associated lung injury. Acknowledgment: This project was supported in part by a NIH grant CA69579 (LBM). Author Disclosure: D. Fried: None. S.M. Miller: None. M. Fayda: None. D.E. Morris: None. T.M. Zagar: None. M. Lawrence: None. E. Schreiber: None. M. Ivanovic: None. W. McCartney: None. L.B. Marks: None.
3216 Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2 I. Nishibuchi,1,2 S. Tashiro,2 H. Suzuki,2 A. Kinomura,2 J. Sun,2 M. Harata,3 T. Fukagawa,4 T. Ikura,5 and Y. Nagata1; 1Department of Radiation Oncology, Hiroshima University, Hiroshima, Japan, 2 Department of Cellular Biology, RIRBM, Hiroshima University, Hiroshima, Japan, 3Department of Biochemistry, Tohoku University, Gr Sch Med., Tohoku, Japan, 4Department of Molecular Genetics, National Institute of Genetics, Mishima, Japan, 5Department of Mutagenesis, RBC., Kyoto University, Kyoto, Japan Purpose/Objective(s): Reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, has been shown to play an important role in the regulation of DNA damage response. It has been shown that yeast H2A.Z is involved in DNA repair. In vertebrates, a recent study has revealed the presence of two H2A.Z isoforms: H2A.Z-1 and H2A.Z-2. However, how each of these vertebrate H2A.Z isoforms functions in the reorganization of damaged chromatin is still unclear. Thus, this study examined the role of human H2A.Z isoforms in the reorganization of chromatin after induction of DNA double strand breaks (DSBs). Materials/Methods: Colony forming assays were performed to compare the survival rates of wild type (WT), H2A.Z-1 -/- (Z-1 k/o) and H2A.Z-2 -/- (Z-2 k/o) DT40 cells after irradiation at 2Gy/4Gy/ 6Gy. Immunofluorescence staining using anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2Gy irradiation of WT, Z-1 k/o and Z-2 k/o. To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing either one of the GFP-H2A.Z isoforms and performed fluorescence recovery after photobleaching (FRAP) analysis in combination with microirradiation. In FRAP analysis, the dynamics of H2A.Z isoform following DNA damage was quantified by determining the fluorescence recovery of GFP signal within two independent strips immediately following microirradiation, i.e., one in the irradiated region and the other in the unirradiated region, of a single nucleus. Results: Survival rate of Z-2 k/o after irradiation was lower than that of WT and Z-1 k/o, suggesting that Z-2 k/o cells were more radiosensitive than WT and Z-1 k/o. RAD51 focus formation after ionizing irradiation was disturbed in Z-2 k/o cells, but not in Z-1 k/o cells. In FRAP analysis, rapid fluorescence recovery of GFP-H2A.Z-2 was observed in the irradiated region, while the fluorescence intensity in the unirradiated region did not change during the observed time. In contrast, fluorescence intensity of GFP-H2A.Z-1 failed to show a significant change after microirradiation. These findings suggest that in vertebrates, exchange of H2A.Z-2 at sites containing DNA damage is involved in the reorganization of damaged chromatin after induction of DSBs. Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of DNA damage response at a very early stage via reorganization of damaged chromatin required for the focus formation of RAD51.
Volume 84 Number 3S Supplement 2012 Materials/Methods: SAMP6 and SAMR1 mice (4 months old) were irradiated to 0 or 20 Gy to the right hind leg with a Linear Accelerator at 300 monitor units per minute using 6 MV photons. The nonirradiated left hind leg was used as a 0 Gy control. Twenty-four hrs later a 2-mm diameter circular unicortical wound was made in each proximal tibia. At day 21, the mice were sacrificed with the tibiae removed, fixed, and radiographed. The x-rays were used to measure the wound diameter as an index of wound healing. Results: Radiographic analysis at day 21 showed healing, smaller wounds in control non-irradiated SAMR1 mice (0.23 mm 0.06, n Z 8) compared with non-irradiated SAMP6 mice (0.48 mm 0.01, p Z 0.0318, n Z 6); this denotes a relative defect in bone formation in the SAMP6 strain. Both SAMR1 and SAMP6 showed delayed bone healing after 20 Gy to one limb. Limb irradiation in SAMR1 mice impaired healing and resulted in 280 % larger wounds (0.64 0.09, p Z 0.0015, n Z 5), compared with non-irradiated mice. Hind limb irradiation of SAMP6 mice further delayed healing and resulted in persistent, 170% large wounds than in non-irradiated SAMP6 mice (0.81 0.07, p Z 0.0240, n Z 5). Conclusion: SAMP6 mice demonstrated delayed bone wound repair compared with control SAMR1 mice and irradiation inhibited wound healing in both strains of mice. Acknowledgement: This project was supported by NIAID U191A168021 and NIH R21AG034254. Author Disclosure: M. Epperly: None. S. Cao: None. T.M. Dixon: None. J. Goff: None. J. Glowacki: None. P. Wipf: None. J.S. Greenberger: None.
3215 Estimating the Alpha/Beta Ratio for Radiation-induced CT Density Changes in Patients Receiving Thoracic Radiation: Comparison of Data from SRS and Conventional Fractionation D. Fried, S.M. Miller, M. Fayda, D.E. Morris, T.M. Zagar, M. Lawrence, E. Schreiber, M. Ivanovic, W. McCartney, and L.B. Marks; University of North Carolina at Chapel Hill, Chapel Hill, NC Purpose: To estimate the Alpha/Beta (a/b) ratio for radiation(RT)-induced lung injury by comparing the density changes observed in patients treated with hypofractionated stereotactic body radiation therapy (SBRT) versus those treated with conventionally-fractionated (i.e., 2 Gy/fraction) radiation. Methods: Images from patients treated with hypofractionated SBRT (48 Gy in 12 fractions through multiple non-axial beams) were compared with the corresponding images from patients treated with conventionally-fractionated radiation treatment (usually with large fields- anteroposterior/posteroanterior and off-cord-obliques, either sequentially or concurrently). For all patients, the pre- and post-computed tomography (CT) images were compared to each other and to the three-dimensional dose distribution, using image fusion and treatment planning software. Therefore, we were able to define changes in regional lung density within different regions of the lung that received different doses of radiation (and hence different radiation fraction sizes). Data from both treatment cohorts was pooled for analysis. Linear regression was used to estimate the a/b ratio by comparing the observed dose response curve (DRC) data in the hypofractionated vs. conventionally-fractionated patients. Results: Considering the data <6 months post-RT, the degree of density change was greater in the hypofractionated patients vs. conventionally-fractionated patients across the entire dose spectrum. Those treated with conventional fractionation, the slope of the dose response curve for radiation-associated density changes was z 1.0, vs. a slope of z2.2 those treated with hypofractionated SBRT. Different values for a/b were estimated for different regions of the lung exposed to different total doses of radiation. For lungs exposed to total doses 12, 24, 36, and 48 Gy, the estimated values for a/ b were 1.3, 2.5, 3.7, and 5.0, respectively. Conclusion: The estimated a/b ratio for acute RT-induced changes in lung density (assessed by CT) is in the range of 1.3-5.0, and appears to
vary with total dose. The lower a/b ratio in the low dose region might reflect the lesser degree of injury observed with these lung doses. Additional work is needed to better define the fractionation sensitivityassociated lung injury, and also the impact of total dose on the fractionation sensitivity. This information might be useful in computing isoeffective dose distributions for competing treatment plans with variable fraction sizes, and for predicting subsequent degree of RT-associated lung injury. Acknowledgment: This project was supported in part by a NIH grant CA69579 (LBM). Author Disclosure: D. Fried: None. S.M. Miller: None. M. Fayda: None. D.E. Morris: None. T.M. Zagar: None. M. Lawrence: None. E. Schreiber: None. M. Ivanovic: None. W. McCartney: None. L.B. Marks: None.
3216 Reorganization of Damaged Chromatin by the Exchange of Histone Variant H2A.Z-2 I. Nishibuchi,1,2 S. Tashiro,2 H. Suzuki,2 A. Kinomura,2 J. Sun,2 M. Harata,3 T. Fukagawa,4 T. Ikura,5 and Y. Nagata1; 1Department of Radiation Oncology, Hiroshima University, Hiroshima, Japan, 2 Department of Cellular Biology, RIRBM, Hiroshima University, Hiroshima, Japan, 3Department of Biochemistry, Tohoku University, Gr Sch Med., Tohoku, Japan, 4Department of Molecular Genetics, National Institute of Genetics, Mishima, Japan, 5Department of Mutagenesis, RBC., Kyoto University, Kyoto, Japan Purpose/Objective(s): Reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, has been shown to play an important role in the regulation of DNA damage response. It has been shown that yeast H2A.Z is involved in DNA repair. In vertebrates, a recent study has revealed the presence of two H2A.Z isoforms: H2A.Z-1 and H2A.Z-2. However, how each of these vertebrate H2A.Z isoforms functions in the reorganization of damaged chromatin is still unclear. Thus, this study examined the role of human H2A.Z isoforms in the reorganization of chromatin after induction of DNA double strand breaks (DSBs). Materials/Methods: Colony forming assays were performed to compare the survival rates of wild type (WT), H2A.Z-1 -/- (Z-1 k/o) and H2A.Z-2 -/- (Z-2 k/o) DT40 cells after irradiation at 2Gy/4Gy/ 6Gy. Immunofluorescence staining using anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2Gy irradiation of WT, Z-1 k/o and Z-2 k/o. To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing either one of the GFP-H2A.Z isoforms and performed fluorescence recovery after photobleaching (FRAP) analysis in combination with microirradiation. In FRAP analysis, the dynamics of H2A.Z isoform following DNA damage was quantified by determining the fluorescence recovery of GFP signal within two independent strips immediately following microirradiation, i.e., one in the irradiated region and the other in the unirradiated region, of a single nucleus. Results: Survival rate of Z-2 k/o after irradiation was lower than that of WT and Z-1 k/o, suggesting that Z-2 k/o cells were more radiosensitive than WT and Z-1 k/o. RAD51 focus formation after ionizing irradiation was disturbed in Z-2 k/o cells, but not in Z-1 k/o cells. In FRAP analysis, rapid fluorescence recovery of GFP-H2A.Z-2 was observed in the irradiated region, while the fluorescence intensity in the unirradiated region did not change during the observed time. In contrast, fluorescence intensity of GFP-H2A.Z-1 failed to show a significant change after microirradiation. These findings suggest that in vertebrates, exchange of H2A.Z-2 at sites containing DNA damage is involved in the reorganization of damaged chromatin after induction of DSBs. Conclusions: We found that vertebrate H2A.Z-2 is involved in the regulation of DNA damage response at a very early stage via reorganization of damaged chromatin required for the focus formation of RAD51.