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Photon Versus Proton Versus Carbon Irradiation of Glioma Initiating Cells
Poster Viewing Abstracts S677
Volume 84 Number 3S Supplement 2012 Conclusion: Results of this study suggest that chromosomal aberration frequency might be associated with cancer development and impairment in repair. This also suggests an association between chromosomal aberration and excess fragments and further chance of toxicity from radiation treatment. Difference in M2% between two groups further validates the association of more in vitro radiation induced DNA damage and clinical radiosensitivity. This can be considered as an in vitro radiosensitivity biomarker. Author Disclosure: S. Samiee: None. L. Beaton: None. L. Eapen: None. C. E: None. C. Ferrarotto: None. S. Grimes: None. K. Malone: None. R. Wilkins: None. S. Malone: None.
3220 SIRT2 Directs the Replication Stress Response Through CDK9 Deacetylation H. Zhang,1 S. Park,2 B.G. Pantazides,1 C.W. Hardy,1 N.T. Seyfried,3 D. Duong,3 and D.S. Yu1; 1Department of Radiation Oncology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, 2Department of Cancer Biology and Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN, 3Department of Biochemistry, Emory University School of Medicine, Atlanta, GA Purpose/Objective(s): SIRT2 is a member of the sirtuin family of NAD+ dependent deacetylases, which regulates genome stability and prevents cancer and aging. Mice deficient in Sirt2 develop breast and liver cancers suggesting that SIRT2 is a tumor suppressor gene. The objective of this study was to determine if SIRT2 has a role in maintaining genome integrity by directing the replication stress response. Materials/Methods: We examined the role of SIRT2 in the replication stress response by assessing hydroxyurea (HU) sensitivity, impairment in cell cycle recovery, induction of RPA foci in the absence of exogenous damage, and a G2/M checkpoint deficit following depletion of SIRT2 in U2OS human osteosarcoma cells. To investigate how SIRT2 functions in the replication stress response, we purified FLAG-SIRT2 from cells and performed proteomic analysis by mass spectrometry to identify potential substrates. To confirm the physical and functional interaction of SIRT2 with CDK9, a protein required for recovery from replication stress, we completed co-immunoprecipitation and in vitro and in vivo deacetylation assays. To identify the sites of deacetylation, we performed mass spectrometry analysis of purified FLAG-CDK9 and confirmed the findings by mutational analyses. We examined if the deacetylation of CDK9 is regulated by replication stress. Finally, to determine if the deacetylation of CDK9 by SIRT2 is significant for replication stress activities, we tested the function of CDK9 in SIRT2 deficiency by complementation assays. Results: We found that SIRT2 deficiency causes sensitivity to replication stress, impairment in recovery from replication arrest, spontaneous accumulation of RPA foci, and a G/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 both in vitro and in vivo at lysine residue 48, promoting its autophosphorylation and localization to chromatin in response to replication stress. In addition, CDK9 complements the replication stress impairment of SIRT2 deficiency. Conclusions: Our results define a novel function for SIRT2 in regulating checkpoint pathways that respond to replication stress through CDK9 deacetylation, providing insight into how SIRT2 maintains genome integrity and loss of SIRT2 results in an in vivo tumor permissive phenotype. Author Disclosure: H. Zhang: None. S. Park: None. B.G. Pantazides: None. C.W. Hardy: None. N.T. Seyfried: None. D. Duong: None. D.S. Yu: None.
3221 Photon Versus Proton Versus Carbon Irradiation of Glioma Initiating Cells S. Chiblak,1 B. Campos,2 Z. Gal,2 Z. Tang,1 S. Brons,3 A. Unterberg,2 J. Debus,1 C. Herold-Mende,2 and A. Abdollahi1,4; 1Max-Eder Molecular Radiation Oncology, Heidelberg Institute of Radiation
Oncology (HIRO), Heidelberg Ion Therapy Center (HIT), University of Heidelberg Medical School & National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany, 2 Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg Medical School, Heidelberg, Germany, 3 Heidelberg Ion Therapy Center (HIT), University of Heidelberg Medical School, Heidelberg, Germany, 4Center of Cancer Systems Biology, St. Elizabeth’s Medical Center, Department of Medicine, Tufts University, Boston, MA Purpose/Objective(s): Radiation therapy is an integral component of Glioblastoma (GBM) treatment. Evidence is provided for tumor cells with self-renewing and stem cell-like features within GBM, referred to as glioma initiating cells (GICs). It was reported that AC133 surface antigen expression on GICs correlates with high tumorigenicity as well as tumor resistance to photon radiation therapy. In contrast to photon irradiation, colorectal tumor initiating cells were recently proposed to be sensitive to heavy ion therapy using carbon particles. Therefore, we sought to investigate the sensitivity of GICs with different AC133 status to photon (X) vs. proton (P) vs. carbon (C) irradiation using classical radiobiology assays. Materials/Methods: Human primary GICs were isolated and established from tumor specimen of six consented GBM patients: NCH601, NCH620, NCH644, NCH441, NCH421k and NCH636. In addition, human U87MG was used to represent a classical GBM cell line. Plating efficiency was tested in soft agar. Prior to plating spheroids were mechanically segregated to generate single cell suspensions. After implementation of cells into the 3D agarose gel, cells were irradiated with X, P and C ions. Spheroid-like colony formation was monitored w 2 weeks post irradiation by phase contrast microscopy and colonies quantified by recovery of cells and fluorescence staining. Cellular surface AC133 expression was measured using FACS. Results: No difference in plating efficacy of GICs exhibiting different AC133 expression in the range of 0.014%-96% were found (p Z 0.24). After an initial photon screen for the most radioresistant cells, irradiation with isodoses and estimated isoeffect doses of photon, proton and carbon ion was performed. As compared to photons, proton irradiation yielded RBE values in the range of 0.7 to 1.11. However, carbon irradiation rendered the radioresistant (photon) GIC cultures sensitive with RBE values of 1.96 (NCH421k), 2.41 (NCH644), 3.44 (NCH636) and 3.75 (NCH441). Of note U87 cells, known to be relatively resistant to irradiation, showed an RBE of 2.00. Potential differences in DNA damage and repair capacity of GICs are currently correlated with CD133 status and survival data and will be presented. Conclusion: The plating efficacy of GICs did not correlate with their CD133 status. GICs were sensitive to carbon ion therapy. In contrast to reported data, we did not observe significant differences in radiosensitivity of GICs as compared to the classical U87 GBM cell lines using standard radiobiology assays. Author Disclosure: S. Chiblak: None. B. Campos: None. Z. Gal: None. Z. Tang: None. S. Brons: None. A. Unterberg: None. J. Debus: None. C. Herold-Mende: None. A. Abdollahi: None.
3222 Oral Delivery of Mitochondrial Targeted GS-Nitroxide JP4-039 Protects Fanconi Anemia (FA) D2 -/- Mice From Irradiation Mucositis H. Berhane,1 M. Epperly,1 T.M. Dixon,1 S. Cao,1 D. Shields,1 P. Wipf,1 S. Li,1 X. Gao,1 E. Guinan,2 and J.S. Greenberger1; 1University of Pittsburgh, Pittsburgh, PA, 2Dana Farber Cancer Institute, Boston, MA Purpose/Objective(s): Fanconi anemia (FA) is an inherited disease characterized by defective DNA repair and cellular sensitivity to DNA cross linking agents. FA is associated with high risk of Human Papilloma Virus (HPV) associated head and neck squamous cell carcinoma (HNSCC), irradiation induced bone marrow failure, and leukemia. There is a need for new ways to protect normal tissues of the oral cavity during radiation therapy of HNSCC.
Volume 84 Number 3S Supplement 2012 Conclusion: Results of this study suggest that chromosomal aberration frequency might be associated with cancer development and impairment in repair. This also suggests an association between chromosomal aberration and excess fragments and further chance of toxicity from radiation treatment. Difference in M2% between two groups further validates the association of more in vitro radiation induced DNA damage and clinical radiosensitivity. This can be considered as an in vitro radiosensitivity biomarker. Author Disclosure: S. Samiee: None. L. Beaton: None. L. Eapen: None. C. E: None. C. Ferrarotto: None. S. Grimes: None. K. Malone: None. R. Wilkins: None. S. Malone: None.
3220 SIRT2 Directs the Replication Stress Response Through CDK9 Deacetylation H. Zhang,1 S. Park,2 B.G. Pantazides,1 C.W. Hardy,1 N.T. Seyfried,3 D. Duong,3 and D.S. Yu1; 1Department of Radiation Oncology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, 2Department of Cancer Biology and Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN, 3Department of Biochemistry, Emory University School of Medicine, Atlanta, GA Purpose/Objective(s): SIRT2 is a member of the sirtuin family of NAD+ dependent deacetylases, which regulates genome stability and prevents cancer and aging. Mice deficient in Sirt2 develop breast and liver cancers suggesting that SIRT2 is a tumor suppressor gene. The objective of this study was to determine if SIRT2 has a role in maintaining genome integrity by directing the replication stress response. Materials/Methods: We examined the role of SIRT2 in the replication stress response by assessing hydroxyurea (HU) sensitivity, impairment in cell cycle recovery, induction of RPA foci in the absence of exogenous damage, and a G2/M checkpoint deficit following depletion of SIRT2 in U2OS human osteosarcoma cells. To investigate how SIRT2 functions in the replication stress response, we purified FLAG-SIRT2 from cells and performed proteomic analysis by mass spectrometry to identify potential substrates. To confirm the physical and functional interaction of SIRT2 with CDK9, a protein required for recovery from replication stress, we completed co-immunoprecipitation and in vitro and in vivo deacetylation assays. To identify the sites of deacetylation, we performed mass spectrometry analysis of purified FLAG-CDK9 and confirmed the findings by mutational analyses. We examined if the deacetylation of CDK9 is regulated by replication stress. Finally, to determine if the deacetylation of CDK9 by SIRT2 is significant for replication stress activities, we tested the function of CDK9 in SIRT2 deficiency by complementation assays. Results: We found that SIRT2 deficiency causes sensitivity to replication stress, impairment in recovery from replication arrest, spontaneous accumulation of RPA foci, and a G/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 both in vitro and in vivo at lysine residue 48, promoting its autophosphorylation and localization to chromatin in response to replication stress. In addition, CDK9 complements the replication stress impairment of SIRT2 deficiency. Conclusions: Our results define a novel function for SIRT2 in regulating checkpoint pathways that respond to replication stress through CDK9 deacetylation, providing insight into how SIRT2 maintains genome integrity and loss of SIRT2 results in an in vivo tumor permissive phenotype. Author Disclosure: H. Zhang: None. S. Park: None. B.G. Pantazides: None. C.W. Hardy: None. N.T. Seyfried: None. D. Duong: None. D.S. Yu: None.
3221 Photon Versus Proton Versus Carbon Irradiation of Glioma Initiating Cells S. Chiblak,1 B. Campos,2 Z. Gal,2 Z. Tang,1 S. Brons,3 A. Unterberg,2 J. Debus,1 C. Herold-Mende,2 and A. Abdollahi1,4; 1Max-Eder Molecular Radiation Oncology, Heidelberg Institute of Radiation
Oncology (HIRO), Heidelberg Ion Therapy Center (HIT), University of Heidelberg Medical School & National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany, 2 Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg Medical School, Heidelberg, Germany, 3 Heidelberg Ion Therapy Center (HIT), University of Heidelberg Medical School, Heidelberg, Germany, 4Center of Cancer Systems Biology, St. Elizabeth’s Medical Center, Department of Medicine, Tufts University, Boston, MA Purpose/Objective(s): Radiation therapy is an integral component of Glioblastoma (GBM) treatment. Evidence is provided for tumor cells with self-renewing and stem cell-like features within GBM, referred to as glioma initiating cells (GICs). It was reported that AC133 surface antigen expression on GICs correlates with high tumorigenicity as well as tumor resistance to photon radiation therapy. In contrast to photon irradiation, colorectal tumor initiating cells were recently proposed to be sensitive to heavy ion therapy using carbon particles. Therefore, we sought to investigate the sensitivity of GICs with different AC133 status to photon (X) vs. proton (P) vs. carbon (C) irradiation using classical radiobiology assays. Materials/Methods: Human primary GICs were isolated and established from tumor specimen of six consented GBM patients: NCH601, NCH620, NCH644, NCH441, NCH421k and NCH636. In addition, human U87MG was used to represent a classical GBM cell line. Plating efficiency was tested in soft agar. Prior to plating spheroids were mechanically segregated to generate single cell suspensions. After implementation of cells into the 3D agarose gel, cells were irradiated with X, P and C ions. Spheroid-like colony formation was monitored w 2 weeks post irradiation by phase contrast microscopy and colonies quantified by recovery of cells and fluorescence staining. Cellular surface AC133 expression was measured using FACS. Results: No difference in plating efficacy of GICs exhibiting different AC133 expression in the range of 0.014%-96% were found (p Z 0.24). After an initial photon screen for the most radioresistant cells, irradiation with isodoses and estimated isoeffect doses of photon, proton and carbon ion was performed. As compared to photons, proton irradiation yielded RBE values in the range of 0.7 to 1.11. However, carbon irradiation rendered the radioresistant (photon) GIC cultures sensitive with RBE values of 1.96 (NCH421k), 2.41 (NCH644), 3.44 (NCH636) and 3.75 (NCH441). Of note U87 cells, known to be relatively resistant to irradiation, showed an RBE of 2.00. Potential differences in DNA damage and repair capacity of GICs are currently correlated with CD133 status and survival data and will be presented. Conclusion: The plating efficacy of GICs did not correlate with their CD133 status. GICs were sensitive to carbon ion therapy. In contrast to reported data, we did not observe significant differences in radiosensitivity of GICs as compared to the classical U87 GBM cell lines using standard radiobiology assays. Author Disclosure: S. Chiblak: None. B. Campos: None. Z. Gal: None. Z. Tang: None. S. Brons: None. A. Unterberg: None. J. Debus: None. C. Herold-Mende: None. A. Abdollahi: None.
3222 Oral Delivery of Mitochondrial Targeted GS-Nitroxide JP4-039 Protects Fanconi Anemia (FA) D2 -/- Mice From Irradiation Mucositis H. Berhane,1 M. Epperly,1 T.M. Dixon,1 S. Cao,1 D. Shields,1 P. Wipf,1 S. Li,1 X. Gao,1 E. Guinan,2 and J.S. Greenberger1; 1University of Pittsburgh, Pittsburgh, PA, 2Dana Farber Cancer Institute, Boston, MA Purpose/Objective(s): Fanconi anemia (FA) is an inherited disease characterized by defective DNA repair and cellular sensitivity to DNA cross linking agents. FA is associated with high risk of Human Papilloma Virus (HPV) associated head and neck squamous cell carcinoma (HNSCC), irradiation induced bone marrow failure, and leukemia. There is a need for new ways to protect normal tissues of the oral cavity during radiation therapy of HNSCC.