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Whole Brain Irradiation-induced Decrease of Histone H3 Acetylation in Hippocampus of Rats
Poster Viewing Abstracts S665
Volume 84 Number 3S Supplement 2012 4/2011 to 3/2012. A program was designed which cross-referenced the frequency of a given Rx within the test period compared to the reference control period. RT Rx were flagged if a dose and fractionation scheme had never been prescribed for that site or was infrequent for that site (i.e., <1% or <5%). Results: There were 4 recurring Rx (3.7%) found to be infrequent at the <1% cutoff level. There were 24 Rx (22%) found to be infrequent at the <5% cutoff level. Interestingly, there were 26 unique Rx which had not been prescribed previously during the reference control time period. Investigation into these unique Rx identified multiple etiologies including: new technique (15/26), unique palliative dosing (7/26), incorrect/multiple ICD9 specifications (3/26), and undeliverable Rx (1/26). Conclusion: Our analysis demonstrates that it is feasible to cross-reference RT Rx to ICD9 linked diagnoses to identify dose-fractionation schemes that are infrequent for a given diagnosis and require further investigation by the RT team. Implementation of a cross-referencing system during order entry of RT Rx could also alert the physician to infrequently used Rx for a given diagnosis thereby providing an additional level of consistency of care across patients and by physicians working in one practice. Author Disclosure: A. Sharabi: None. T. McNutt: None. T. DeWeese: None.
3188 Next Generation Sequencing as a Tool to Track Low-dose Effects Linked to Transformation A. Vaughan,1 S. Shih,1 and D. Lin2; 1University of California, Davis, Comprehensive Cancer Center, Sacramento, CA, 2University of California, Davis, Bioinformatics Core, Davis, CA Purpose/Objective(s): The assessment of risk from low doses of irradiation has been a multi-decade concern, mostly informed by data collated from large population exposures to irradiation, such as those at Hiroshima and Nagasaki. Over time, the nature of such low dose exposures has changed within the US population. Thus individuals are increasingly exposed to irradiation from routine CT scans; in addition, the distribution of dose external to the radiation therapy treatment field has also altered, in terms of both dose and dose gradient, across peripherally involved normal tissues. Screening for the presence of randomly deposited DNA damage, such as that detected by gH2AX foci, or altered levels of arbitrarily chosen gene transcripts, ignores damage that occurs within key genes that are potential cooperating agents in the transformation process. Here, a strategy for the detection of radiation-induced damage to key genes related to transformation is described. Materials/Methods: To assess the consequences of irradiation on either a single gene, or groups of genes, parallel sequencing was employed to detect rearrangements in a pro-leukemogenic gene, MLL (Mixed Lineage Leukemia). This gene is involved in the generation of both therapy related AML and IAL through fusion to a multiplicity of partners. The analytical methodology employed is applicable, with suitable modification, to all genes that are known, or suspected, to be involved in transformation. Here, a human lymphoblastoid cell culture isolated from a patient with hereditary spherocytosis, the TK6 cell line, was irradiated with 4 Gy and returned to culture for 24 hours. At this time all mis-repair events will have been fixed. MLL rearrangements were analyzed within one million cells using inverse PCR targeting a hot spot for such aberrations. Subsequently, the amplified material containing both normal and aberrant MLL was fragmented, capped with machine specific adapters, and sequenced using single end reads. A custom algorithm was designed to detect sequence disjunctions indicative of a rearrangement. Results: The strategy generated approximately 1 Gbp of sequenced material. Using a custom algorithm to detect MLL rearrangements, individual fusions to Chromosomes 3, 7, 13, and 18 were detected from the two million copies of DNA analyzed. These data show that aberrations within a single gene linked to neoplastic transformation can be detected, rapidly and accurately, after irradiation.
Conclusions: A simple strategy for screening gene-specific aberrations is presented from potentially very low doses of radiation. The focus on the analysis of key genes associated with transformation may provide a more robust interpretation of low dose radiation effects, and complement endpoints such as DNA damage markers and the radiation-induced alteration of gene transcription. Author Disclosure: A. Vaughan: None. S. Shih: None. D. Lin: None.
3189 Whole Brain Irradiation-induced Decrease of Histone H3 Acetylation in Hippocampus of Rats S. Ji, R. Sun, L. Zhang, and Y. Tian; The Second Affiliated Hospital of Soochow University, SuZhou, China Purpose/Objective(s): The aim is to investigate the role of epigenetics in the irradiation-induced cognitive deficiency by observing the alterations of histone acetylation in the hippocampus of rats after irradiation. Materials/Methods: A signal dose of 2Gy and 30Gy 4MV electron beam were given to adult male Sprague-Dawley rats (150-200g, 4-5 weeks), this dose covered a dose range of clinical and experimental applications, and the animals were divided into 2Gy group, 30Gy group, and control group. After whole brain irradiation, the total protein was extracted from irradiated hippocampus and whole brain 30mm cryosections were cut by using a cryostat at day 7 and day 30. Western blot was performed and the brain sections were immunohistochemical stained. The alterations of histone H3 acetylation was observed and analyzed. To evaluate the consequence of whole brain irradiation on hippocampus-dependent memory formation, Morris water maze test, passive-avoidance test and open field test were performed. Results: We show that both 2Gy and 30Gy 4MV electron beam whole brain irradiation induced significant decrease in histone H3 acetylation and different alterations of histone deacetylases (HDACs) in the hippocampus. We found a 25% reduction in histone H3 acetylation at day 7 (p < 0.01) and 5% reduction at day 30 (p < 0.05) after 2Gy irradiation. Compare to control, 30 Gy induced 5% reduction at day 7 and 13% reduction at day 30. Interestingly, we found that irradiation did not impair the expression of HDAC2, which has been indicated negatively regulates memory formation and synaptic plasticity. However, HDAC1 was increased. In Morris water maze test, passive-avoidance test and open field test, we confirmed whole brain irradiation lead to notable memory impairment at day 30 after irradiation. Conclusion: This study suggests that the alterations of histone H3 acetylation and HDACs after whole brain irradiation. Decreased histone H3 acetylation suggests altered hippocampal epigenetic signaling after whole brain irradiation. These results indicate epigenetics was involved in irradiation-induced memory deficiency, and alterations in chromatin structure may be a new possible molecular correlation of irradiation-induced cognitive deficiency. Acknowledgment: This study was supported by the National Natural Scientific Foundation of China grants 30870740, 81172128 and 81102077. Author Disclosure: S. Ji: None. R. Sun: None. L. Zhang: None. Y. Tian: None.
3190 Examining the Cellular Mechanisms of Chromosomal Instability as Means for Enhanced Radiation-induced Cytotoxicity S. Bakhoum, B.I. Zaki, and D.A. Compton; Dartmouth Medical School, Hanover, NH Purpose/Objective(s): Chromosomal instability (CIN) is a hallmark of human neoplasms. Increased frequencies of whole-chromosome missegregation have been shown to lead to tumorigenesis and is thought to alter tumor growth characteristics, adaptation, and response to therapy. Recent reports show that ionizing radiation can directly lead to CIN and resultant aneuploidy. However the cellular mechanisms leading to
Volume 84 Number 3S Supplement 2012 4/2011 to 3/2012. A program was designed which cross-referenced the frequency of a given Rx within the test period compared to the reference control period. RT Rx were flagged if a dose and fractionation scheme had never been prescribed for that site or was infrequent for that site (i.e., <1% or <5%). Results: There were 4 recurring Rx (3.7%) found to be infrequent at the <1% cutoff level. There were 24 Rx (22%) found to be infrequent at the <5% cutoff level. Interestingly, there were 26 unique Rx which had not been prescribed previously during the reference control time period. Investigation into these unique Rx identified multiple etiologies including: new technique (15/26), unique palliative dosing (7/26), incorrect/multiple ICD9 specifications (3/26), and undeliverable Rx (1/26). Conclusion: Our analysis demonstrates that it is feasible to cross-reference RT Rx to ICD9 linked diagnoses to identify dose-fractionation schemes that are infrequent for a given diagnosis and require further investigation by the RT team. Implementation of a cross-referencing system during order entry of RT Rx could also alert the physician to infrequently used Rx for a given diagnosis thereby providing an additional level of consistency of care across patients and by physicians working in one practice. Author Disclosure: A. Sharabi: None. T. McNutt: None. T. DeWeese: None.
3188 Next Generation Sequencing as a Tool to Track Low-dose Effects Linked to Transformation A. Vaughan,1 S. Shih,1 and D. Lin2; 1University of California, Davis, Comprehensive Cancer Center, Sacramento, CA, 2University of California, Davis, Bioinformatics Core, Davis, CA Purpose/Objective(s): The assessment of risk from low doses of irradiation has been a multi-decade concern, mostly informed by data collated from large population exposures to irradiation, such as those at Hiroshima and Nagasaki. Over time, the nature of such low dose exposures has changed within the US population. Thus individuals are increasingly exposed to irradiation from routine CT scans; in addition, the distribution of dose external to the radiation therapy treatment field has also altered, in terms of both dose and dose gradient, across peripherally involved normal tissues. Screening for the presence of randomly deposited DNA damage, such as that detected by gH2AX foci, or altered levels of arbitrarily chosen gene transcripts, ignores damage that occurs within key genes that are potential cooperating agents in the transformation process. Here, a strategy for the detection of radiation-induced damage to key genes related to transformation is described. Materials/Methods: To assess the consequences of irradiation on either a single gene, or groups of genes, parallel sequencing was employed to detect rearrangements in a pro-leukemogenic gene, MLL (Mixed Lineage Leukemia). This gene is involved in the generation of both therapy related AML and IAL through fusion to a multiplicity of partners. The analytical methodology employed is applicable, with suitable modification, to all genes that are known, or suspected, to be involved in transformation. Here, a human lymphoblastoid cell culture isolated from a patient with hereditary spherocytosis, the TK6 cell line, was irradiated with 4 Gy and returned to culture for 24 hours. At this time all mis-repair events will have been fixed. MLL rearrangements were analyzed within one million cells using inverse PCR targeting a hot spot for such aberrations. Subsequently, the amplified material containing both normal and aberrant MLL was fragmented, capped with machine specific adapters, and sequenced using single end reads. A custom algorithm was designed to detect sequence disjunctions indicative of a rearrangement. Results: The strategy generated approximately 1 Gbp of sequenced material. Using a custom algorithm to detect MLL rearrangements, individual fusions to Chromosomes 3, 7, 13, and 18 were detected from the two million copies of DNA analyzed. These data show that aberrations within a single gene linked to neoplastic transformation can be detected, rapidly and accurately, after irradiation.
Conclusions: A simple strategy for screening gene-specific aberrations is presented from potentially very low doses of radiation. The focus on the analysis of key genes associated with transformation may provide a more robust interpretation of low dose radiation effects, and complement endpoints such as DNA damage markers and the radiation-induced alteration of gene transcription. Author Disclosure: A. Vaughan: None. S. Shih: None. D. Lin: None.
3189 Whole Brain Irradiation-induced Decrease of Histone H3 Acetylation in Hippocampus of Rats S. Ji, R. Sun, L. Zhang, and Y. Tian; The Second Affiliated Hospital of Soochow University, SuZhou, China Purpose/Objective(s): The aim is to investigate the role of epigenetics in the irradiation-induced cognitive deficiency by observing the alterations of histone acetylation in the hippocampus of rats after irradiation. Materials/Methods: A signal dose of 2Gy and 30Gy 4MV electron beam were given to adult male Sprague-Dawley rats (150-200g, 4-5 weeks), this dose covered a dose range of clinical and experimental applications, and the animals were divided into 2Gy group, 30Gy group, and control group. After whole brain irradiation, the total protein was extracted from irradiated hippocampus and whole brain 30mm cryosections were cut by using a cryostat at day 7 and day 30. Western blot was performed and the brain sections were immunohistochemical stained. The alterations of histone H3 acetylation was observed and analyzed. To evaluate the consequence of whole brain irradiation on hippocampus-dependent memory formation, Morris water maze test, passive-avoidance test and open field test were performed. Results: We show that both 2Gy and 30Gy 4MV electron beam whole brain irradiation induced significant decrease in histone H3 acetylation and different alterations of histone deacetylases (HDACs) in the hippocampus. We found a 25% reduction in histone H3 acetylation at day 7 (p < 0.01) and 5% reduction at day 30 (p < 0.05) after 2Gy irradiation. Compare to control, 30 Gy induced 5% reduction at day 7 and 13% reduction at day 30. Interestingly, we found that irradiation did not impair the expression of HDAC2, which has been indicated negatively regulates memory formation and synaptic plasticity. However, HDAC1 was increased. In Morris water maze test, passive-avoidance test and open field test, we confirmed whole brain irradiation lead to notable memory impairment at day 30 after irradiation. Conclusion: This study suggests that the alterations of histone H3 acetylation and HDACs after whole brain irradiation. Decreased histone H3 acetylation suggests altered hippocampal epigenetic signaling after whole brain irradiation. These results indicate epigenetics was involved in irradiation-induced memory deficiency, and alterations in chromatin structure may be a new possible molecular correlation of irradiation-induced cognitive deficiency. Acknowledgment: This study was supported by the National Natural Scientific Foundation of China grants 30870740, 81172128 and 81102077. Author Disclosure: S. Ji: None. R. Sun: None. L. Zhang: None. Y. Tian: None.
3190 Examining the Cellular Mechanisms of Chromosomal Instability as Means for Enhanced Radiation-induced Cytotoxicity S. Bakhoum, B.I. Zaki, and D.A. Compton; Dartmouth Medical School, Hanover, NH Purpose/Objective(s): Chromosomal instability (CIN) is a hallmark of human neoplasms. Increased frequencies of whole-chromosome missegregation have been shown to lead to tumorigenesis and is thought to alter tumor growth characteristics, adaptation, and response to therapy. Recent reports show that ionizing radiation can directly lead to CIN and resultant aneuploidy. However the cellular mechanisms leading to