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The Dynamics of Histone Variant H2A.Z upon DNA Damage
Proceedings of the 53rd Annual ASTRO Meeting
44
‘‘Large Mediastinal Tumor Mass’’ is a Prognostic Factor in Hodgkin Lymphoma (HL): Is the Definition on the Basis of a Chest Radiograph in the Era of CT Obsolete?
J. Kriz, R. Mueller, H. Mueller, C. Kobe, A. Engert, H. Eich University of Cologne, 50937, Germany Purpose/Objective(s): The risk factor ‘‘large mediastinal tumor mass’’ is an internationally accepted unfavorable prognostic factor for patients with HL. Measurements of the mediastinal mass size on chest radiograph have been correlated with prognosis and are used to assign treatment in clinical trials including chemotherapy plus involved field or involved node radiotherapy. The definition of this risk factor varies considerably between large cooperative study groups. The German Hodgkin Study Group (GHSG) has defined a ‘‘large mediastinal tumor mass’’ as a mediastinal lymph node involvement of $1/3 of the thoracic diameter measured on the basis of a chest radiograph. The maximal transverse diameter of the mediastinal tumor is divided by the maximal diameter of the chest. In the era of CT and PET-guided imaging the question arises whether this definition can be transferred offhand to the cross sectional imaging. The purpose of the current analysis is to determine whether the measured data obtained from chest radiographs correlate with those from CT scans. Materials/Methods: A total of 145 patients with de novo HL in early unfavorable and advanced stages were included in this study. The size of the mediastinal involvement and the thoracic diameter were measured on a chest radiograph and on the corresponding CT-scan. The measured values were evaluated with correlated statistics, specificity, and sensitivity. Results: The patient’s group consisted of 76 men and 69 women with a mean age of 29 years (range, 18 – 60 years). Ninety-four patients had a ‘‘large mediastinal tumor mass’’ according to the guidelines of the GHSG and 51 had a mediastinal lymph node involvement only. The mean measured tumor size obtained from the chest radiograph was 10.7 cm and for the CT 10.3 cm. The mean thoracic diameter was 29.1 cm on the chest radiographs and 27.5 cm on the CT. The correlation of the measured values on the chest radiographs compared to those of the CT scans was 0.95 for the tumor size and 0.77 for the thoracic diameter. The correlation of the total analysis was 0.81. In 90.3% the measured values within the radiograph and CT were equal. Taken the chest radiograph as the gold standard, the sensitivity is 0.87 and the specificity 0.96. Conclusions: These results show that there is no correlation between the measurements of the radiographs and corresponding CTscans in 10%. This can be explained by the different patient positioning during the examinations. Since the correct definition of the risk factor ‘‘large mediastinal tumor mass’’ is necessary for the adequate multimodal treatment (chemotherapy and radiotherapy), none of the imaging techniques, conventional radiograph nor CT, should be omitted at the moment. Author Disclosure: J. Kriz: None. R. Mueller: None. H. Mueller: None. C. Kobe: None. A. Engert: None. H. Eich: None.
45
DNA Double-strand Break Repair in the Context of Chromatin
C. E. Ruebe, Y. Lorat, S. Schanz, N. Schuler, C. Ruebe Dept. of Radiation Oncology, Saarland University, Homburg/Saar, Germany Purpose/Objective(s): DNA double-strand breaks (DSBs) generated by ionizing radiation represent an extremely cytolethal form of DNA damage and thus pose a serious threat to the preservation of genetic and epigenetic information. DSB repair is a highly coordinated process that requires the unraveling of the compacted chromatin structure to facilitate repair machinery access and then restoration of the original undamaged chromatin state. Materials/Methods: Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic vs. heterochromatic regions. Results: While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of gH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Moreover, we show that the persisting accumulation of gH2AX, MDC1, and 53BP1 in heterochromatin at late repair-times (48 hours, 72 hours, and 1 week post-irradiation) do not co-localize with pKu70 or DNA-PKcs and thus, may represent incomplete or incorrect restoration of chromatin structure (rather than unrepaired DNA damage), leaving a DSB-induced epigenetic memory of damage with potentially pathological repercussions. Conclusions: The elaborate higher-order organization of chromatin appears to be important in assembling the repair machinery, improving the accessibility of DNA lesions to repair complexes, and potentially influencing how readily a lesion is detected and how the repair machinery responds to the lesion. Author Disclosure: C. E. Ruebe: None. Y. Lorat: None. S. Schanz: None. N. Schuler: None. C. Ruebe: None.
46
The Dynamics of Histone Variant H2A.Z upon DNA Damage
I. Nishibuchi1, S. Tashiro2, H. Shima3, T. Kimura1, Y. Murakami1, M. Kenjo1, Y. Kaneyasu1, Y. Nagata1 1 3
Dept. of Radiation Oncology, Hiroshima, Japan, 2Dept. of Cellular Biology, RIRBM, Hiroshima University, Hiroshima, Japan, Dept. of Biochemistry, Tohoku University, Graduate School of Medicine, Sendai, Japan
Purpose/Objective(s): Ionizing irradiation induces lethal genome damages like DNA double strand breaks (DSBs) in human cells. Reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, has been shown to play a role in the regulation of DNA damage response. Histone variant H2A.Z in yeast is deposited close to the DSBs early but transiently and directs DNA resection, single DSBs-induced checkpoint activation, and DSBs anchoring. However, the role of
S23
I. J. Radiation Oncology d Biology d Physics
S24
Volume 81, Number 2, Supplement, 2011
human H2A.Z in the reorganization of damaged chromatin is still unclear. So, we investigated that the role of H2A.Z in the reorganization of chromatin after induction of DNA double strand breaks (DSBs). Materials/Methods: We constructed GM06237 cells stably expressing GFP-H2A.Z. Then we performed inverse fluorescence recovery after photobleaching (iFRAP) analysis of GFP-H2A.Z in combination with microirradiation. In iFRAP experiments, immediately following microirradiation, all fluorescence except small regions in irradiated and unirradiated areas were bleached and the remaining GFP-H2A.Z fluorescence was chased. The dynamics of H2A.Z following DNA damage was quantified by determining the fluorescence recovery of GFP-H2A.Z within two independent strips immediately following microirradiation, one in the irradiated region and the other in unirradiated region, of a single nucleus (FRAP analysis). Then, to investigate the pathway which regulates H2A.Z dynamics in the damaged chromatin, we performed iFRAP with cells expressing siRNA against tip60 (a histone acetyltransferase) or UBC13 (a histone ubiquitination enzyme). Results: In iFRAP analysis, a slow diffusion of GFP-H2A.Z into the photobleached area was observed within the irradiated area, but not in the unirradiated area. In FRAP analysis, rapid fluorescence recovery was observed in the irradiated region, while the fluorescence intensity in the unirradiated region did not change during the observed time. Dynamics of H2A.Z in microirradiated area was repressed by knockdown of UBC13, but not Tip60. Conclusions: We found that H2A.Z is released from damaged chromatin immediately after induction of DSBs. This suggests that human H2A.Z is involved in the regulation of DNA damage response at the very early stage via reorganization of damaged chromatin and UBC13 may be affected in the regulation of H2A.Z dynamics upon DSBs. Author Disclosure: I. Nishibuchi: None. S. Tashiro: None. H. Shima: None. T. Kimura: None. Y. Murakami: None. M. Kenjo: None. Y. Kaneyasu: None. Y. Nagata: None.
47
EGFR Inhibition Radiosensitizes NSCLC Cells via DNA Double-strand Break Inducible Senescence
H. Willers1, M. Wang1, F. Morsbach1, D. Sander1, A. Nanda2, M. Kriegs3, M. Krause4, E. Dikomey3, M. Baumann4, J. Settleman1 Massachusetts General Hospital, Boston, MA, 2Harvard Radiation Oncology Program, Boston, MA, 3University of Hamburg, Hamburg, Germany, 4Technical University Dresden, Dresden, Germany 1
Purpose/Objective(s): The subset of patients with Non-small cell lung cancer (NSCLC) sensitive to combined ionizing radiation (IR) and epidermal growth factor receptor (EGFR) inhibitors is not defined. To this end, large cell line panels are useful for ‘‘personalizing’’ targeted agents through the identification of sensitive tumor subsets and associated predictive biomarkers. However, the clonogenic survival assay is not suitable for large-scale cell line screens and whether short-term survival/proliferation is an appropriate surrogate endpoint for measuring radiosensitization is not established. Here, we evaluated the radiosensitizing properties of erlotinib (ERL) and cetuximab (CET) in a large panel of NSCLC cell lines resistant to ERL alone. Materials/Methods: Forty-two cell lines were tested for sensitivity to IR 0 – 8 Gy with ERL 2 mM or CET 100 nM using a Syto60 stain-based 72-hour proliferation assay and colony formation. Follow-up analysis included FACS, cell-based staining for g-H2AX foci as a surrogate for double-strand breaks (DSB), p21, b-galactosidase, and Western blot for H3K9me3, p53, and various EGFR dependent signaling pathway components. Results: ERL and CET sensitized 38% and 47% of cell lines to IR in the Syto60 proliferation assay, respectively. The assay was 96% accurate in predicting radiosensitization in a clonogenic assay. Follow-up study of a subset of 11 cell lines revealed cellular senescence as a cause of radiosensitization in 83% of lines. ERL- or CET-induced senescence occurred within 72 hours after 2 Gy and corresponded to the reduction in cell number seen in the Syto60 assay. In vivo confirmation in xenografts is ongoing. In cells with wild-type p53, ERL or CET increased DSB in the G1 and G2 phases of the cell cycle at 24 hours, resulting in prolonged p21mediated G1 arrest but not G2 arrest. Senescence was not only correlated with residual g-H2AX foci but also a decrease in the contribution of sublethal damage repair to loss of clonogenicity, and this observation extended to cell lines with mutant p53. Senescence and increased DSB levels were dependent on a disruption of the MEK-ERK pathway irrespective of p53 or KRAS status, suggesting a common mechanism of radiosensitization across diverse genetic backgrounds. Conclusions: Cellular senescence is unexpectedly a prominent mechanism of cell inactivation following combined EGFR inhibition and low-dose irradiation, which should guide the identification of NSCLC subsets sensitive to this treatment combination. Our data also provide an important validation of short-term proliferation assays that may facilitate future testing of the efficacy of radiosensitizing agents in large cell line screens. Acknowledgment: Supported by the DF/HCC SPORE in Lung Cancer, P50 CA090578, RO1 CA115830, a RSNA Roentgen Resident Research Award, and DFG PAK 190, DI 457/8-1. Author Disclosure: H. Willers: None. M. Wang: None. F. Morsbach: None. D. Sander: None. A. Nanda: None. M. Kriegs: None. M. Krause: None. E. Dikomey: None. M. Baumann: None. J. Settleman: A. Employment; Genentech.
48
BRCA1 Nuclear Export Sensitizes Prostate Cancer Cells to Poly (ADP-ribose) Polymerase (PARP) Inhibition via the CRM1/exportin Pathway
L. C. Klepczyk1, S. Nowsheen1, F. Xia2, E. S. Yang1 1 University of Alabama Birmingham, Birmingham, AL, 2Vanderbilt University, Nashville, TN Purpose/Objective(s): Poly (ADP-ribose) polymerase (PARP) inhibitors have been shown to be effective in the treatment of tumors with defective homologous recombination (HR). BRCA1 is a protein intimately involved with HR repair when located in the nucleus near its repair substrates. In this study, we investigated whether targeting the shuttling of BRCA1 to the cytoplasm will render human prostate cancer cells susceptible to PARP inhibition. The dependence of this strategy on the chromosome region maintenance 1 (CRM1)/exportin pathway was also assessed. As the majority of sporadic prostate cancers possess wild-type (WT) BRCA1, making tumors susceptible to PARP inhibition could have a large clinical impact. Materials/Methods: WT BRCA1 in various human prostate cancer cell lines was transported out of the nucleus using either irradiation or expression of tr-BRCA1, a truncated form of BRCA1. Subcellular location of BRCA1 was verified using immunohistochemistry (IHC). HR capacity was assessed by a GFP-based HR repair assay. Cell survival using colony formation assays was measured after BRCA1 export out of the nucleus and treatment with the PARP1 inhibitor ABT888. To specifically analyze the
44
‘‘Large Mediastinal Tumor Mass’’ is a Prognostic Factor in Hodgkin Lymphoma (HL): Is the Definition on the Basis of a Chest Radiograph in the Era of CT Obsolete?
J. Kriz, R. Mueller, H. Mueller, C. Kobe, A. Engert, H. Eich University of Cologne, 50937, Germany Purpose/Objective(s): The risk factor ‘‘large mediastinal tumor mass’’ is an internationally accepted unfavorable prognostic factor for patients with HL. Measurements of the mediastinal mass size on chest radiograph have been correlated with prognosis and are used to assign treatment in clinical trials including chemotherapy plus involved field or involved node radiotherapy. The definition of this risk factor varies considerably between large cooperative study groups. The German Hodgkin Study Group (GHSG) has defined a ‘‘large mediastinal tumor mass’’ as a mediastinal lymph node involvement of $1/3 of the thoracic diameter measured on the basis of a chest radiograph. The maximal transverse diameter of the mediastinal tumor is divided by the maximal diameter of the chest. In the era of CT and PET-guided imaging the question arises whether this definition can be transferred offhand to the cross sectional imaging. The purpose of the current analysis is to determine whether the measured data obtained from chest radiographs correlate with those from CT scans. Materials/Methods: A total of 145 patients with de novo HL in early unfavorable and advanced stages were included in this study. The size of the mediastinal involvement and the thoracic diameter were measured on a chest radiograph and on the corresponding CT-scan. The measured values were evaluated with correlated statistics, specificity, and sensitivity. Results: The patient’s group consisted of 76 men and 69 women with a mean age of 29 years (range, 18 – 60 years). Ninety-four patients had a ‘‘large mediastinal tumor mass’’ according to the guidelines of the GHSG and 51 had a mediastinal lymph node involvement only. The mean measured tumor size obtained from the chest radiograph was 10.7 cm and for the CT 10.3 cm. The mean thoracic diameter was 29.1 cm on the chest radiographs and 27.5 cm on the CT. The correlation of the measured values on the chest radiographs compared to those of the CT scans was 0.95 for the tumor size and 0.77 for the thoracic diameter. The correlation of the total analysis was 0.81. In 90.3% the measured values within the radiograph and CT were equal. Taken the chest radiograph as the gold standard, the sensitivity is 0.87 and the specificity 0.96. Conclusions: These results show that there is no correlation between the measurements of the radiographs and corresponding CTscans in 10%. This can be explained by the different patient positioning during the examinations. Since the correct definition of the risk factor ‘‘large mediastinal tumor mass’’ is necessary for the adequate multimodal treatment (chemotherapy and radiotherapy), none of the imaging techniques, conventional radiograph nor CT, should be omitted at the moment. Author Disclosure: J. Kriz: None. R. Mueller: None. H. Mueller: None. C. Kobe: None. A. Engert: None. H. Eich: None.
45
DNA Double-strand Break Repair in the Context of Chromatin
C. E. Ruebe, Y. Lorat, S. Schanz, N. Schuler, C. Ruebe Dept. of Radiation Oncology, Saarland University, Homburg/Saar, Germany Purpose/Objective(s): DNA double-strand breaks (DSBs) generated by ionizing radiation represent an extremely cytolethal form of DNA damage and thus pose a serious threat to the preservation of genetic and epigenetic information. DSB repair is a highly coordinated process that requires the unraveling of the compacted chromatin structure to facilitate repair machinery access and then restoration of the original undamaged chromatin state. Materials/Methods: Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic vs. heterochromatic regions. Results: While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of gH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Moreover, we show that the persisting accumulation of gH2AX, MDC1, and 53BP1 in heterochromatin at late repair-times (48 hours, 72 hours, and 1 week post-irradiation) do not co-localize with pKu70 or DNA-PKcs and thus, may represent incomplete or incorrect restoration of chromatin structure (rather than unrepaired DNA damage), leaving a DSB-induced epigenetic memory of damage with potentially pathological repercussions. Conclusions: The elaborate higher-order organization of chromatin appears to be important in assembling the repair machinery, improving the accessibility of DNA lesions to repair complexes, and potentially influencing how readily a lesion is detected and how the repair machinery responds to the lesion. Author Disclosure: C. E. Ruebe: None. Y. Lorat: None. S. Schanz: None. N. Schuler: None. C. Ruebe: None.
46
The Dynamics of Histone Variant H2A.Z upon DNA Damage
I. Nishibuchi1, S. Tashiro2, H. Shima3, T. Kimura1, Y. Murakami1, M. Kenjo1, Y. Kaneyasu1, Y. Nagata1 1 3
Dept. of Radiation Oncology, Hiroshima, Japan, 2Dept. of Cellular Biology, RIRBM, Hiroshima University, Hiroshima, Japan, Dept. of Biochemistry, Tohoku University, Graduate School of Medicine, Sendai, Japan
Purpose/Objective(s): Ionizing irradiation induces lethal genome damages like DNA double strand breaks (DSBs) in human cells. Reorganization of damaged chromatin, such as posttranslational modification and/or exchange of histones, has been shown to play a role in the regulation of DNA damage response. Histone variant H2A.Z in yeast is deposited close to the DSBs early but transiently and directs DNA resection, single DSBs-induced checkpoint activation, and DSBs anchoring. However, the role of
S23
I. J. Radiation Oncology d Biology d Physics
S24
Volume 81, Number 2, Supplement, 2011
human H2A.Z in the reorganization of damaged chromatin is still unclear. So, we investigated that the role of H2A.Z in the reorganization of chromatin after induction of DNA double strand breaks (DSBs). Materials/Methods: We constructed GM06237 cells stably expressing GFP-H2A.Z. Then we performed inverse fluorescence recovery after photobleaching (iFRAP) analysis of GFP-H2A.Z in combination with microirradiation. In iFRAP experiments, immediately following microirradiation, all fluorescence except small regions in irradiated and unirradiated areas were bleached and the remaining GFP-H2A.Z fluorescence was chased. The dynamics of H2A.Z following DNA damage was quantified by determining the fluorescence recovery of GFP-H2A.Z within two independent strips immediately following microirradiation, one in the irradiated region and the other in unirradiated region, of a single nucleus (FRAP analysis). Then, to investigate the pathway which regulates H2A.Z dynamics in the damaged chromatin, we performed iFRAP with cells expressing siRNA against tip60 (a histone acetyltransferase) or UBC13 (a histone ubiquitination enzyme). Results: In iFRAP analysis, a slow diffusion of GFP-H2A.Z into the photobleached area was observed within the irradiated area, but not in the unirradiated area. In FRAP analysis, rapid fluorescence recovery was observed in the irradiated region, while the fluorescence intensity in the unirradiated region did not change during the observed time. Dynamics of H2A.Z in microirradiated area was repressed by knockdown of UBC13, but not Tip60. Conclusions: We found that H2A.Z is released from damaged chromatin immediately after induction of DSBs. This suggests that human H2A.Z is involved in the regulation of DNA damage response at the very early stage via reorganization of damaged chromatin and UBC13 may be affected in the regulation of H2A.Z dynamics upon DSBs. Author Disclosure: I. Nishibuchi: None. S. Tashiro: None. H. Shima: None. T. Kimura: None. Y. Murakami: None. M. Kenjo: None. Y. Kaneyasu: None. Y. Nagata: None.
47
EGFR Inhibition Radiosensitizes NSCLC Cells via DNA Double-strand Break Inducible Senescence
H. Willers1, M. Wang1, F. Morsbach1, D. Sander1, A. Nanda2, M. Kriegs3, M. Krause4, E. Dikomey3, M. Baumann4, J. Settleman1 Massachusetts General Hospital, Boston, MA, 2Harvard Radiation Oncology Program, Boston, MA, 3University of Hamburg, Hamburg, Germany, 4Technical University Dresden, Dresden, Germany 1
Purpose/Objective(s): The subset of patients with Non-small cell lung cancer (NSCLC) sensitive to combined ionizing radiation (IR) and epidermal growth factor receptor (EGFR) inhibitors is not defined. To this end, large cell line panels are useful for ‘‘personalizing’’ targeted agents through the identification of sensitive tumor subsets and associated predictive biomarkers. However, the clonogenic survival assay is not suitable for large-scale cell line screens and whether short-term survival/proliferation is an appropriate surrogate endpoint for measuring radiosensitization is not established. Here, we evaluated the radiosensitizing properties of erlotinib (ERL) and cetuximab (CET) in a large panel of NSCLC cell lines resistant to ERL alone. Materials/Methods: Forty-two cell lines were tested for sensitivity to IR 0 – 8 Gy with ERL 2 mM or CET 100 nM using a Syto60 stain-based 72-hour proliferation assay and colony formation. Follow-up analysis included FACS, cell-based staining for g-H2AX foci as a surrogate for double-strand breaks (DSB), p21, b-galactosidase, and Western blot for H3K9me3, p53, and various EGFR dependent signaling pathway components. Results: ERL and CET sensitized 38% and 47% of cell lines to IR in the Syto60 proliferation assay, respectively. The assay was 96% accurate in predicting radiosensitization in a clonogenic assay. Follow-up study of a subset of 11 cell lines revealed cellular senescence as a cause of radiosensitization in 83% of lines. ERL- or CET-induced senescence occurred within 72 hours after 2 Gy and corresponded to the reduction in cell number seen in the Syto60 assay. In vivo confirmation in xenografts is ongoing. In cells with wild-type p53, ERL or CET increased DSB in the G1 and G2 phases of the cell cycle at 24 hours, resulting in prolonged p21mediated G1 arrest but not G2 arrest. Senescence was not only correlated with residual g-H2AX foci but also a decrease in the contribution of sublethal damage repair to loss of clonogenicity, and this observation extended to cell lines with mutant p53. Senescence and increased DSB levels were dependent on a disruption of the MEK-ERK pathway irrespective of p53 or KRAS status, suggesting a common mechanism of radiosensitization across diverse genetic backgrounds. Conclusions: Cellular senescence is unexpectedly a prominent mechanism of cell inactivation following combined EGFR inhibition and low-dose irradiation, which should guide the identification of NSCLC subsets sensitive to this treatment combination. Our data also provide an important validation of short-term proliferation assays that may facilitate future testing of the efficacy of radiosensitizing agents in large cell line screens. Acknowledgment: Supported by the DF/HCC SPORE in Lung Cancer, P50 CA090578, RO1 CA115830, a RSNA Roentgen Resident Research Award, and DFG PAK 190, DI 457/8-1. Author Disclosure: H. Willers: None. M. Wang: None. F. Morsbach: None. D. Sander: None. A. Nanda: None. M. Kriegs: None. M. Krause: None. E. Dikomey: None. M. Baumann: None. J. Settleman: A. Employment; Genentech.
48
BRCA1 Nuclear Export Sensitizes Prostate Cancer Cells to Poly (ADP-ribose) Polymerase (PARP) Inhibition via the CRM1/exportin Pathway
L. C. Klepczyk1, S. Nowsheen1, F. Xia2, E. S. Yang1 1 University of Alabama Birmingham, Birmingham, AL, 2Vanderbilt University, Nashville, TN Purpose/Objective(s): Poly (ADP-ribose) polymerase (PARP) inhibitors have been shown to be effective in the treatment of tumors with defective homologous recombination (HR). BRCA1 is a protein intimately involved with HR repair when located in the nucleus near its repair substrates. In this study, we investigated whether targeting the shuttling of BRCA1 to the cytoplasm will render human prostate cancer cells susceptible to PARP inhibition. The dependence of this strategy on the chromosome region maintenance 1 (CRM1)/exportin pathway was also assessed. As the majority of sporadic prostate cancers possess wild-type (WT) BRCA1, making tumors susceptible to PARP inhibition could have a large clinical impact. Materials/Methods: WT BRCA1 in various human prostate cancer cell lines was transported out of the nucleus using either irradiation or expression of tr-BRCA1, a truncated form of BRCA1. Subcellular location of BRCA1 was verified using immunohistochemistry (IHC). HR capacity was assessed by a GFP-based HR repair assay. Cell survival using colony formation assays was measured after BRCA1 export out of the nucleus and treatment with the PARP1 inhibitor ABT888. To specifically analyze the