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p21 Functions to Prevent Radiation-induced Myocardial Injury in Mice
Poster Viewing Abstracts S673
Volume 84 Number 3S Supplement 2012 Materials/Methods: We determined the minimal nucleotide recognition element (RE) of the prototypical IMP protein, IMP1, using gel mobility shift assays, in-vitro selection, and NMR spectroscopy. We identified conserved transcripts containing the IMP1 RE by Sequel query of the UCSC 3´UTR database (hg19/mm8); we further performed immunoprecipitation and qRT-PCR detection of IMP1 associated mRNAs from mouse embryonic cells. Ingenuity pathway analysis v9.0 was used to explore functional relationships between proteins encoded by the mRNA ligands of IMP1; P values are based on a right-tailed Fisher’s exact test. Results: The third and fourth hnRNP K homology domains of IMP1 specifically recognize a bipartite RE: a 5´ element, CGGAC, followed by a variable 3´ element, C/A-CA-C/U. The IMP1 consensus REs are evolutionarily conserved in the 3´UTRs of 114 transcripts. Approximately 70% of these transcripts (17 of 25 tested) are enriched over fourfold in IMP1 immunoprecipitates. Gene ontology analysis of predicted IMP1 ligands reveals enrichment of gene-networks controlling cell survival (n Z 33, p < 0.0001) and differentiation (n Z 28, p < 0.005). Aberrant transcription of IMP1 RNA ligands (n Z 13) has been demonstrated in neoplastic conditions including ovarian cancer, papillary thyroid carcinoma, NSCLC, DLBCL, and melanoma (p < 0.0050.01, Table).
IMP1 ligand
P value
CAMK2N2, COL4A1, CTDSPL, DTNA, EFNB2, ESR1, FOSB, TIMP1 EFNB2, NTRK2, TIMP1 HES1, MAFB, TIMP1
0.004
checkpoints at least partially via the cyclin-dependent kinase inhibitor p21, we have utilized p21+/- and p21-/- mice to investigate how p21 regulates RIMI in vivo. Materials/Methods: p21-/- and p21+/- mice were exposed to WHI using a small-field micro-irradiator. Serial transthoracic echocardiography was performed on the mice with a high-resolution image system to measure changes in ventricular mass and cardiac function. Histopathological examination was performed to assess the size of cardiomyocytes, microvessel density and vascular permeability in the myocardium. Primary CECs were isolated from the ventricles of the heart and irradiated with ionizing radiation in vitro at 0, 3, 6, 12 and 24 Gy. Cell death after 96 hours was determined with propidium iodide exclusion and a cleaved caspase-3 assay. DNA damage in mitosis was measured by phospho-histone H3 (pHH3) and gH2AX double staining. Results: After 12 Gy WHI, p21-/- mice developed myocardial injury characterized by myocardial necrosis, systolic dysfunction, and cardiac hypertrophy. In addition, p21-/- mice showed a reduction in microvessel density and increased vascular permeability prior to the onset of cardiac dysfunction. Moreover, studies using primary CECs from p21+/- and p21-/mice irradiated in vitro indicate that p21 protects CECs from post-mitotic cell death after 12 Gy. Conclusion: Collectively, our results demonstrate that the p53/p21 axis functions to prevent radiation-induced myocardial injury in mice. Acknowledgment: This project was supported by ASTRO: Minority Summer Fellowship Award (to IW) and R01 AI080488 (to DGK). Author Disclosure: I. Washington: None. C. Lee: None. E.J. Moding: None. Y. Li: None. L. Mao: None. Y. Kim: None. H.A. Rockman: None. D.G. Kirsch: None.
0.006 0.009
3211
BRD2
0.006
BRSK2
0.01
Poster Viewing Abstract 3209; Table Aberrant Transcription of IMP1 RNA Ligands in Neoplastic Conditions where IMP1 is Overexpressed Cancer type Ovarian carcinoma
Papillary thyroid carcinoma Non-small cell lung carcinoma (NSCLC) Diffuse large B-cell lymphoma (DLBCL) Melanoma
Conclusion: Our results suggest altered post-transcriptional regulation of IMP1 mRNA ligands may facilitate tumor cell survival in neoplastic conditions where IMP1 is upregulated. Therefore, IMP1 may represent a potential therapeutic target for enhancing tumor sensitivity to IR. Author Disclosure: V. Patel: None. J.A. Chao: None. R.H. Singer: None. C. Guha: None.
3210 p21 Functions to Prevent Radiation-induced Myocardial Injury in Mice I. Washington,1 C. Lee,2 E.J. Moding,2 Y. Li,1 L. Mao,3 Y. Kim,4 H.A. Rockman,3 and D.G. Kirsch1; 1Department of Radiation Oncology, Duke University Medical Center, Durham, NC, 2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 3Department of Medicine, Duke University Medical Center, Durham, NC, 4Laboratory of Veterinary Clinical Pathology, College of Veterinary Medicine, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul, Korea, Republic of Korea Purpose/Objective(s): Radiation-related heart disease is an important cause of treatment-associated mortality for cancer patients after thoracic radiation therapy. Although it has been established that microvascular loss precedes myocardial necrosis in radiation-induced myocardial injury (RIMI), the molecular mechanisms controlling the loss of the myocardial capillaries following radiation therapy remain to be fully defined. Recently our lab has utilized the Cre-loxP system to specifically delete p53 in endothelial cells and demonstrated that p53 functions in cardiac endothelial cells (CECs) to prevent RIMI after 12 Gy whole-heart irradiation (WHI). Because p53 controls the G1/S and G2/M cell cycle
Irradiation Induction of mRNA for Redox-Sensitive Promoters and Inflammatory Cytokines in Conditional Manganese Superoxide Dismutase (MnSOD) Tet on -/- Mouse Bone Marrow Stromal Cells R. Kalash, J.R. Chaillet, F. Houghton, X. Zhang, M. Epperly, S. Cao, and J.S. Greenberger; University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Bioavailability of the mitochondrial targeted antioxidant enzyme MnSOD in normal tissues counteracts irradiation induced apoptosis. To determine the effect of MnSOD on gene expression, we developed a mouse line in which expression of one or both endogenous MnSOD alleles is activated by doxycycline (DOX) administration. Homozygous (tet/tet) mice have very low levels of MnSOD in the absence of DOX. Both tet/tet and tet/+ mice show an increase in MnSOD expression in the presence of DOX. Materials/Methods: Bone marrow stromal cell lines derived from MnSOD tet/tet, tet/+ and +/+ mice were compared with those from unconditional knockout, embryonic-lethal (MnSOD -/-) mouse embryonic fibroblasts and control C57BL/6NHsd mice using irradiation survival curves, western blots, MnSOD biochemical activity, and gene expression using Real Time Polymerase Chain Reaction (RT-PCR). Results: In the absence of DOX (0.25mg/ml), the MnSOD tet/tet cells showed decreased MnSOD mRNA by RT-PCR, decreased protein by Western analysis, decreased MnSOD biochemical activity and increased radiosensitivity. To determine if MnSOD level altered gene expression after irradiation, MnSOD tet/tet and MnSOD+/+ cells were incubated in the presence or absence of 0.25 mg/ml DOX for 24 hr and then irradiated to either 0 or 10 Gy, and RNA was extracted at 0, 1, or 6 hr after irradiation. RT-PCR quantitated gene expression for MnSOD, NFK-b, Nrf2 and TGFb (genes responsive to irradiation) was performed. At 1 and 6 hr after irradiation, MnSOD+/+ cells showed a twofold increased expression of NRK-b and a 2 to 3 fold increased expression of MnSOD and TGF-b, independent of DOX. Nrf2 expression increased by 50% at 1 hr and twofold by 6 hr after irradiation, all independent of DOX. In marked contrast, irradiation of MnSOD tet/tet cells without DOX showed no transcript induction, while irradiated cells in DOX showed a twofold irradiation induced increase in MnSOD gene expression. The MnSOD tet/ tet cells, even in DOX, showed persistent 50% reduction in expression at 1
Volume 84 Number 3S Supplement 2012 Materials/Methods: We determined the minimal nucleotide recognition element (RE) of the prototypical IMP protein, IMP1, using gel mobility shift assays, in-vitro selection, and NMR spectroscopy. We identified conserved transcripts containing the IMP1 RE by Sequel query of the UCSC 3´UTR database (hg19/mm8); we further performed immunoprecipitation and qRT-PCR detection of IMP1 associated mRNAs from mouse embryonic cells. Ingenuity pathway analysis v9.0 was used to explore functional relationships between proteins encoded by the mRNA ligands of IMP1; P values are based on a right-tailed Fisher’s exact test. Results: The third and fourth hnRNP K homology domains of IMP1 specifically recognize a bipartite RE: a 5´ element, CGGAC, followed by a variable 3´ element, C/A-CA-C/U. The IMP1 consensus REs are evolutionarily conserved in the 3´UTRs of 114 transcripts. Approximately 70% of these transcripts (17 of 25 tested) are enriched over fourfold in IMP1 immunoprecipitates. Gene ontology analysis of predicted IMP1 ligands reveals enrichment of gene-networks controlling cell survival (n Z 33, p < 0.0001) and differentiation (n Z 28, p < 0.005). Aberrant transcription of IMP1 RNA ligands (n Z 13) has been demonstrated in neoplastic conditions including ovarian cancer, papillary thyroid carcinoma, NSCLC, DLBCL, and melanoma (p < 0.0050.01, Table).
IMP1 ligand
P value
CAMK2N2, COL4A1, CTDSPL, DTNA, EFNB2, ESR1, FOSB, TIMP1 EFNB2, NTRK2, TIMP1 HES1, MAFB, TIMP1
0.004
checkpoints at least partially via the cyclin-dependent kinase inhibitor p21, we have utilized p21+/- and p21-/- mice to investigate how p21 regulates RIMI in vivo. Materials/Methods: p21-/- and p21+/- mice were exposed to WHI using a small-field micro-irradiator. Serial transthoracic echocardiography was performed on the mice with a high-resolution image system to measure changes in ventricular mass and cardiac function. Histopathological examination was performed to assess the size of cardiomyocytes, microvessel density and vascular permeability in the myocardium. Primary CECs were isolated from the ventricles of the heart and irradiated with ionizing radiation in vitro at 0, 3, 6, 12 and 24 Gy. Cell death after 96 hours was determined with propidium iodide exclusion and a cleaved caspase-3 assay. DNA damage in mitosis was measured by phospho-histone H3 (pHH3) and gH2AX double staining. Results: After 12 Gy WHI, p21-/- mice developed myocardial injury characterized by myocardial necrosis, systolic dysfunction, and cardiac hypertrophy. In addition, p21-/- mice showed a reduction in microvessel density and increased vascular permeability prior to the onset of cardiac dysfunction. Moreover, studies using primary CECs from p21+/- and p21-/mice irradiated in vitro indicate that p21 protects CECs from post-mitotic cell death after 12 Gy. Conclusion: Collectively, our results demonstrate that the p53/p21 axis functions to prevent radiation-induced myocardial injury in mice. Acknowledgment: This project was supported by ASTRO: Minority Summer Fellowship Award (to IW) and R01 AI080488 (to DGK). Author Disclosure: I. Washington: None. C. Lee: None. E.J. Moding: None. Y. Li: None. L. Mao: None. Y. Kim: None. H.A. Rockman: None. D.G. Kirsch: None.
0.006 0.009
3211
BRD2
0.006
BRSK2
0.01
Poster Viewing Abstract 3209; Table Aberrant Transcription of IMP1 RNA Ligands in Neoplastic Conditions where IMP1 is Overexpressed Cancer type Ovarian carcinoma
Papillary thyroid carcinoma Non-small cell lung carcinoma (NSCLC) Diffuse large B-cell lymphoma (DLBCL) Melanoma
Conclusion: Our results suggest altered post-transcriptional regulation of IMP1 mRNA ligands may facilitate tumor cell survival in neoplastic conditions where IMP1 is upregulated. Therefore, IMP1 may represent a potential therapeutic target for enhancing tumor sensitivity to IR. Author Disclosure: V. Patel: None. J.A. Chao: None. R.H. Singer: None. C. Guha: None.
3210 p21 Functions to Prevent Radiation-induced Myocardial Injury in Mice I. Washington,1 C. Lee,2 E.J. Moding,2 Y. Li,1 L. Mao,3 Y. Kim,4 H.A. Rockman,3 and D.G. Kirsch1; 1Department of Radiation Oncology, Duke University Medical Center, Durham, NC, 2Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 3Department of Medicine, Duke University Medical Center, Durham, NC, 4Laboratory of Veterinary Clinical Pathology, College of Veterinary Medicine, Seoul National University, Gwanak-ro, Gwanak-gu, Seoul, Korea, Republic of Korea Purpose/Objective(s): Radiation-related heart disease is an important cause of treatment-associated mortality for cancer patients after thoracic radiation therapy. Although it has been established that microvascular loss precedes myocardial necrosis in radiation-induced myocardial injury (RIMI), the molecular mechanisms controlling the loss of the myocardial capillaries following radiation therapy remain to be fully defined. Recently our lab has utilized the Cre-loxP system to specifically delete p53 in endothelial cells and demonstrated that p53 functions in cardiac endothelial cells (CECs) to prevent RIMI after 12 Gy whole-heart irradiation (WHI). Because p53 controls the G1/S and G2/M cell cycle
Irradiation Induction of mRNA for Redox-Sensitive Promoters and Inflammatory Cytokines in Conditional Manganese Superoxide Dismutase (MnSOD) Tet on -/- Mouse Bone Marrow Stromal Cells R. Kalash, J.R. Chaillet, F. Houghton, X. Zhang, M. Epperly, S. Cao, and J.S. Greenberger; University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Bioavailability of the mitochondrial targeted antioxidant enzyme MnSOD in normal tissues counteracts irradiation induced apoptosis. To determine the effect of MnSOD on gene expression, we developed a mouse line in which expression of one or both endogenous MnSOD alleles is activated by doxycycline (DOX) administration. Homozygous (tet/tet) mice have very low levels of MnSOD in the absence of DOX. Both tet/tet and tet/+ mice show an increase in MnSOD expression in the presence of DOX. Materials/Methods: Bone marrow stromal cell lines derived from MnSOD tet/tet, tet/+ and +/+ mice were compared with those from unconditional knockout, embryonic-lethal (MnSOD -/-) mouse embryonic fibroblasts and control C57BL/6NHsd mice using irradiation survival curves, western blots, MnSOD biochemical activity, and gene expression using Real Time Polymerase Chain Reaction (RT-PCR). Results: In the absence of DOX (0.25mg/ml), the MnSOD tet/tet cells showed decreased MnSOD mRNA by RT-PCR, decreased protein by Western analysis, decreased MnSOD biochemical activity and increased radiosensitivity. To determine if MnSOD level altered gene expression after irradiation, MnSOD tet/tet and MnSOD+/+ cells were incubated in the presence or absence of 0.25 mg/ml DOX for 24 hr and then irradiated to either 0 or 10 Gy, and RNA was extracted at 0, 1, or 6 hr after irradiation. RT-PCR quantitated gene expression for MnSOD, NFK-b, Nrf2 and TGFb (genes responsive to irradiation) was performed. At 1 and 6 hr after irradiation, MnSOD+/+ cells showed a twofold increased expression of NRK-b and a 2 to 3 fold increased expression of MnSOD and TGF-b, independent of DOX. Nrf2 expression increased by 50% at 1 hr and twofold by 6 hr after irradiation, all independent of DOX. In marked contrast, irradiation of MnSOD tet/tet cells without DOX showed no transcript induction, while irradiated cells in DOX showed a twofold irradiation induced increase in MnSOD gene expression. The MnSOD tet/ tet cells, even in DOX, showed persistent 50% reduction in expression at 1