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Nuclear Translocation of Caspase 3 and Its Role in Potentiating Apoptosis
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A5722 - Nuclear Translocation of Caspase 3 and Its Role in Potentiating Apoptosis
Author Block: L. Johnston, R. Torres, K. Carino, T. Nicholson, K. Suresh, L. Servinsky, M. Damarla; Pulmonary and Critical Care, Johns Hopkins University, Baltimore, MD, United States.
Rationale: Endothelial cell apoptosis is a key pathogenic process in the development of pulmonary vascular permeability. As an executioner caspase, caspase 3 plays a key role in the terminal stages of apoptosis. Recent reports identified defective nuclear translocation of active caspase 3 as a potential mechanism of apoptosis resistance. We sought to determine if nuclear translocation of caspase 3 could potentiate apoptosis.
Methods: We generated stable cell lines by transfecting, wild type caspase 3 (casp 3), a nuclear localization sequence tagged caspase 3 (NLS-casp 3) and a nuclear export sequence tagged caspase 3 (NES-casp 3). The MCF-7 cell line (because of its lack of caspase 3) and the H23 cell line (because of defective nuclear translocation of caspase 3) were utilized. Additionally, primary mouse microvascular endothelial cells from wild type and caspase 3 knockout mice were transiently transfected with caspase 3 plasmids. Cells were exposed to etoposide, a topoisomerase inhibitor, for induction of apoptosis. After treatment, control and treated cells were either harvested for protein analysis, or fractionated into nuclear and cytoplasmic extracts. Cells were assessed for nuclear fragmentation as a marker of apoptosis.
Results: After etoposide treatment, activation of the apoptotic cascade was seen as evidenced by cleavage of caspase 3, in cells with caspase 3. Further, there was a decrease in total PARP1 and an increase in cleaved PARP1 in total cell lysates and nuclear extracts from the casp 3 and NLS-casp 3 expressing cell lines. DNA staining with Hoechst 33342 showed an increase in apoptosis in etoposide-treated cells, most significantly in the casp 3 and NLS-casp 3 expressing cell lines, the latter having the highest percent of increase.
Conclusions: Taken together our results suggest the nuclear translocation of caspase 3 plays an important role in the potentiation of apoptosis. Further, pathways that mediate nuclear translocation of caspase 3 may be a therapeutic target for the treatment of acute lung injury.
Author Block: L. Johnston, R. Torres, K. Carino, T. Nicholson, K. Suresh, L. Servinsky, M. Damarla; Pulmonary and Critical Care, Johns Hopkins University, Baltimore, MD, United States.
Rationale: Endothelial cell apoptosis is a key pathogenic process in the development of pulmonary vascular permeability. As an executioner caspase, caspase 3 plays a key role in the terminal stages of apoptosis. Recent reports identified defective nuclear translocation of active caspase 3 as a potential mechanism of apoptosis resistance. We sought to determine if nuclear translocation of caspase 3 could potentiate apoptosis.
Methods: We generated stable cell lines by transfecting, wild type caspase 3 (casp 3), a nuclear localization sequence tagged caspase 3 (NLS-casp 3) and a nuclear export sequence tagged caspase 3 (NES-casp 3). The MCF-7 cell line (because of its lack of caspase 3) and the H23 cell line (because of defective nuclear translocation of caspase 3) were utilized. Additionally, primary mouse microvascular endothelial cells from wild type and caspase 3 knockout mice were transiently transfected with caspase 3 plasmids. Cells were exposed to etoposide, a topoisomerase inhibitor, for induction of apoptosis. After treatment, control and treated cells were either harvested for protein analysis, or fractionated into nuclear and cytoplasmic extracts. Cells were assessed for nuclear fragmentation as a marker of apoptosis.
Results: After etoposide treatment, activation of the apoptotic cascade was seen as evidenced by cleavage of caspase 3, in cells with caspase 3. Further, there was a decrease in total PARP1 and an increase in cleaved PARP1 in total cell lysates and nuclear extracts from the casp 3 and NLS-casp 3 expressing cell lines. DNA staining with Hoechst 33342 showed an increase in apoptosis in etoposide-treated cells, most significantly in the casp 3 and NLS-casp 3 expressing cell lines, the latter having the highest percent of increase.
Conclusions: Taken together our results suggest the nuclear translocation of caspase 3 plays an important role in the potentiation of apoptosis. Further, pathways that mediate nuclear translocation of caspase 3 may be a therapeutic target for the treatment of acute lung injury.
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