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Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

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HCV infection induces apoptosis-like cell death in MIN6 cells.MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. (B) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. (C) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. (D) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). (A, B, D) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). **P<0.01, compared with respective controls.
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pone-0038522-g004: HCV infection induces apoptosis-like cell death in MIN6 cells.MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. (B) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. (C) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. (D) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). (A, B, D) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). **P<0.01, compared with respective controls.

Mentions: To further characterize HCV-induced cell death, transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. There were significantly fewer TUNEL-stained nuclei in the control groups, as compared to the groups incubated with 1.0 MOI HCV for 96 h, where nuclei fluoresced brightly, indicating fragmentation of DNA (Fig. 4A). The percentage of TUNEL-positive cells was significantly higher in HCV-infected MIN6 cells as compared to mock-infected cells (47.1+7.5% vs. 5.1+1.0%, P<0.01). It is important to note that, when the nuclei were stained with TUNEL, cells had intact cytoplasm and nuclear membranes.


Hepatitis C virus induced a novel apoptosis-like death of pancreatic beta cells through a caspase 3-dependent pathway.

Wang Q, Chen J, Wang Y, Han X, Chen X - PLoS ONE (2012)

HCV infection induces apoptosis-like cell death in MIN6 cells.MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. (B) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. (C) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. (D) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). (A, B, D) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). **P<0.01, compared with respective controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366942&req=5

pone-0038522-g004: HCV infection induces apoptosis-like cell death in MIN6 cells.MIN6 cells were mock-infected or infected with 1.0 MOI of HCV. (A) Confocal image of cells stained with TUNEL at 96 hpi. Scale bar, 10 µm. Quantitative summary of the TUNEL-positive staining is provided on the left. (B) Caspase 3 activity levels at 24 and 48 hpi. The caspase 3 activity of the control cells at 0 h after treatment was arbitrarily expressed as 1.0. (C) Immunoblot analysis of caspase 3 and PARP at 48, 72, 96 hpi. Cells treated with CHX (50 ng/ml) for 48 h were served as an apoptosis positive control. Immunoblots are representative of at least three independent experiments. Amounts of actin were measured as an internal control to verify equivalent sample loading. (D) Diagrams of FITC-Annexin V/PI flow cytometry in a representative experiment at 48 hpi. Numbers in the quadrants indicate the proportions of cells in the corresponding areas (left panel). (A, B, D) All measurements were done in triplicates. Data represent means + SD of three independent experiments (n = 9). **P<0.01, compared with respective controls.
Mentions: To further characterize HCV-induced cell death, transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. There were significantly fewer TUNEL-stained nuclei in the control groups, as compared to the groups incubated with 1.0 MOI HCV for 96 h, where nuclei fluoresced brightly, indicating fragmentation of DNA (Fig. 4A). The percentage of TUNEL-positive cells was significantly higher in HCV-infected MIN6 cells as compared to mock-infected cells (47.1+7.5% vs. 5.1+1.0%, P<0.01). It is important to note that, when the nuclei were stained with TUNEL, cells had intact cytoplasm and nuclear membranes.

Bottom Line: Pancreatic beta cell failure is central to the progression of type 2 diabetes.Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells.However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs.

View Article: PubMed Central - PubMed

Affiliation: State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.

ABSTRACT
Epidemiological and experimental studies have suggested that Hepatitis C virus (HCV) infection is associated with the development of type 2 diabetes. Pancreatic beta cell failure is central to the progression of type 2 diabetes. Using virus infection system, we investigate the influence of HCV infection on the fate of the insulinoma cell line, MIN6. Our experiments demonstrate that the HCV virion itself is indispensable and has a dose- and time-dependent cytopathic effect on the cells. HCV infection inhibits cell proliferation and induces death of MIN6 cells with apoptotic characteristics, including cell surface exposure of phosphatidylserine, decreased mitochondrial membrane potential, activation of caspase 3 and poly (ADP-ribose) polymerase, and DNA fragmentation in the nucleus. However, the fact that HCV-infected cells exhibit a dilated, low-density nucleus with intact plasma and nuclear membrane indicates that a novel apoptosis-like death occurs. HCV infection also causes endoplasmic reticulum (ER) stress. Further, HCV RNA replication was detected in MIN6 cells, although the infection efficiency is very low and no progeny virus particle generates. Taken together, our data suggest that HCV infection induces death of pancreatic beta cells through an ER stress-involved, caspase 3-dependent, special pathway.

Show MeSH
Related in: MedlinePlus