Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.

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Abstract 268: Transforming Growth Factor-β1 Increases Resistance of Fibroblasts to Apoptotic Cell Death

Circulation Research ◽

10.1161/res.115.suppl_1.268 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Ulugbek Negmadjanov ◽

Zarko Godic ◽

Mahek Mirza ◽

Larisa Emelyanova ◽

Farhan Rizvi ◽

...

Keyword(s):

Heart Failure ◽

Cell Death ◽

Caspase 3 ◽

Fas Ligand ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

3T3 Cells ◽

Nih 3T3 ◽

Tgf Β1 ◽

Nih 3T3 Cells

Introduction: Cardiac injury results in the death of cardiac myocytes and subsequent scar formation through extracellular matrix (ECM) deposition by fibroblasts (FB) and myofibroblasts (myoFB). Excessive fibrosis results in pathological scarring that predisposes to arrhythmogenesis and heart failure, particularly in the elderly. Strategies to limit adverse ECM remodeling are urgently needed to curtail the growing epidemic of atrial fibrillation and heart failure in the aging population. Persistence of myoFB and resistance to apoptotic cell death has been proposed to underlie the mechanism of excessive fibrosis, yet is not fully characterized. Methods: Cultured NIH/3T3 cells (control and TGF-β1 treated) have been challenged with activators of extrinsic (FAS-Ligand, 1 μg/mL) or intrinsic (Thapsigargin 10 μM and Staurosporine 5 μM) apoptotic pathways and Caspase-3 activity was measured in cellular lysate. Results: FAS-L exposure induced ~40-fold suppression of Caspase-3 activity in TGF-β1 treated cells as compared with control (17±12 vs 686±5 nmol AMC/min/106 cells, respectively). Similarly, Staurosporine activated Caspase-3 in TGF-β1 treated cells ~3-fold (171±38 vs 536±29 nmol AMC/min/106 cells), and Thapsigargin ~10-fold (73±33 vs 742±8 nmol AMC/min/106 cells). Conclusion: TGF-β1 treatment increased the sensitivity of NIH/3T3 cells toward extrinsic and intrinsic apoptotic stimuli. Although, TGF-β1 treatment increased overall resistance of NIH/3T3 cells to apoptosis, the responsiveness of cells to extrinsic vs intrinsic pathways was differentially affected. This data support the hypothesis that persistence of myoFB results in pathological scarring.

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Cytotoxicity, Antiproliferative Effects, and Apoptosis Induction of Methanolic Extract ofCynometra caulifloraLinn. Whole Fruit on Human Promyelocytic Leukemia HL-60 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2012/127373 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 5

Author(s):

T-Johari S. A. Tajudin ◽

Nashriyah Mat ◽

Abu Bakar Siti-Aishah ◽

A. Aziz M. Yusran ◽

Afnani Alwi ◽

...

Keyword(s):

Cell Death ◽

Methanolic Extract ◽

Apoptotic Cell ◽

Mouse Fibroblast ◽

Promyelocytic Leukemia ◽

Apoptotic Cell Death ◽

Apoptotic Cells ◽

3T3 Cells ◽

Nih 3T3 ◽

Nih 3T3 Cells

Methanolic extract ofCynometra cauliflorawhole fruit was assayed for cytotoxicity against the human promyelocytic leukemia HL-60 and the normal mouse fibroblast NIH/3T3 cell lines by using the MTT assay. The CD50of the extract for 72 hours was 0.9 μg/mL whereas the value for the cytotoxic drug vincristine was 0.2 μg/mL. The viability of the NIH/3T3 cells was at 80.0% when treated at 15.0 μg/mL. The extract inhibited HL-60 cell proliferation with dose dependence. AO/PI staining of HL-60 cells treated with the extract revealed that majority of cells were in the apoptotic cell death mode. Flow cytometry analysis of HL-60 cells treated at CD50of the extract showed that the early apoptotic cells were 31.0, 26.3 and 19.9% at 24, 48, and 72 hours treatment, respectively. The percentage of late apoptotic cells was increased from 62.0 at 24 hours to 64.1 and 70.2 at 48 and 72 hours, respectively. Meanwhile, percent of necrotic cells were 4.9, 6.6, and 8.5 at 24, 48, and 72 hours, respectively. This study has shown that the methanolic extract ofC. cauliflorawhole fruit was cytotoxic towards HL-60 cells and induced the cells into apoptotic cell death mode, but less cytotoxic towards NIH/3T3 cells.

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Caveola-Dependent Endocytic Entry of Amphotropic Murine Leukemia Virus

Journal of Virology ◽

10.1128/jvi.79.16.10776-10787.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10776-10787 ◽

Cited By ~ 57

Author(s):

Christiane Beer ◽

Ditte S. Andersen ◽

Aleksandra Rojek ◽

Lene Pedersen

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

3T3 Cells ◽

Coated Pits ◽

Caveolin 1 ◽

Amphotropic Murine Leukemia Virus ◽

Triton X ◽

Nih 3T3 ◽

Murine Leukemia ◽

Nih 3T3 Cells

ABSTRACT Early results suggested that the amphotropic murine leukemia virus (A-MLV) does not enter cells via endocytosis through clathrin-coated pits and this gammaretrovirus has therefore been anticipated to fuse directly with the plasma membrane. However, here we present data implicating a caveola-mediated endocytic entry route for A-MLV via its receptor Pit2. Caveolae belong to the cholesterol-rich microdomains characterized by resistance to nonionic detergents such as Triton X-100. Extraction of murine fibroblastic NIH 3T3 cells in cold Triton X-100 showed the presence of the A-MLV receptor Pit2 in detergent-insoluble microdomains. Using coimmunoprecipitation of cell extracts, we were able to demonstrate direct association of Pit2 with caveolin-1, the structural protein of caveolae. Other investigations revealed that A-MLV infection in contrast to vesicular stomatitis virus infection is a slow process (t ≈5 h), which is dependent on plasma membrane cholesterol but independent of NH4Cl treatment of cells; NH4Cl impairs entry via clathrin-coated pits. Furthermore, expression of dominant-negative caveolin-1 decreased the susceptibility to infection via Pit2 by approximately 70%. These results show that A-MLV can enter cells via a caveola-dependent entry route. Moreover, increase in A-MLV infection by treatment with okadaic acid as well as entry of fusion-defective fluorescent A-MLV virions in NIH 3T3 cells further confirmed our findings and show that A-MLV can enter mouse fibroblasts via an endocytic entry route involving caveolae. Finally, we also found colocalization of fusion-defective fluorescent A-MLV virions with caveolin-1 in NIH 3T3 cells. This is the first time substantial evidence has been presented implicating the existence of a caveola-dependent endocytic entry pathway for a retrovirus.

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