Forced cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells - PubMed (original) (raw)
Forced cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells
Santanu Dasgupta et al. Int J Cancer. 2009.
Abstract
Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The proapoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an antiapoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.
Figures
Figure 1
Number and volume of mitochondria in mtCYTB transfected SV-HUC-1 cells. a) Mitochondrial concentration was significantly higher in mtCYTB compared to empty vector and wtCYTB cells (P<.001) as determined by Mitotracker red dye. Scale bar: 100μm. b) the percent of cytoplasm occupied by mitochondria was similar in the all the groups, c) the number of mitochondria increased significantly (P<0.001) in the mtCYTB expressing cells compared to the controls, d) the mitochondrial volume decreased significantly (P<0.001) in the mtCYTB expressing cells compared to the controls. e) Representative photomicrograph showing an increased number of mitochondria in the mtCYTB group. Magnification X 6000. Representative example of 3 different clones analyzed has been shown. Scale bar: 2 micron.
Figure 2
Increased mtDNA content in mtCYTB transfected HUC-1 cells. a) MtDNA content measured as the ratio of mtDNA sequence to nDNA sequence was significantly higher (P<0.02) in the mtCYTB transfected HUC-1 cells compared to the wtCYTB and empty vector treated cells. Representative example of 3 different clones analyzed has been shown. b) Immunofluorescence analysis revealed marked increase in COX-I protein expression in mtCYTB- HUC-1 cells compared to empty vector and wtCYTB transfected cells. Magnification X 400. Scale bar: 100μm. Each experiment was repeated two times. c) Western blot analysis demonstrated similar level of expression of mitochondria encoded COX-I in all the experimental groups. Nuclear encoded COX-IV antibody was used as control.
Figure 3
Distribution of Bax protein into the cytoplasm of CYTB transfected cells. a) Immunofluorescence analysis by anti-Bax-FITC antibodies demonstrated localization of Bax protein into the cytoplasm in mtCYTB overexpressing HUC-1 cells (Arrows), where as Bax remained mostly in the mitochondria in empty vector and wtCYTB transfected cells. Magnification X 400. Scale bar: 100μm. b) Bax remained translocated in the cytoplasm of the CYTB transfected cells. Immunofluorescence analysis using a combination of Mitotracker red dye and anti-Bax-FITC antibodies detected considerable amount of Bax in the cytoplasm of mtCYTB transfected HUC-1 cells (Arrows). Magnification X 400. Representative example of 3 different clones analyzed has been shown. Scale bar: 100μm.
Figure 4
Distribution of Bax and Cytochrome C in the CYTB transfected cells. a) Western blot analysis showed a markedly higher expression of Bax protein in the mitochondria of the mtCYTB expressing HUC-1 cells compared to the control wt-CYTB transfected cells. b) Western blot and c) Immunofluorescence analysis showed the presence of intact Cytochrome C in the mitochondria of all the experimental groups. Magnification X 400. Scale bar: 100μm. Representative example of 3 different clones analyzed has been shown. Mitochondrial Complex-III- Core-II and ACTIN were used as loading controls for mitochondria and cytosol respectively.
Figure 5
Expression of PARP and Lamin B1 in the CYTB transfected cells. Western blot analysis showed uncleaved PARP in all the experimental groups (Panel 1) and a considerable increase in Lamin B1 in the mtCYTB transfected HUC-1 cells. ACTIN was used as normal loading control.
Figure 6
Apoptotic resistance of the CYTB transfected cells. a) The mtCYTB cells were significantly resistant to killing by Staurosporine compared to wtCYTB and empty vector treated cells (P<.0001). b) Hydrogen peroxide treated mtCYTB cells were also significantly resistance (P<0.0001) compared to wtCYTB and empty vector treated cells (P<.02). *P <0.05 versus Control. Representative example of 3 different clones analyzed has been shown.
Figure 7
Translocation of Bax to the mitochondria in mtCYTB transfected HUC-1 cells. The mtCYTB transfected HUC-1 cells were treated with NFκB activation inhibitor 6-amino-4 (4phenoxyphenylethylamino) for 6 hr. Western blot analysis revealed a marked increase of the Bax protein expression in the mitochondria and Cytochrome C in the cytoplasm (Fig. 7). The expression of Bax decreased considerably in the cytosol of the 6-amino-4 (4 phenoxyphenylethylamino) treated _mtCYTB_-HUC-1 cells. Mitochondrial Complex-III- Core-II and ACTIN were used as loading controls for mitochondria and cytosol respectively.
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