Cellular redox imbalance and changes of protein S-glutathionylation patterns are associated with senescence induced by oncogenic H-ras

Abstract

H-Ras oncogene requires deregulation of additional oncogenes or inactivation of tumor suppressor proteins to increase cell proliferation rate and transform cells. In fact, the expression of the constitutively activated H-RasV12 induces cell growth arrest and premature senescence, which act like barriers in pre-neoplastic lesions. In our experimental model, human fibroblasts transfected with H-RasV12 show a dramatic modification of morphology. H-RasV12 expressing cells also show premature senescence followed by cell death, induced by autophagy and apoptosis. In this context, we provide evidence that in H-RasV12 expressing cells, the premature senescence is associated with cellular redox imbalance as well as with altered post-translation protein modification. In particular, redox imbalance is due to a strong reduction of total antioxidant capacity, and significant decrease of glutathione level. As the reversible addition of glutathione to cysteinyl residues of proteins is an important post-translational regulative modification, we investigated S-glutathionylation in cells expressing active H-Ras. In this contest we observed different S-glutathionylation patterns in control and H-RasV12 expressing cells. Particularly, the GAPDH enzyme showed S-glutathionylation increase and significant enzyme activity depletion in H-Ras V12 cells. In conclusion, we proposed that antioxidant defense reduction, glutathione depletion and subsequent modification of S-glutathionylation of target proteins contribute to arrest cell growth, leading to death of fibroblasts expressing constitutively active H-Ras oncogene, thus acting as oncogenic barriers that obstacle the progression of cell transformation.

Figure 1. Analysis of cell growth and cell cycle.

A) Immunoblotting analysis of H-RasV12 transfected cells. Cell extracts were incubated with an anti-H-Ras antibody. As internal control an anti-β actin antibody was used. The increased expression of H-Ras in transfected cells compared to empty vector transfected cells (control) is shown. B) Growth curve of HuDe fibroblasts transfected with empty vector and H-RasV12. The cell number increase after the end of the selection with blasticidin-S (4 µg/ml) is reported. Mean values were calculated on 5 replicates and mean ± s.d. was indicated for each sample. C) Cell morphology analysis by light microscopy, 200× total magnification. D) Flow cytometry cell cycle analysis of transfected cells using PI (40 µg/ml) as probe. Percentage of fluorescent cells for sub G0, G0/G1, S and G2/M phases were reported in the bottom panel. Mean ± s.e.m. of three analyses is reported. E) Cell proliferation measured by BrdU incorporation in newly synthesized DNA using immunostaining and flow citometry analysis. Control and H-RasV12 transfected cells were treated with 40 µM BrdU for 30 minutes and analyzed after 3, 6, 18 and 24 h. Cells without BrdU were used as negative control according to the manufacture’s instruction. Values of the BrdU positive cells are given as mean ± s.e.m. of three independent experiments subtracting the relative negative control. **P<0.01.

Figure 2. Effect of H-RasV12 on cell vitality.

A) Semi-thin sections of control and H-RasV12 cells obtained by light micrographs, 40× original magnification. Black arrows indicate the presence of vacuolization. B) Ultrastructural analysis of control (left photo) and H-RasV12 cells (right photo) obtained by transmission electron microscope (Philips; Endhoven, the Netherlands) at 100 kV. White arrows indicate macro-vacuolization and undigested material. C) Annexin-V FITC flow cytometry analysis for apoptosis. Control cells are indicated in grey, H-RasV12 transfected cells are indicated in black. In the table results are the mean ± s.e.m of percentage values for normal, apoptotic and necrotic cells in three independent experiments. D) Senescence-associated β-galactosidase staining of control and H-RasV12 cells. Percentages of SA-β-gal positive cells were counted at least on five different fields in three independent experiments. **P<0.01.

Figure 3. Analysis of cell redox state.

A) Measurement of ROS production. Carboxy-H2DCFDA (C400) probe was used to analyze ROS concentration in control and H-RasV12 transfected cells by flow cytometry. In the left panel % of ROS production was reported as fluorescence arbitrary units, obtained by subtraction of PI to carboxy-H2DCFDA. In the right panel cytometric graphs were reported for control cells (black), H-RasV12 cells (dark grey), in comparison with positive control cells treated with 250 µM H₂O₂ (light grey). B) Total antioxidant capacity for control and H-RasV12 transfected cells. Results reported are the mean values ± s.d. of three different experiments. C) Quantification of thiol groups which are present in total protein fraction. *P<0.05; **P<0.01.

Figure 4. Measurement of glutathione concentration, glutathione dependent enzymes and GAPDH.

Total cellular GSH (A) and GSSG (B) were determined for control and H-RasV12 cells by DTNB-GSSG reductase recycling assay. The amount of GSH or GSSG was normalized for proteins content. Values are the mean ± s.d. of six different experiments. Enzyme activity of C) glutathione reductase, D) glutathione-S-transferase and E) glyceraldehyde 3-phosphate dehydrogenase was determined spectrophotometrically at 340 nm. Results reported are mean values ± s.d. of six different experiments. *P<0.05; **P<0.01.

Figure 5. Analysis of protein S-glutathionylation.

A) Immunoblotting with anti-GSH antibody of control (lanes 1, 3) and H-RasV12 expressing cells (lanes 2, 4). Proteins (30 µg) were electrophoresed on 10% acrylamide/bis-acrylamide gel. Cell extracts were obtained either in non-reducing (lanes 1, 2) or in reducing (lanes 3, 4) conditions, by means of 25 mM DTT addition prior to loading. An anti-β actin antibody was used as internal control. B) Densitometric analysis of total bands intensity of immunoblotting was reported. No statistical difference of total glutathionylated proteins were observed in H-RasV12 expressing cells in comparison with the control cells C) Dot blot analysis was reported. 3µg of protein extract were serially diluted 1∶2 till 1∶256, spotted into a nitrocellulose membrane and immunoblotted with anti GSH antibody. D) Densitometric analysis of dot blot showed no statistical difference in H-RasV12 expressing cells in comparison with the control cells.

Figure 6. Evaluation of protein S-glutathionylation patterns.

Evaluation of protein S-glutathionylation patterns was performed by 2D-immunoblotting with anti-GSH antibody: A) control cells. B) H-RasV12 expressing cells. 100 µg of total cell extracts were separated on non- linear pH 3–10 strips (7 cm) for first dimension and then loaded on 10% acrylamide/bis-acrylamide gels for second dimension. Gels were blotted on PVDF membrane. Black rounds highlight protein spots which present a different level of S-glutathionylation. C) Total loading control. A total of 20 µg of cellular extract proteins used for 2D-immunoblotting was loaded on 10% acrylamide/bis-acrylamide gel and stained with Coomassie Blu.

Figure 7. Effect of antioxidant mix on H-RasV12 cell population.

Growth curve of HuDe fibroblasts transfected with H-RasV12 treated with 0.5 mM GSH ethyl ester, 50 µM α-tocopherol and 1.5 mM vitamin C. The cell number increase after the end of selection with Blasticidin-S is reported and results were compared with growth curve obtained for control and H-RasV12 transfected cells. Mean values were calculated on 5 replicates and mean ± s.d. was indicated for each sample. *P<0.05.