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The sensitivity of renal cell carcinoma cells to interferon alpha correlates with p53-induction and involves Bax

European Cytokine Network. Volume 16, Numéro 2, 123-7, June 2005, Research papers

Summary

Auteur(s) : S Wittnebel, A Jalil, J Thiery, S DaRocha, E Viey, B Escudier, S Chouaib, A Caignard , INSERM Unité 487, Cytokines et immunologie des tumeurs humaines, Institut Gustave-Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif Cedex, Unité des thérapies innovantes, Institut Gustave-Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif Cedex.

Illustrations

Figure 1 HLA class-I induction following IFN-α treatment in RCC.The renal cell carcinoma cell lines RCC5 and RCC7 were treated with IFN-α (750 U/mL). Control cells were grown in medium without IFN-α. After 48 hours, cells were harvested and labeled with anti-HLA-I (W6/32). Basal HLA-I expression on control cells is shown as solid profiles, HLA-I expression after IFN-α treatment is shown as an open profile. Isotypic controls are depicted by a thin open profile. MFI indicates mean fluorescence intensity in a logarithmic scale.

Figure 2 INF-α increases p53-expression in RCC7.p53 expression following the time course of IFN-α-treatment of RCC cell lines. RCC5 and RCC7 were treated with INF-α (750 U/mL) and p53-expression was probed by Western blot at the indicated time points. Top: Western blot. Bottom: quantitative display of the induction levels of p53 protein. Specific band intensities were measured using a densitometer and p53 induction was calculated as p53/ β-actin ratio.

Figure 3 INF-α enhances γ-irradiation-induced cell death in RCC7.Effect of IFN-α treatment on the sensitivity to γ-irradiation of RCC5 and RCC7. Cells were pretreated with INF-α (750 U/mL) and γ-irradiated (5Gy). Following additional incubation for 72h, floating and adherent cells were harvested and stained for cell-cycle analysis using PI staining. The percentage of dead cells was determined by the percentages of cells in sub-G1. A) cell cycle analysis. B) display of cells in sub-G1.

Figure 4 Bax/Bcl-2 ratio in RCC5 and RCC7.Expression of Bcl-2 and Bax in RCC5 and RCC7 after γ-irradiation (5Gy) alone or in combination with IFN-α (750 U/mL). Top: Western blot. Bottom: display of the Bax/Bcl-2 ratio. The quantities of both proteins were determined at the indicated time points using a densitometer.

Figure 5 Determination of sub-cellular Bax-location.Intracellular staining for Bax and cytochrome c in RCC5 and RCC7 after γ-irradiation (5Gy) alone or in combination with IFN-α (750 U/mL). Immunostaining was performed with anti-Bax mAb (green), anti-cytochrome c mAb (red) and TO-PRO-3 for the nucleus (blue). Note the enforced shift of Bax to the mitochondria, and an initial release of cytochrome c in RCC7 following pretreatment with IFN-α and γ-irradiation.