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.
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