Figure 3.

The p53 pathway modulates CD46 expression and sensitivity to MV. Cells were treated for 24 hours with 5 or 10 μM nutlin3a (in TP53^wt and TP53^abn cell lines, respectively). (A-B) DR5 (A) and CD46 (B) staining was performed at 24 hours in 10 TP53^wt and 5 TP53^abn cell lines. DR5 and CD46 expression was assessed by flow cytometry and expressed as the MFIR. Results are expressed as the fold changes of MFIR (nutlin3a-treated cells over control cells). Histograms represent the expression of DR5, CD46, and GFP after treatment with nutlin3a in the TP53^wt BCN and TP53^abn LP1 cell lines. Thin line, control staining; thick line, specific staining. r, MFIR. Statistical analyses were performed using the Mann-Whitney U test. (C) After 24 hours of nutlin3a treatment, cells (5 TP53^wt and 4 TP53^abn cell lines) were washed and incubated with MV-GFP (MOI = 1). The percentage of GFP-positive cells was assessed 2 days later, and results are expressed as the fold changes of the percentage of GFP-positive cells in nutlin3a-treated over control (cont) cells. The histograms represent the expression of GFP after treatment with nutlin3a (duplicate wells are shown) in the TP53^wt BCN and TP53^abn LP1 cell lines (MOI = 2 or MOI = 0.5, respectively). Thin line, GFP in noninfected cells; thick line, GFP in MV-GFP infected cells. Statistical analyses were performed using the Mann-Whitney U test. (D) CD46 and TNFRSF10B (DR5) mRNA expression in nutlin3a-treated HMCLs. Cells were treated for 24 hours with 5 or 10 μM nutlin3a (in TP53^wt and TP53^abn cell lines, respectively). mRNA expression was assessed using quantitative RT-PCR and results are expressed as the fold change of treated over control cells. Each point represents the mean of 2 experiments performed in triplicate wells in each cell line. Statistical analyses were performed using the Mann-Whitney U test. (E) Western blotting analysis of p53 expression in TP53-silenced BCN cells. p53 expression and p53 pathway activation were assessed with and without nutlin3a (24 hours, 5 μM). (F) TP53 silencing induced a decrease in DR5 expression and an increase in CD46 expression. BCN cells were transfected with siControl or siTP53, and expression of receptors was assessed by flow cytometry after 24 hours. Statistical analyses were performed using the paired Student t test. (G) TP53 silencing increased MV-GFP infection in TP53^wt BCN cells. BCN cells were transfected with siControl or siTP53 for 2 days. For the last 24 hours, nutlin3a was added prior to washes and MV-GFP infection. GFP expression was assessed 24 hours later. The data represent 2 independent experiments. Statistical analyses were performed using the paired Wilcoxon matched-paired signed rank test. *P < .05. siCt, siControl mRNA.