Figure 3. Proteasome inhibition with bortezomib induces functional p53 protein in HPV-positive cells but not HPV-negative cells. Three HPV-negative HNSCC cell lines (A) and three HPV-positive HNSCC cell lines (B) were left untreated, treated for 24 h with 0.1% DMSO, or treated for 24 or 48 h with 50 nM (UM-SCC-22A, UM-SCC-1, 1483, UPCI:SCC090, UM-SCC-47) or 200 nM (UD-SCC-2) bortezomib. Following treatment, immunoblotting was used to detect expression of p53, p21 and β-actin. Ratios of p53/β-actin and p21/β-actin were determined by densitometric scanning. Similar results were obtained in three independent experiments. (C) Suppression of p53 expression/activity in bortezomib-treated HPV-positive cells using p53 siRNA. HPV-positive HNSCC cells were either left untransfected or were transfected with 100 nM nonspecific siRNA or p53 siRNA. After 24 h, untransfected cells were treated with 0.1% DMSO, 50 nM (UPCI:SCC090, UM-SCC-47) or 200 nM (UD-SCC-2) bortezomib, and transfected cells were treated with bortezomib (50 or 200 nM, as above). After 48 h of treatment, immunoblotting was used to detect p53, p21, HDM2 and β-actin. Bortezomib induction of p53 and the products of p53 target genes were inhibited by p53 siRNA, but not nonspecific siRNA. The experiment was performed three times with similar results.