Figure 5.

The p73-mediated induction of bak-promoter driven reporter gene expression, DNA binding, and repression by Δ133p53, DNp73, and p73 knockdown. (a) The HCT116/p53^−/− cells were co-transfected with the indicated reporter gene plasmids and a p73α expression vector. After 48 h cells were collected and luciferase activity was assessed. Values are expressed as fold induction normalized to the activity induced from pGL3-Basic alone. Reporter gene induction was reduced by the single mutation of each of the putative p53-binding sites, whereas mutation of all three binding sites had an additive effect. (b) The HCT116/p53^−/− cells were treated with 50 μM etoposide for 48 h and EMSAs were performed on nuclear extracts using ³²P-labeled ds-oligonucleotides for the p53 consensus sequence (p53con), BS1, BS2, BS3, and mutated BS3 (mutBS3). Addition of a p73 antibody produces a supershift (^*) of p53con, BS1, BS2, and BS3, thus indicating binding of p73α to these oligonucleotides. (c) The HeLa and HCT116/p53^−/− cells were co-transfected with p73 and Δ133p53 or DNp73 together with the bak promoter reporter construct. After 48 h, cells were collected and luciferase activity detected luminometrically. Induction of luciferase activity by p73 was reduced to basal level in cells co-transfected with the DNp73 expression plasmid, whereas no reduction was seen in Δ133p53 co-expressing cells. (d) The HeLa and HCT116/p53^−/− cells were co-transfected with p53 and Δ133p53 or DNp73 together with the bak promoter reporter construct. Induction of luciferase activity by p53 was measured at 48 h after transfection and reduced in both Δ133p53 and DNp73 co-expressing cells. (e) The HCT116/p53^−/− cells were transduced with 50 MOI of Ad-p73α and at 72 h, the samples were analyzed for Bak mRNA expression by qRT-PCR. The Bak mRNA expression was upregulated by 2.5-fold in Ad-p73 transduced cells compared with control cells. (f) The HCT116/wt and HCT116/p53^−/− cells were transfected with a vector for the expression of p73-shRNA and incubated in the absence or presence of 50 μM etoposide for 72 h. The qRT-PCR revealed inhibition of etoposide-induced Bak mRNA expression by the p73-specific shRNA as compared with the control in both wild-type and p53-deficient HCT116 cells