FIG. 4.

Activity of p53 mutated at T81. (a) p53 null cells were transfected with p21^WAF1/CIP1-Luc, a constitutively active form of MEKK1 or JNKK2 and wt or mutant forms of p53. (Inset) Expression levels of the p53 forms in extracts used for transcriptional analysis. Basal transcriptional activities of wt p53 were five times higher than those seen with either the T81A mutant or empty vector. The data shown reflect fold increases over basal activity of the construct. C, control; M, ΔMEKK1; J, JNKK^CAA. (b) Comparison of T81A transcriptional activities with those of different transcriptionally dead p53 mutants. Mouse 10.1 p53 null cells were transfected with p21^WAF1/CIP1-Luc and wt or mutant forms of p53. Cells were treated with UV (15 J/m²) 36 h later, and proteins were prepared after an additional 6 h. Analysis was carried out as indicated in panel a. Bottom, Western blots of proteins prepared from the same experiment using antibodies to p53 and p21^WAF1/CIP. (c) p53 vectors and GFP were transfected into 10.1 cells, and 24 h later cells were treated with Taxol or nocodazole (5 μg/ml). PI staining and flow cytometry analyses of GFP⁺ cells were performed 16 h after treatment as detailed in Materials and Methods. (d) H1299 cells were transfected with the constructs and 24 h later were treated with anisomycin (10 μg/ml) or UV-C irradiation (15 J/m²). Analysis of cell death was performed after an additional 36 h. The fraction of dead cells shown includes annexin-V- and PI-positive cells, thereby including apoptotic and necrotic death. (e) 3T3 mouse fibroblasts were transfected with P7 (corresponding to the JNK binding site on p53) or C7 (P7 sequence in scrambled order) cloned into pcDNA3_HA. UV irradiation was administered 24 h later. The degree of apoptosis was calculated 24 or 72 h after irradiation based on fluorescence-activated cell sorter analysis of PI-positive cells. The inset depicts immunocytochemistry analysis of P7 expression.