Increased apoptosis induction by 121F mutant p53

Abstract

p53 mutants in tumours have a reduced affinity for DNA and a reduced ability to induce apoptosis. We describe a mutant with the opposite phenotype, an increased affinity for some p53-binding sites and an increased ability to induce apoptosis. The apoptotic function requires transcription activation by p53. The mutant has an altered sequence specificity and selectively fails to activate MDM2 transcription. Loss of MDM2 feedback results in overexpression of the mutant, but the mutant kills better than wild-type p53 even in MDM2-null cells. Thus the apoptotic phenotype is due to a combination of decreased MDM2 feedback control and increased or unbalanced expression of other apoptosis-inducing p53 target genes. To identify these genes, DNA chips were screened using RNA from cells expressing the apoptosis-inducing mutant, 121F, and a sequence-specificity mutant with the reciprocal phenotype, 277R. Two potential new mediators of p53-dependent apoptosis were identified, Rad and PIR121, which are induced better by 121F than wild-type p53 and not induced by 277R. The 121F mutant kills untransformed MDM2-null but not wild-type mouse embryo fibroblasts and kills tumour cells irrespective of p53 status. It may thus expand the range of tumours which can be treated by p53 gene therapy.

TAT-RasGAP317-326 Requires p53 and PUMA to Sensitize Tumor Cells to Genotoxins

“…Figure 2B shows that the parental HCT116 cells were efficiently sensitized by the TAT-RasGAP 317-326 peptide to cisplatin-induced death but that the variant lacking p53 remained unaffected by the peptide. To confirm these results, we used the H1299 lung carcinoma cell line in which p53 can be repressed by tetracycline (20). As shown in Fig.…”

“…We next used p53 proteins bearing various alanine substitutions at sites known to be phosphorylated following DNA damage (27), including some (e.g., Ser 15 and Ser 20 ) that have been shown in vivo to play important role in p53-induced apoptosis and tumor suppression (28). Cells expressing p53 mutants with one or two of these point mutations (at position 33-37, 15, and 20) were less sensitive to cisplatin than wildtype p53-expressing cells (Fig.…”

“…HCT116 p53 +/+ , HCT116 p53 À/À , HCT116 PUMA À/À , and HCT116 p21 À/À have been described earlier (41,42) and were cultured as described above. The H1299 cell line encoding a p53 construct under the control of a tetracycline-repressible promoter (Tet-off system) was described earlier (20). Experiments using cells were done in 2 mL in six-well plates containing 1.5 Â 10 5 cells per well for U2OS, SAOS, and H1299 cells or 4 Â 10 5 cells per well for HCT116 cells.…”

CYFIP2, a Direct p53 Target, is Leptomycin-B Sensitive

“…These results are consistent with those from previously published microarray studies. 11,24,25 Saller et al first identified CYFIP2 (also known as PIR121) as a candidate p53 target gene whose mRNA was up regulated 5.3X by wild-type p53, 15.3X by a pro-apoptotic p53-121F mutant, and not induced in response to an anti-apoptotic p53-277R mutant, respectively, when stably expressed under TET control in H1299 cells or adenovirally expressed in Saos-2 (null for p53) or U2OS (expressing WT p53) cells. Using the nucleotide analog 5-fluorouracil, a DNA damage inducing chemotherapeutic drug, Kho et al detected a 2X increase in CYFIP2 mRNA by microarray in HCT116 cells in a p53-dependent manner, suggesting that CYFIP2 can be induced by endogenous p53.…”

“…5 The retrieved cDNA band from DD was subcloned into the pCR-TRAP vector and utilized for sequencing and Northern blotting as previously described. 5 The pRD1-215 plasmid, obtained from R. Iggo, 11 containing the full-length CYFIP2/PIR121 cDNA was used as a probe for the CYFIP2 Northern blot.…”