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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.

SUBMITTER: Saller E

PROVIDER: S-EPMC1171517 | biostudies-other | 1999 Aug

REPOSITORIES: biostudies-other

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Increased apoptosis induction by 121F mutant p53.

Saller E E Tom E E Brunori M M Otter M M Estreicher A A Mack D H DH Iggo R R

The EMBO journal 19990801 16

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 ...[more]

PMID: 10449408

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Project description:Prostaglandin (PG) A2, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA2 has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical pro-apoptotic genes, on PGA2-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA2 was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA2-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA2 induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-? (PFT-?), a chemical inhibitor of p53's transcriptional activity, interfered with the induction of apoptosis in PGA2-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA2-induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA2-induced apoptosis. In the meanwhile, in HCT116 p53-/- cells, PGA2 induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA2 in HCT116 p53-/- cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT-? or NU7441. Collectively, these results suggest the following. PGA2 may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA2 may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/- cells.

Project description:Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-? (an inhibitor of p53 transcriptional activity), pifithrin-? (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL specific pathway.

Project description:The tumor suppressor protein p53 induces apoptosis, cell cycle arrest, and DNA repair along with other functions in a transcription-dependent manner [Vousden, K. H. Cell 2000, 103(5), 691-694]. The selection of these functions depends on sequence-specific recognition of p53 to a target decameric sequence of gene promoters [Kitayner, M.; et al. Mol. Cell 2006, 22(6), 741-753]. Amino acid residues in p53 that directly bind to DNA were analyzed, and the replacement of A276 in p53 with selected amino acids elucidated its importance in promoter transcription. For most apoptotic and cell cycle gene promoters, position 9 of the target decameric sequence is a cytosine, while for DNA repair gene promoters, thymine is found instead. Therefore, selective binding to the cytosine at the ninth position may transcribe apoptotic gene promoters and thus can induce apoptosis and cell cycle arrest. Molecular modeling with PyMOL indicated that substitution of a hydrophilic residue, A276S, would prefer binding to cytosine at the ninth position of the target decameric sequence, whereas substitution of a hydrophobic residue (A276F) would fail to do so. Correspondingly, A276S demonstrated higher transcription of PUMA, PERP, and p21(WAF1/CIP1)gene promoters containing a cytosine at the ninth position and lower transcription of GADD45 gene promoter containing a thymine at the ninth position compared to wild-type p53. Cell cycle analysis showed that A276S maintained similar G1/G0 phase arrest as wild-type p53. Additionally, A276S induced higher apoptosis than wild-type p53 as measured by DNA segmentation and 7-AAD assay. Since the status of endogenous p53 can influence the activity of the exogenous p53, we examined the activity of A276S in HeLa cells (wild-type endogenous p53) in addition to T47D cells (mutated and mislocalized endogenous p53). The same apoptotic trend in both cell lines suggested A276S can induce cell death regardless of endogenous p53 status. Cell proliferation assay depicted that A276S efficiently reduced the viability of T47D cells more than wild-type p53 over time. We conclude that the predicted preferred binding of A276S to cytosine at the ninth position better transactivates a number of apoptotic gene promoters. Higher induction apoptosis than wild-type p53 makes A276S an attractive candidate for therapy to eradicate cancer.

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Increased apoptosis induction by 121F mutant p53.

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Increased apoptosis induction by 121F mutant p53.

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