The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53␤ can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while ⌬133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.[Keywords: Splice; promoter; Drosophila; cancer; p73; p63] Supplemental material is available at http://www.genesdev.org.

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

We have previously reported that the human p53 gene encodes at least nine different p53 isoforms, including D133p53a, which can modulate p53 transcriptional activity and apoptosis. In this study, we aimed to investigate the regulation of D133p53a isoform expression and its physiological role in modulating cell cycle arrest and apoptosis. We report here that in response to a low dose of doxorubicin (which induces cell cycle arrest without promoting apoptosis), p53 directly transactivates the human p53 internal promoter, inducing D133p53a protein expression. The induced D133p53a then inhibits p53-dependent apoptosis and G1 arrest without inhibiting p53-dependent G2 arrest. Therefore, endogenous D133p53a does not exclusively function in a dominant-negative manner toward p53, but differentially regulates cell cycle arrest and apoptosis.

a b s t r a c t p53 gene expresses several protein isoforms modulating p53-mediated responses through regulation of gene expression. Here, we identify a novel p53 isoform, D160p53, lacking the first 159 residues. By knockdown experiments and site-directed mutagenesis, we show that D160p53 is encoded by D133p53 transcript using ATG160 as translational initiation site. This hypothesis is supported by endogenous expression of D160p53 in U2OS, T47D and K562 cells, the latter ones carrying a premature stop codon that impairs p53 and D133p53 protein expression but not the one of D160p53. Overall, these results show that the D133p53 transcript generates two different p53 isoforms, D133p53 and D160p53.

In addition to the tumor suppressor p53 protein, also termed p53a, the TP53 gene produces p53b and p53c through alternative splicing of exons 9b and 9c located within TP53 intron 9. Here we report that both TG003, a specific inhibitor of Cdc2-like kinases (Clk) that regulates the alternative splicing pre-mRNA pathway, and knockdown of SFRS1 increase expression of endogenous p53b and p53c at mRNA and protein levels. Development of a TP53 intron 9 minigene shows that TG003 treatment and knockdown of SFRS1 promote inclusion of TP53 exons 9b/9c. In a series of 85 primary breast tumors, a significant association was observed between expression of SFRS1 and a variant, supporting our experimental data. Using siRNA specifically targeting exons 9b/9c, we demonstrate that cell growth can be driven by modulating p53b and p53c expression in an opposite manner, depending on the cellular context. In MCF7 cells, p53b and p53c promote apoptosis, thus inhibiting cell growth. By transient transfection, we show that p53b enhanced p53a transcriptional activity on the p21 and Bax promoters, while p53c increased p53a transcriptional activity on the Bax promoter only. Moreover, p53b and p53c co-immunoprecipitate with p53a only in the presence of p53-responsive promoter. Interestingly, although p53b and p53c promote apoptosis in MCF7 cells, p53b and p53c maintain cell growth in response to TG003 in a p53a-dependent manner. The dual activities of p53b and p53c isoforms observed in non-treated and TG003-treated cells may result from the impact of TG003 on both expression and activities of p53 isoforms. Overall, our data suggest that p53b and p53c regulate cellular response to modulation of alternative splicing pre-mRNA pathway by a small drug inhibitor. The development of novel drugs targeting alternative splicing process could be used as a novel therapeutic approach in human cancers. including C-terminal isoforms produced by alternative splicing of intron 9 3 (Figure 1a). Exclusion of the entire intron 9 generates the canonical full-length p53 protein (or p53a), a transcription factor activated in response to diverse intracellular and extracellular signals (Figure 1b). Hence, p53 regulates gene expression to modulate cell-fate outcome by regulating biological processes, including apoptosis and cell cycle progression.5 Inclusion of alternative exons 9b and 9g contained in intron 9 gives rise to p53b and p53g protein isoforms that present new residues in place of the usual oligomerization domain present in p53a. Early studies reported that p53b and p53g retain characteristics of a tumor suppressor. 4 Indeed, p53b modulates p53a transcriptional activity in a promoter-dependent manner in response to stress 3 and promotes apoptosis and senescence, indicating that p53b can modulate p53a suppressive activity. 3,6 The biological significance of p53b and p53g isoforms is emphasized by clinical data. Both p53b and p53g expression is lost in B60% of breast cancer tumors.3,7 Furthermore, breast cancer patients expressing both mutant p53 and p53g ha...

p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after γ-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis.

No abstract

IntroductionNormal function of the p53 network is lost in most cancers, often through p53 mutation. The clinical impact of p53 mutations in breast cancer remains uncertain, especially where p53 isoforms may modify the effects of these p53 mutations.MethodsExpression of p53β and p53γ isoforms, the isoforms identified in normal breast tissue, was detected by reverse transcription polymerase chain reaction from a cohort of 127 primary breast tumours. Expression of p53β and p53γ isoforms was analysed in relation to clinical markers and clinical outcomes (5 years) by binary logistic regression, Cox proportional hazards regression and Kaplan-Meier survival analyses.Resultsp53β and p53γ were not randomly expressed in breast cancer. p53β was associated with tumour oestrogen receptor (ER) expression, and p53γ was associated with mutation of the p53 gene. The patient group with the mutant p53 breast tumour-expressing p53γ isoform had low cancer recurrence and an overall survival as good as that of patients with wild-type p53 breast cancer. Conversely, patients expressing only mutant p53, without p53γ isoform expression, had a particularly poor prognosis.ConclusionsThe determination of p53γ expression may allow the identification, independently of the ER status, of two subpopulations of mutant p53 breast cancer patients, one expressing p53γ with a prognosis as good as the wild-type p53 breast cancer patients and a second one not expressing p53γ with a particularly poor prognosis. The p53γ isoform may provide an explanation of the hitherto inconsistent relationship between p53 mutation, treatment response and outcome in breast cancer.