The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells.

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs.

The mouse p53 gene generates two alternative splice products encoding p53 protein and a naturally occurring protein (p53as) with changes at the C-terminus. In p53as the negative regulatory region for DNA binding and PAb421 antibody binding site are replaced, and p53as is constitutively active for sequence-specific DNA binding. Using the technique of randomized synthetic oligonucleotide in cyclic amplification and selection of targets, we have found that p53as and p53 proteins have the same DNA binding specificities but that these specificities frequently diverge from the consensus of two copies of PuPuPuCATGPyPyPy. The importance of tetranucleotide CATG was confirmed but there was a less rigorous requirement for patterns of flanking or intervening sequences. In particular, the three purines upstream and three pyrimidines downstream of CATG are not required for p53 or p53as binding, 29 or more intervening nucleotides are tolerated, and one CATG is sufficient where adjacent nucleotides contain a region of homology with certain previously reported non-consensus p53 binding sequences. These results suggested further definition of the non-consensus motifs, and database searches with these uncovered additional candidate genes for p53 protein binding. We conclude that p53as and perhaps other activated forms of p53 exert their effects on the same genes and that differential activities of p53 protein forms are not due to inherently different sequence selectivities of DNA binding.     

Mitochondrial dysfunction impairs tumor suppressor p53 expression/function

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.     

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3′ splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis-acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3′ splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.     

The p53 tumor suppressor gene

p53 was originally considered to be a nuclear oncogene, but several convergent lines of research have indicated that the wild-type gene functions as a tumor suppressor gene negatively regulating the cell cycle. Mutations in the p53 gene have been detected in many tumor types and seem to be the most common genetic alterations in human cancer. In this preliminary study, sera of 92 patients (pts) with breast disease were analyzed for the presence of the mutant p53 protein (mp53) with a selective immunoenzyme assay employing a monoclonal antibody (PAb 240) specific for the majority of mammalian m p53 but not for the wild-type protein. Of the 10 patients with benign breast disease, only two (20%) showed detectable m p53 levels in the serum. In the breast cancer group, sera from 7 of the 30 pts (23%) without lymph node involvement were positive for m p53, as were 7 out of the 45 pts (15%) with metastatic lymph nodes and 1 out of the 7 pts (14%) with disseminated disease. The specifity of m p53 assay evaluated in 20 healthy controls was 100%. These preliminary results showed that serum positivity for m p53 is not related to breast disease extension. Further studies to assess the utility of m p53 as a possible prognosis factor in breast cancer are currently in progress.     

Structure of the rat p53 tumor suppressor gene.

Aberration within the p53 tumor suppressor gene is the most frequently identified genetic damage in human cancer. Regulatory functions proposed for the p53 protein include modulation of the cell cycle, cellular differentiation, signal transduction, and gene expression. Additionally, the p53 gene product may guard the genome against incorporation of damaged DNA. To facilitate study of its role in carcinogenesis using a common animal model, we determined the structure of the rat p53 gene. We identified 18 splice sites and defined 25 bases of the intervening sequences adjacent to these sites. We also discovered an allelic polymorphism that occurs within intron 5 of the gene. The rat gene approximates the mouse ortholog. It is 12 kb in length with the non-coding exon 1 separated from exon 2 by 6.2 kb of intervening sequence. The location and size of all rat gene introns approximate those of the mouse. Whereas the mouse and human genes each contain 11 exons, the rat p53 gene is composed of only 10. No intervening sequence occurs between the region of the rat gene corresponding to exons 6 and 7 of the mouse and human p53 genes. This implies intron 6 may be functionally insignificant for species in which it is retained. To extrapolate to p53 involvement in human tumorigenesis, we suggest that mutational events within intron 6 may not be of pathological significance unless splicing is hindered.     

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.     

Preferential binding of tumor suppressor p53 to positively or negatively supercoiled DNA involves the C-terminal domain.

The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings.     

Activation of the p53 tumor suppressor protein

The p53 tumor suppressor gene plays an important role in preventing cancer development, by arresting or killing potential tumor cells. Mutations within the p53 gene, leading to the loss of p53 activity, are found in about half of all human cancers, while many of the tumors that retain wild type p53 carry mutations in the pathways that allow full activation of p53. In either case, the result is a defect in the ability to induce a p53 response in cells undergoing oncogenic stress. Significant advances have been made recently in our understanding of the molecular pathways through which p53 activity is regulated, bringing with them fresh possibilities for the design of cancer therapies based on reactivation of the p53 response.