Tissue-specific regulation of Fas/APO-1/CD95 expression by p53

The regulation of Fas/APO-1(CD95), an important member of the tumor necrosis factor (TNF) superfamily involved in membrane-mediated apoptosis, has been a subject of recent research. Ligation of Fas by Fas ligand or an anti-Fas cross-linking antibody triggers receptor trimerization followed by recruitment of FADD to the cytoplasmic domain of the receptor and the activation of the caspase cascade. The tumor suppressor p53 has been shown to upregulate Fas expression under numerous pro-apoptotic stimuli in vitro. Using the p53 knockout mouse model, we demonstrate by Western blot analysis, immunohistochemistry, and semi-quantitative RT-PCR that Fas expression is reduced in spleen and liver from p53-/- mice compared to p53+/+ controls, while similar expression levels were observed in brain, heart, kidney, lung, skin, testis, and thymus between the two groups. While Fas protein was abundant in brain, heart, liver, and spleen, low levels of endogenous expression was observed in other tissues from the p53+/+ and p53-/- mice. These data indicate that p53 regulates Fas expression in a tissue-specific manner.     

Tissue-specific induction of p53 targets in vivo

The in vivo response to radiotherapy is not well understood but appears to involve the p53 tumor suppressor protein. We investigated the expression of apoptosis-inducing p53 target genes during gamma-irradiation-induced cell death in p53(+/+) or p53(-/-) mouse tissues using in situ hybridization. Our results reveal striking tissue specificity with distinct regulation of target p53-induced genes in different cells and tissue compartments, as well as variations in dependence on p53 for basal expression. p53-dependent induction of Puma occurred in the splenic white pulp, whereas Noxa and Bid were induced in the red pulp. These patterns correlated with activation of caspase-3 in both compartments. All apoptotic targets of p53 studied here (DR5, Bid, Puma, Noxa) were induced in the jejunum and ileum, which appeared to be the tissues most sensitive to irradiation. We also observed unexpected differences in p53 target gene activation between the transverse and descending colon. Finally, in the liver where irradiation did not lead to caspase-3 activation, we primarily observed p21(WAF1) induction as the major p53-dependent target gene response. Our findings indicate that the selectivity of p53 in transactivation following DNA damage in vivo results in unique tissue and cell type specificity, which may correlate with growth arrest or variable sensitivity to gamma-irradiation.     

Co-transduction of Apaf-1 and caspase-9 highly enhances p53-mediated apoptosis in gliomas

Mutation of the p53 gene plays a critical role in the development of cancer and response to cancer therapy. To analyze the mechanism of cancer development and to improve cancer therapy, it is important to assess which genes are downstream components of p53 in cancers, and whether the expression levels of these genes affect p53-mediated apoptosis. In this study, we transduced the wild type p53 gene along with the Apaf-1 and caspase-9 genes via adenovirus vectors into U251 and U-373MG glioma cells harbouring a mutated p53, and evaluated the degree of apoptosis. Co-induction of Apaf-1 and caspase-9 genes highly enhanced p53-mediated apoptosis in glioma cells. Induction of wild type p53 enhanced the expression levels of Bax, p21/WAF1, and Fas protein. To determine which gene is activated by wild type p53 induction and, in turn, activates Apaf-1 and caspase-9, we transduced the Bax, p21/WAF1 or Fas gene via adenovirus vector to U251 cells to achieve a similar expression level as that induced by the Adv for p53 in U251 cells. U251 cells transduced with Fas concomitant with the Apaf-1 and caspase-9 genes underwent drastic apoptosis. This suggests that induction of wild type p53 upregulates Fas, which in turn may play a role in the activation of Apaf-1 and caspase-9. These results are important for analyzing the mechanism of tumour development and for predicting the therapeutic effect of p53 replacement gene therapy in a particular patient.     

Tissue-specific expression of the p53 tumor-suppressor gene in the intestine of transgenic mice exposed to DMH and p53 antibodies.

We studied the tissue-specific expression of the p53 gene in different parts of the intestine of mice treated with low doses of a carcinogen and exposed to different p53 antibodies. The human p53 promoter-CAT transgenic mice were immunized with different p53 antibodies (monoclonal - PAb 421 and DO1, and polyclonal - H-p53 and anti-soluble p53 IgG) and then exposed to low doses of dimethylhydrazine (DMH). Enzymatic CAT activity was determined in the ileum and colon 8 weeks later after the final injection of DMH. Expression of the p53 transgene in the normal ileum was twice as high as in the colon. Treatment with DMH significantly decreased the expression of the p53 transgene both in the ileum (from 18% to 100%) and in the colon (from 10% to 52%). Vaccination of mice protected at least in part such a decrease. The most effective results were found after exposure of mice to polyclonal H-p53 and to a lesser extent to anti-p53 IgG. No difference was found in the effects of antibodies on the small and large intestines. We concluded that polyclonal antibodies were more effective than monoclonal ones in protection against anti-p53 action of DMH. The observation of these effects may make it possible to explain the higher antitumor activity of polyclonal antibodies.     

Differential expression and regulation of p53 in human prostatic cells

Although genetic analysis has convincingly shown the association possibly existing between alterations in p53 tumor suppressor gene and a broad spectrum of human tumors including prostate cancer, surprisingly little is known about ways in which p53 at the protein level is controlled. To determine factors that may play a role in its regulation and expression, changes in p53 protein was investigated by using the androgen-insensitive JCA-1, DU-145, PC-3 and the androgen-responsive LNCaP cells. With the exception of PC-3 cells in which p53 is missing, multiple distinct forms of p53 were found in the other 3 prostate cell lines. A single p53 band was detected in the JCA-1 cell extracts, whereas two and three p53 immunoreactive bands were correspondingly observed in the DU-145 and LNCaP cells. The relative abundance and distribution of the different forms of p53 in the latter two cell types varied with proliferation of cells in culture. In the presence of charcoal-stripped fetal bovine serum (cFBS), LNCaP took on the morphology of neuroendocrine cells, a phenotypic change which was accompanied by a greater than 80% reduction in p53 expression, concurrent with elimination of the two slow migrating forms of p53. Induction of apoptosis in JCA-1 cells by treatment with the retinoid 4-HPR caused the virtual disappearance of p53, which coincided with specific processing of p53 into lower molecular weight 28 kD fragments. We propose that rapid and dynamic posttranslational changes in p53 may actively participate in determining mutually exclusive functional cellular events such as proliferation, differentiation, and apoptosis.     

p53 controls hPar1 function and expression

Human protease-activated receptor 1 (hPar1) is a bona fide receptor of the hemostatic protease thrombin, and has a central function in tumor progression. Inactivation of the tumor suppressor gene p53 is one of the most common genomic alterations occurring in cancer. Here, we address the interrelations between p53 and hPar1 in cancer. We demonstrate an inverse correlation between hPar1 gene expression and wild-type (wt) p53 levels, and a direct correlation with levels of the mutant (mt) p53. Bioinformatic search revealed the presence of at least two p53 motifs in the hPar1 promoter. Indeed, temperature-sensitive (ts) p53 forms reduced hPar1 promoter activity on wt p53 expression. Ectopic introduction of the p53R175H mutant into cells lacking p53 caused a moderate two-fold induction of hPar1 promoter activity. Chromatin immunoprecipitation (ChIP) analyses confirmed a physical association between the p53 protein and hPar1 chromatin fragments. In parallel, PAR1 function is attenuated by p53, as shown by inhibition of pFAK levels and a Matrigel invasion assay. Ectopic reinforcement of hPar1 rescued the inhibition conferred by p53, confirming that p53 directly affects hPar1 expression and function. Altogether, we provide evidence for a direct binding between p53 and hPar1 chromatin, and assign hPar1 as a target of p53.     

The regulation of energy generating metabolic pathways by p53

The function of p53 as a tumor suppressor remains undisputed. p53 has a central role in cellular stress responses as well as affecting cancer development and progression. The word "central", however, is becoming increasingly more of an understatement as the list of p53-regulated pathways and processes is ever expanding. Although much focus continues to center on p53-mediated signaling cascades that control cell growth arrest and/or apoptosis, recent work has begun to define a role for p53 in the regulation of metabolic pathways typically thought of as essential for maintaining life. With the first potential link between p53 and glycolysis reported nearly ten years ago, the topic has gained a renewed interest. Recent studies now demonstrate the ability of p53 to regulate the expression of several novel genes including PGM (phosphoglycerate mutase), TIGAR (TP53-induced glycolysis and apoptosis regulator) and, SCO2 (synthesis of cytochrome c oxidase 2), each intimately linked to the processes of glycolysis and oxidative phosphorylation. With this discovery, yet another novel means by which p53 carries out its tumor suppressor function is brought into light.     

TIGAR induces p53-mediated cell-cycle arrest by regulation of RB-E2F1 complex

Background:

p53 induces cell-cycle arrest and apoptosis in cancer cells and negatively regulates glycolysis via TIGAR. Glycolysis is crucial for cancer progression although TIGAR provides protection from reactive oxygen species and apoptosis. The relation between TIGAR-mediated inhibition of glycolysis and p53 tumour-suppressor activity is unknown.

Methods:

RT–PCR, western blot, luciferase and chromatin immunoprecipitation assays were used to study TIGAR gene regulation. Co-IPP was used to determine the role of TIGAR protein in regulating the protein–protein interaction between retinoblastoma (RB) and E2F1. MCF-7 tumour xenografts were utilised to study the role of TIGAR in tumour regression.

Results:

Our study shows that TIGAR promotes p21-independent, p53-mediated G1-phase arrest in cancer cells. p53 activates the TIGAR promoter only in cells exposed to repairable doses of stress. TIGAR regulates the expression of genes involved in cell-cycle progression; suppresses synthesis of CDK-2, CDK-4, CDK-6, Cyclin D, Cyclin E and promotes de-phosphorylation of RB protein. RB de-phosphorylation stabilises the complex between RB and E2F1 thus inhibiting the entry of cell cycle from G1 phase to S phase.

Conclusion:

TIGAR mediates de-phosphorylation of RB and stabilisation of RB–E2F1 complex thus delaying the entry of cells in S phase of the cell cycle. Thus, TIGAR inhibits proliferation of cancer cells and increases drug-mediated tumour regression by promoting p53-mediated cell-cycle arrest.     

Reduced Apaf-1 expression in human cutaneous melanomas

Malignant melanoma is a life-threatening skin cancer due to its highly metastatic character and resistance to radio- and chemotherapy. It is believed that the ability to evade apoptosis is the key mechanism for the rapid growth of cancer cells. However, the exact mechanism for failure in the apoptotic pathway in melanoma cells is unclear. p53, the most frequently mutated tumour suppressor gene in human cancers, is a key apoptosis inducer. However, p53 mutation is only found in 15-20% of melanoma biopsies. Recently, it was found that Apaf-1, a downstream target of p53, is inactivated in metastatic melanoma. Specifically, loss of heterozygosity (LOH) of the Apaf-1 gene was found in 40% of metastatic melanoma. To determine if loss of Apaf-1 expression is indeed involved in melanoma progression, we employed the tissue microarray technology and examined Apaf-1 expression in 70 human primary malignant melanoma biopsies by immunohistochemistry. Our data showed that Apaf-1 expression is significantly reduced in melanoma cells compared with normal nevi (chi(2)=6.02, P=0.014). Our results also revealed that loss of Apaf-1 was not associated with the tumour thickness, ulceration or subtype, patient's gender, age and 5-year survival. In addition, our in vitro apoptosis assay revealed that overexpression of Apaf-1 can sensitise melanoma cells to anticancer drug treatment. Taken together, our data indicate that Apaf-1 expression is significantly reduced in human melanoma and that Apaf-1 may serve as a therapeutic target in melanoma.