Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143

The p53 mutant 143Ala is a human temperature-sensitive mutant with two conformational states.To definitively determine whether the Fas signal transduction pathway and the function of the pathway are dependent on p53 status, we have established stable transfectants of p53 mutant 143Ala in two human cancer cell lines: H1299 (lung cancer line) and PC-3 (prostate cancer line), the native state of which contains null p53 status and can grow at 37 degrees C and 32.5 degrees C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed inhibition of the growth of cells overexpressing p53 mutant 143Ala in the wild-type p53 form at 32.5 degrees C because of induction of G0/G1 arrest. Transfected cells had increased protein expression of p21, Fas, and MDM2 at the wild-type p53 conformation at 32.5 degrees C, but not in the mutant p53 form at 37 degrees C. However, there was no change in protein expression of FADD, FAP-1, Bcl-2, or Bax at 32.5 or 37 degrees C. Assays for apoptosis demonstrated that anti-Fas antibody CH-11 and FasL induced apoptosis only in cells that overexpress p53 mutant 143Ala at 32.5 degrees C with the wild-type p53 form. Both caspase-3 and caspase-8 activities were increased by anti-Fas antibody CH-11 only in cells at 32.5 degrees C with wild-type p53. Our results demonstrated that Fas-mediated apoptosis in H1299 and PC-3 cells expressing p53 mutant 143Ala occurred only with the wild-type p53 phenotype. These results support the hypothesis that Fas-mediated apoptosis is dependent, at least partially, on the presence of a functional wild-type p53 state. This model may be a useful tool for dissecting the specific interactions between wild-type p53 and the Fas signal transduction pathway in human cancer cells.     

p53 and apoptosis

One of the several biological functions attributed to p53 is the ability to induce apoptotic cell suicide. It has become clear that this apoptotic activity of p53 is central to its role as a tumor suppressor. A summary of current knowledge concerning the mechanisms of p53-mediated apoptosis is presented. The pivotal 'choice' between p53-induced viable growth arrest and apoptosis is discussed.     

Elevated susceptibility of the p53 knockout mouse esophagus to methyl-N-amylnitrosamine carcinogenesis

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers, including those in the esophagus. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (-/-), heterozygous (+/-) and wild-type (+/+) mice to methyl-N-amylnitrosamine (MNAN), which specifically induces esophageal tumors in mice and rats. The p53 (+/-) and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An additional group of p53 (-/-) mice were given 5 p.p.m. MNAN for 8 weeks and killed at week 15. At 15 weeks in the 5 p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (-/-) and 1/15 (6.7%) p53 (+/-) mice, but in none of the p53 (+/+) mice. In the animals receiving 15 p.p.m., 2/14 (14.3%) p53 (+/-) and 1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidence of SCCs was 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/-) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice at 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR-single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/-) and 50% (6/12) from p53 (+/+) mice demonstrated mutations in the p53 gene (exons 5-8). These results indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (-/-) > heterozygotes (+/-) > wild-type (+/+), and provide strong evidence of involvement of p53 mutations in the development of esophageal SCCs.     

Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice

We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.     

肿瘤抑制基因p53与自噬

近年来研究表明,p53是一种抑癌基因,作为一种重要的转录因子,其在细胞周期阻滞、凋亡中都发挥着重要作用。自噬是在细胞营养物质匮乏条件下蛋白质动态降解的过程,自噬性细胞死亡是不同于细胞凋亡的另一种细胞程序性死亡模式,被称之为II型程序性死亡,细胞凋亡为I型程序性死亡。研究表明,肿瘤的发生发展与自噬性细胞死亡相关,作为肿瘤抑制基因p53也参与了自噬活性的调节。本文对此做一简要概述。     

肿瘤抑制基因p53与自噬

近年来研究表明,p53是一种抑癌基因,作为一种重要的转录因子,其在细胞周期阻滞、凋亡中都发挥着重要作用。自噬是在细胞营养物质匮乏条件下蛋白质动态降解的过程,自噬性细胞死亡是不同于细胞凋亡的另一种细胞程序性死亡模式,被称之为II型程序性死亡,细胞凋亡为I型程序性死亡。研究表明,肿瘤的发生发展与自噬性细胞死亡相关,作为肿瘤抑制基因p53也参与了自噬活性的调节。本文对此做一简要概述。     

Reversal of p53 epigenetic silencing in multiple myeloma permits apoptosis by a p53 activator

Inactivation of the p53 pathway is a common feature of neoplasia. Dysregulation of the p53 pathway has been shown to involve mutations of p53, increased expression of the p53 inhibitor HDM-2, or epigenetic silencing of the p53 promoter. In multiple myeloma, a neoplasia of terminally differentiated B cells, p53 mutations and deletions are relatively rare and occur in late stage disease. Here, we show that the p53 promoter is hypermethylated in several multiple myeloma cell lines in comparison to normal plasma cells. Two cell lines containing mutant p53, Lp-1 and OPM-2, show a methylation pattern that suggests that they contain one methylated and one unmethylated mutant allele. Two other cell lines, KMS-11 and OPM-2, show hypermethylation of p53 with a lack of expression. In all cell lines tested, treatment with a demethylating agents results in higher expression of p53. Furthermore, following increased expression of p53, treatment of the myeloma cell lines with a p53 activating peptide induces apoptosis. Therefore, combinatorial treatment with demethylating agents followed by delivery of a p53 activating peptide may be an effective therapeutic strategy against multiple myeloma.     

Induction of p53-mediated apoptosis and recovery of chemosensitivity through p53 transduction in human glioblastoma cells by cisplatin

Cisplatin is a DNA-damaging chemotherapeutic drug that may have a role in the adjuvant chemotherapy of several solid tumors, such as malignant glioblastoma, and the status of p53 tumor suppressor protein is a critical determinant of cisplatin chemosensitivity. In the present study, we showed the relationship of p53 status and chemosensitivity of cisplatin between two human malignant glioblastoma cell lines, A172 and T98G, harboring wild-type and mutant-type p53, respectively. Cisplatin was found to be more cytotoxic to A172 than T98G cells in a time- and concentration-dependent manner. Cisplatin-induced cytotoxicity manifested as apoptosis, characterized by genomic DNA fragmentation, nuclear condensation and an increase in sub-G1 population. Cisplatin induced the accumulation of p53 and p21 proteins in A172 cells, but not in T98G cells. The introduction of the adenovirus-mediated wild-type p53 gene into T98G cells resulted in the decrease of viability as well as the increase in sub-G1 population with p53 accumulation, activation of caspase-3 protease and release of cytochrome c from the mitochondria. These data strongly suggest that the expression of p53 is essential for the cytotoxic effect of cisplatin in human malignant glioblastoma cells, A172 and T98G, and the introduction of apoptotic signal molecules, such as p53, will be beneficial to achieve chemosensitivity in malignant glioma.     

Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.     

Adenoviral-mediated p53 transgene expression sensitizes both wild-type and null p53 prostate cancer cells in vitro to radiation

Purpose/Objective: The effect of adenoviral-mediated p53 transgene expression on the radiation response of two human prostate cancer cell lines, the p53^wild-type LNCaP and p53^null PC3 lines, was examined. The objective was to determine if this vector sensitizes cells to radiation independently of their p53 status.

Methods and Materials: A recombinant adenovirus-5 vector (RPR/INGN 201, Introgen Therapeutics, Houston, TX) containing a CMV promoter and wild-type p53-cDNA (Ad5-p53) was used to facilitate p53 transgene expression. A multiplicity of infection (MOI) of 10–40 viral particles per cell was used, based on Ad5/CMV/lacz infection and staining for the β-galactosidase reporter gene product. Clonogenic assays were performed to evaluate the degree of sensitization to radiation of viral-transduced cells compared with irradiated nontransduced controls. The relative efficacy of these treatments to induce apoptotic cell death was determined using the TUNEL assay.

Results: The delivery of Ad5-p53 (10 MOI) reduced control plating efficiency from 36.5% to 0.86% in the LNCaP cell line and from 75.1% to 4.1% in the PC3 cell line. After correcting for the effect of Ad5-p53 on plating efficiency, the surviving fraction after 2 Gy (SF2) of gamma-irradiation was reduced over 2.5-fold, from 0.187 to 0.072, with transgene p53 expression in the LNCaP cell line. Surviving fraction after 4 Gy (SF4) was reduced over 4.5-fold, from 0.014 to 0.003, after Ad5-p53 treatment. In the PC3 cell line, Ad5-p53 (40 MOI) reduced SF2 over 1.9-fold from 0.708 to 0.367, and SF4 over 6-fold from 0.335 to 0.056. In both the LNCaP and PC3 cell lines, the combination of Ad5-p53 plus radiation (2 Gy) resulted in supra-additive apoptosis (20% for LNCaP and 15% for PC3 at 50 MOI), above that seen from the addition of the controls; control vector Ad5-pA plus RT (0.15% for LNCaP and 1.44% for PC3), Ad5-p53 alone (28.6% for LNCaP and 21.7% for PC3), RT alone (0% for LNCaP and 0.23% for PC3), or Ad5-pA alone (0.1% for LNCaP and 0.29% for PC3).

Conclusion: The clonogenic survival and apoptosis data demonstrate that p53 transgene expression sensitizes human prostate adenocarcinoma cells in vitro to irradiation. As this effect was observed in both the p53^wild-type LNCaP and p53^null PC3 lines, radiosensitization was independent of p53 status.     

siRNA-mediated down-regulation of iASPP promotes apoptosis induced by etoposide and daunorubicin in leukemia cells expressing wild-type p53

Oncoprotein inhibitory member of the ASPP family (iASPP) is a key inhibitor of tumor suppressor p53. Our previous study revealed that the expression of iASPP in acute leukemia (AL) patients was higher than that of normal control which implied that iASPP might play an important role in the pathogenesis and/or disease progression of AL. In this study, the iASPP expression was blocked by RNA interference (RNAi) in two leukemic cell lines, Nalm6 and K562, to explore the effects of iASPP on leukemia cells. The results indicated that down-regulation of endogenous iASPP increased p53-dependent apoptosis of leukemia cells. Thus, iASPP could be a molecular target in leukemia therapy.     

p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis

DNA degradation is one of the biochemical hallmarks detected in apoptotic cells, and several nucleases have been reported to function cooperatively in this process. It has also been suggested that different sets of nucleases are activated by different stimuli, and induce distinct patterns of DNA degradation. Here we report that apoptosis-enhancing nuclease (AEN) is a novel direct target gene of p53. AEN is induced by p53 with various DNA damage, and its expression is regulated by the phosphorylation status of p53. We demonstrate that AEN is a typical exonuclease with conserved exonuclease domains Exo I-III, and it targets both single- and double-stranded DNA and RNA. AEN induces apoptosis by itself, and the conserved domains are essential for both AEN nuclease activity and its apoptosis-inducing ability. AEN possesses nuclear and nucleolar localization signals, and it translocates from the nucleolus to nucleoplasm upon apoptosis induction. We also show the dislocation of nucleophosmin in conjunction with the translocation of AEN to the nucleoplasm, indicating the ability of AEN in nucleolus disruption. In addition, AEN is shown to be required for efficient DNA fragmentation in p53-dependent apoptosis. These results suggest that AEN is an important downstream mediator of p53 in apoptosis induction.     

PPAR gamma ligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells

We have recently demonstrated that the PPARγ ligand troglitazone induced cell growth arrest and evoked apoptosis in a gastric cancer cell line, MKN–45. Since in general, p53 plays an important role in the induction of apoptosis and growth inhibition, we tried to clarify whether or not p53 mediates troglitazone-induced apoptosis and growth arrest in gastric cancer cells. Troglitazone increased the number of apoptoic cells in MKN-28, MKN-45 and MKN-74, but not in KATO-III cells. The troglitazone-induced apo-ptotic change was significantly reduced by coincubation with bisphenol A digycidyl ether (BADGE), a synthetic PPARy antagonist, in MKN-74 cells, suggesting that PPARγ mediates the apo-ptotic effect of troglitazone. Since KATO-III lacks the p53 gene, we speculated that p53 might be implicated in the PPARγ ligand-induced apoptosis. Western blot analysis revealed that p53 expression was increased by troglitazone in a time-dependent manner in MKN-74 cells, further suggesting that p53 may mediate the ap-optotic process induced by troglitazone. We next established a dominant-negative p53 mutant by stable transfection of p53 mutant into MKN-74 cells. In the dominant-negative p53 mutant cells, troglitazone failed to induce apoptosis, strongly supporting the hypothesis that p53 indeed mediates the process of the troglitazone-induced apoptosis. In the dominant-negative p53 mutant cells, troglitazone significantly induced cell growth arrest and increased expression of p27^Kip1 protein, which is thought to be the key molecule to evoke growth arrest, suggesting that p53 is not involved in the growth inhibition by troglitazone. All these results suggest that p53 mediates the PPARy ligand-induced apoptosis, but not the cell growth inhibition. (Cancer Sci 2003; 94: 338–343)     

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53 -/- normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53 +/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53-/- normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.     

Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21^{WAF 1/CIP1} protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.