The promise and obstacle of p53 as a cancer therapeutic agent

p53 is a tumor suppressor gene that is mutated in greater than 50% of human cancers. The action of p53 as a tumor suppressor involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Loss of p53 function therefore allows the uncontrolled proliferation associated with cancerous cells. While design of most anti-cancer agents has focused on targeting and inactivating cancer promoting targets, such as oncogenes, recent attention has been given to restoring the lost activity of tumor suppressor genes. Because the loss of p53 function is so prevalent in human cancer, this protein is an ideal candidate for such therapy. Several gene therapeutic strategies have been employed in the attempt to restore p53 function to cancerous cells. These approaches include introduction of wild-type p53 into cells with mutant p53; the use of small molecules to stabilize mutant p53 in a wild-type, active conformation; and the introduction of agents to prevent degradation of p53 by proteins that normally target it. In addition, because mutant p53 has oncogenic gain of function activity, several approaches have been investigated to selectively target and kill cells harboring mutant p53. These include the introduction of mutant viruses that cause cell death only in cells with mutant p53 and the introduction of a gene that, in the absence of functional p53, produces a toxic product. Many obstacles remain to optimize these strategies for use in humans, but, despite these, restoration of p53 function is a promising anti-cancer therapeutic approach.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Enhanced gene transfer and cell death following p53 gene transfer using photochemical internalisation of glucosylated PEI-DNA complexes

BACKGROUND: p53 is frequently mutated in many cancers including human head and neck squamous cell carcinoma and pancreatic cancer. In tumor models, wild-type (wt) p53 gene transfer induces apoptosis and tumor regression in vivo, justifying the extensive clinical investigation of p53 gene therapy. METHODS: p53 nonviral-mediated gene transfer was achieved using glucosylated polyethylenimine (PEI) in conjunction with photochemical internalisation (PCI). Experimental conditions were optimised using the green fluorescent protein (GFP) as a reporter. p53 gene transfer was then evaluated using semi-quantitative RT-PCR in p53-deleted PANC3 and p53-mutated FaDu cell lines. Following gene transfer, induction of apoptosis was investigated using phosphatidylserine externalisation and nuclear fragmentation assays. Induction of long-term cell death was analysed using colony-forming assays. RESULTS: PCI was found to enhance GFP gene transfer after 48 h in both cell lines. Whether using glucosylated-PEI alone or associated with PCI, p53 gene transfer was achieved with subsequent recovery of p53 mRNA expression in PANC3 cells and a significant 4-fold increase in p53 mRNA expression in FaDu cells. PCI was found to further enhance p53 mRNA expression by 2.3-fold in PANC3 cells. Spontaneous induction of apoptosis following wt-p53 gene transfer was achieved in both cell lines. PCI was found to enhance apoptosis up to levels similar to those achieved with chemotherapy. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used in both cell lines, yielding up to 60% cell death. CONCLUSIONS: PCI increases glucosylated-PEI-mediated p53 gene transfer, apoptosis as well as cell death in mutant p53 human cancer cells.     

A carbazole compound, 9-ethyl-9H-carbazole-3-carbaldehyde,plays an antitumor function through reactivation of the p53 pathway in human melanoma cells

p53, the major tumor suppressor, is frequently mutated in many cancers, and up to 84% of human melanomas harbor wild-type p53, which is considered to be an ideal target for melanoma therapy. Here, we evaluated the antitumor activity of a carbazole derivative, 9-ethyl-9H-carbazole-3-carbaldehyde (ECCA), on melanoma cells. ECCA had a selectively strong inhibitory activity against the growth of BRAF-mutated and BRAF-wild-type melanoma cells but had little effect on normal human primary melanocytes. ECCA inhibited melanoma cell growth by increasing cell apoptosis, which was associated with the upregulation of caspase activities and was significantly abrogated by the addition of a caspase inhibitor. In vivo assays confirmed that ECCA suppressed melanoma growth by enhancing cell apoptosis and reducing cell proliferation, and importantly ECCA did not have any evident toxic effects on normal tissues. RNA-Seq analysis identified several pathways related to cell apoptosis that were affected by ECCA, notably, activation of the p53 signaling pathway. Biochemical assays demonstrated that ECCA enhanced the phosphorylation of p53 at Ser15 in melanoma cells harboring wild-type p53, and importantly, the knockdown or deletion of p53 in those cells counteracted the ECCA-induced apoptosis, as well as senescence. Further investigations revealed that ECCA enhanced the phosphorylation of p38-MAPK and c-Jun N-terminal kinase (JNK), and treatment with either a p38-MAPK or a JNK inhibitor rescued the cell growth inhibition elicited by ECCA, which depended on the expression of the p53 gene. Finally, the combination of ECCA with a BRAF inhibitor significantly enhanced the growth inhibition of melanoma cells. In summary, our study demonstrates that the carbazole derivative, ECCA, induces melanoma cell apoptosis and senescence through the activation of p53 to significantly and selectively suppress the growth of melanoma cells without affecting normal human melanocytes, suggesting its potential to develop a new drug for melanoma therapy.Subject terms: Melanoma, Apoptosis, Biologics     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Gain of Function of p53 Cancer Mutants in Disrupting Critical DNA Damage Response Pathways

Loss of the tumor suppression activity of p53 is required for the progression of most human cancers. In this context, p53 gene is somatically mutated in about half of all human cancers; in the rest human cancers, p53 is mostly inactivated due to the disruption of pathways important for its activation. Most p53 cancer mutations are missense mutations within the core domain, leading to the expression of full-length mutant p53 protein. The expression of p53 mutants is usually correlated with the poor prognosis of the cancer patients. Accumulating evidence has indicated that p53 cancer mutants not only lose the tumor suppression activity of WT p53, but also gain novel oncogenic activities to promote tumorigenesis and drug resistance. Therefore, to improve current cancer therapy, it is critical to elucidate the gain-of-functions of p53 cancer mutants. By analyzing the humanized p53 mutant knock-in mouse models, we have identified a new gain of function of the common p53 cancer mutants in inducing genetic instability by disrupting ATM-mediated cellular responses to DNA double-stranded break (DSB) damage. Considering that some current cancer therapies such as radiotherapy kills the cancer cells by inducing DSBs in their genome DNA, our findings will have important implications on the treatment of human cancers that express common p53 mutants.     

Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8⁺ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8⁺ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8⁺ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8⁺ T cells.     

Validation of a genotoxicity test based on p53R2 gene expression in human lymphoblastoid cells

Genotoxicity assessment is important for predicting the carcinogenicity of chemical substances. p53R2 is a p53-regulated gene that is induced by various genotoxic stresses. We previously developed a p53R2-dependent luciferase reporter gene assay in the MCF-7 human breast adenocarcinoma cell line, and demonstrated its ability to detect genotoxic agents. In this paper, we investigate the applicability of the p53R2-based genotoxicity test in the human lymphoblastoid cell line TK6. TK6 cells that express wild-type p53 have been widely used for genetic toxicology studies. To evaluate the performance of the test system in TK6 cells, we referred to 61 of the chemicals on the list of 20 genotoxic and 42 non-genotoxic chemicals recommended for the evaluation of modified or new mammalian cell genotoxicity tests by the European Centre for the Validation of Alternative Methods. The overall accordance, sensitivity, and specificity of our results with the ECVAM list were 90% (55/61), 85% (17/20), and 93% (38/41), respectively. These results indicate that the p53R2-based genotoxicity test can detect various types of genotoxic chemicals without compromising its specificity. This test will be a valuable tool for rapid screen for identifying chemicals that may be genotoxic to humans.     

Mutant p53 exerts oncogenic effects through microRNAs and their target gene networks

MicroRNAs are potent regulators of gene expression and modulate multiple cellular processes including proliferation, differentiation and apoptosis. A number of microRNAs have been shown to be regulated by p53, the most frequently mutated gene in human cancer. It is has been demonstrated that some mutant p53 proteins not only lose tumor suppressor activity, but also acquire novel oncogenic functions that are independent of wild-type p53. In this review, we highlight recent evidences suggesting that some mutant p53 proteins regulate the expression of specific microRNAs to gain oncogenic functions and identify a gene network regulated by the microRNAs downstream of mutant p53.     

The p53 tumor suppressor: Critical regulator of life & death in cancer

p53 is the most commonly mutated or deleted known gene in human cancer. The consequences of its disruption are profound, either in the germlines of patients with Li-Fraumeni Syndrome, or in mice with targeted gene knockouts. Abundant evidence suggests that p53 exerts regulation of cell cycle progression as well as apoptotic cell death, both in response to identical environmental or metabolic stressors. The specific decision of cell cycle arrest vs. death may underlie p53's differential ability to trigger death in cancer cells and arrest with repair in non-cancer cells, thus producing a therapeutic index pertinent to cancer therapy. Indeed, p53 status is likely to correlate with prognosis in many human cancers and in multiple animal tumor models. The mechanistic basis for p53's functions are still emerging, and will hopefully yield new therapeutic strategies applicable to treatment of the many poor-prognosis, p53-deficient human malignancies.     

Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.     

Inhibition of p53 by Lentiviral Mediated sh RNA Abrogates G1 Arrest and Apoptosis in Retinal Pigmented Epithelial Cell Line

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G1 arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.     

p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM

Tp53 is the most commonly mutated tumour-suppressor gene in human cancers. In addition to the loss of tumour-suppression function, some missense mutants gain novel oncogenic activities. To elucidate the nature of the gain of function, we introduced the most common p53 cancer mutations (R248W and R273H) independently into the humanized p53 knock-in (HUPKI) allele in mice. Tumour-suppressor functions of p53 are abolished in p53-mutant mice. Several lines of evidence further indicate gain-of-function of p53 mutants in promoting tumorigenesis. p53(R248W) mice rapidly succumb to certain types of cancers not commonly observed in p53(-/-) mice. Interchromosomal translocations, a type of genetic instability rarely observed in p53(-/-) cells, are readily detectable in p53-mutant pre-tumor thymocytes. Although normal in p53(-/-) mouse cells, the G(2)-M checkpoint is impaired in p53-mutant cells after DNA damage. These acquired oncogenic properties of mutant p53 could be explained by the findings that these p53 mutants interact with the nuclease Mre11 and suppress the binding of the Mre11-Rad50-NBS1 (MRN) complex to DNA double-stranded breaks (DSBs), leading to impaired Ataxia-telangiectasia mutated (ATM) activation. Therefore, p53 gain-of-function mutants promote tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.