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The Oncogenic Activity of p53 Mutants
Abstract
Single mutations in the DNA binding domain of p53 cause a radical shift in function from tumor suppressor to oncogene. The
mutated proteins lose the negative feedback regulation mediated by MDM2. Their oncogenic activity consists of a dominant negative
inhibition of the remaining wild-type p53 protein, and a gain of function (GOF) activity independent of wild-type p53 inhibition.
An understanding of the properties of these very common oncogenes is yielding promising therapeutic approaches, and is predicted
to offer more clinical applications as the field develops.
The p53 gene family, a well-known group of genes, is the primary propagator of tumor-suppressing mechanisms in multicellular organisms. Although they are currently critical drug targets in cancer, the p53 family also serves specific functions in the development of multicellular organisms. In this paper, the current function, origin, and evolutionary purpose of the p53 family are reviewed in the evolution of multicellular organisms. The TP53 gene induces cellular responses such as apoptosis as a way to combat detrimental environmental and cellular factors that can damage the integrity of a cell’s DNA. The other two members of the p53 family are the TP63/TP73 genes. The TP63/TP73 genes are involved in the embryonic development of limbs and the neuronal system, respectively. It has been discovered that these three genes originated as an ancestral gene that later separated individually throughout the evolution of higher functioning vertebrates from invertebrates. The p53 family provides complex networks of tumor-suppressor genes that allow for the prevention of oncogenesis in multicellular organisms. The ultimate evolutionary goal of the p53 family is to preserve the integrity of their organisms from unwanted mutations, which in turn also assists their own fitness. However, the genomic evidence presented in the current literature contains gaps. Further research is needed to fill in these gaps in the genomes of multicellular organisms, in order to have a cohesive understanding of the evolutionary purpose of the p53 family, not only for the benefit of humans but for other species as well.
In tumors that retain wild-type p53, its tumor-suppressor function is often impaired as a result of the deregulation of HDM-2, which binds to p53 and targets it for proteasomal degradation. We have screened a chemical library and identified a small molecule named RITA (reactivation of p53 and induction of tumor cell apoptosis), which bound to p53 and induced its accumulation in tumor cells. RITA prevented p53–HDM-2 interaction in vitro and in vivo and affected p53 interaction with several negative regulators. RITA induced expression of p53 target genes and massive apoptosis in various tumor cells lines expressing wild-type p53. RITA suppressed the growth of human fibroblasts and lymphoblasts only upon oncogene expression and showed substantial p53-dependent antitumor effect in vivo. RITA may serve as a lead compound for the development of an anticancer drug that targets tumors with wild-type p53.
Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3σ is strongly induced by γ irradiation and other DNA-damaging agents. The induction of 14-3-3σ is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3σ into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.
The involvement of p53 protein in cell differentiation has been recently suggested by some observations made with tumor cells and the correlation found between differentiation and increased levels of p53. However, the effect of p53 on differentiation is in apparent contrast with the normal development of p53-null mice. To test directly whether p53 has a function in cell differentiation, we interfered with the endogenous wt-p53 protein of nontransformed cells of two different murine histotypes: 32D myeloid progenitors, and C2C12 myoblasts. A drastic inhibition of terminal differentiation into granulocytes or myotubes, respectively, was observed upon expression of dominant-negative p53 proteins. This inhibition did not alter the cell cycle withdrawal typical of terminal differentiation, nor p21(WAF1/CIP1) upregulation, indicating that interference with endogenous p53 directly affects cell differentiation, independently of the p53 activity on the cell cycle. We also found that the endogenous wt-p53 protein of C2C12 cells becomes transcriptionally active during myogenesis, and this activity is inhibited by p53 dominant-negative expression. Moreover, we found that p53 DNA-binding and transcriptional activities are both required to induce differentiation in p53-negative K562 cells. Taken together, these data strongly indicate that p53 is a regulator of cell differentiation and it exerts this role, at least in part, through its transcriptional activity.
Compounds that stabilize the DNA binding domain of p53 in the active conformation were identified. These small synthetic molecules
not only promoted the stability of wild-type p53 but also allowed mutant p53 to maintain an active conformation. A prototype
compound caused the accumulation of conformationally active p53 in cells with mutant p53, enabling it to activate transcription
and to slow tumor growth in mice. With further work aimed at improving potency, this class of compounds may be developed into
anticancer drugs of broad utility.
Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.
The p53 tumor suppressor is the most commonly mutated gene in human cancer. p53 protein is stabilized in response to different checkpoints activated by DNA damage, hypoxia, viral infection, or oncogene activation resulting in diverse biological effects, such as cell cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis. The stable p53 protein is activated by phosphorylation, dephosphorylation and acetylation yielding a potent sequence-specific DNA-binding transcription factor. The wide range of p53's biological effects can in part be explained by its activation of expression of a number of target genes including p21WAF1, GADD45, 14-3-3σ, bax, Fas/APO1, KILLER/ DR5, PIG3, Tsp1, IGF-BP3 and others. This review will focus on the transcriptiona l targets of p53, their regulation by p53, and their relative importance in carrying out the biological effects of p53.
Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.
The MDM2 oncogene encodes an inhibitor of the p53 tumor suppressor protein that regulates p53 in a negative feedback loop. MDM2 gene amplification and overexpression occur in several types of tumors and are often associated with poor prognosis. An MDM2 antisense phosphorothioate oligodeoxynucleotide has been identified that effectively inhibits MDM2 expression in tumor cells containing MDM2 gene amplifications. Antisense inhibition of MDM2 is associated with a decrease in MDM2–p53 complex formation, increase in p53-inducible gene expression, increase in p53 transcriptional
activity, and apoptosis. Significantly, inhibition of MDM2 expression enhances the activation of p53 by a DNA-damaging cancer chemotherapy agent in a synergistic fashion. Therefore,
the MDM2 negative feedback pathway is an important limiting factor in DNA damage-induced p53 activation. MDM2 antisense oligonucleotides may be useful as antitumor agents alone or as enhancers of other conventional DNA-damaging drugs.