p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression

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Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Oncogenic H-ras induces cyclin B1 expression in a p53-independent manner

Alcohol abuse is a major risk factor for cancer of the upper alimentary tract, the upper respiratory tract, and liver. Chromosome damage is used as early effect biomarker in the surveillance of human exposure to genotoxic carcinogens. In the present study, two genetic markers, namely chromosome aberrations (CAs) and micronuclei (MN), were used to evaluate genetic damage in peripheral lymphocytes from 20 alcoholics, 20 abstinent alcoholics, and 20 controls. Composition of the three groups was fairly similar as regards sex, age and smoking habits. A highly significant increase was observed in the frequencies of CA and MN in lymphocytes of alcoholics as compared both with controls and abstinent alcoholics. However, no correlation was found between the length of alcohol abuse and the frequencies of either biomarkers in alcoholics. CA and MN frequencies in abstinent alcoholics were similar than those in controls.Our data indicate that CA and MN can be two useful biomarkers to assess genetic damage associated with alcohol abuse. They could be included in programs for cancer prevention in alcoholics. Abstinence appears to normalize the frequency of both MN and CA. This could offer therapists another tool to help alcoholics change their lifestyle.     

p73-Mediated Chemosensitivity: A Preferential Target of Oncogenic Mutant p53

No abstract available.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.     

Wrapping Effects within a Proposed Function-Rescue Strategy for the Y220C Oncogenic Mutation of Protein p53

Soluble proteins must protect their structural integrity from water attack by wrapping interactions which imply the clustering of nonpolar residues around the backbone hydrogen bonds. Thus, poorly wrapped hydrogen bonds constitute defects which have been identified as promoters of protein associations since they favor the removal of hydrating molecules. More specifically, a recent study of our group has shown that wrapping interactions allow the successful identification of protein binding hot spots. Additionally, we have also shown that drugs disruptive of protein-protein interfaces tend to mimic the wrapping behavior of the protein they replace. Within this context, in this work we study wrapping three body interactions related to the oncogenic Y220C mutation of the tumor suppressor protein p53. Our computational results rationalize the oncogenic nature of the Y220C mutation, explain the binding of a drug-like molecule already designed to restore the function of p53 and provide clues to help improve this function-rescue strategy and to apply in other drug design or re-engineering techniques.     

Variable Mutations at the p53-R273 Oncogenic Hotspot Position Leads to Altered Properties

Mutations in p53 protein, especially in the DNA-binding domain, is one of the major hallmarks of cancer. The R273 position is a DNA-contact position and has several oncogenic variants. Surprisingly, cancer patients carrying different mutant variants of R273 in p53 have different survival rates, indicating that the DNA-contact inhibition may not be the sole reason for reduced survival with R273 variants. Here, we probed the properties of three major oncogenic variants of the wild-type (WT) p53: [R273H]p53, [R273C]p53, and [R273L]p53. Using a series of biophysical, biochemical, and theoretical simulation studies, we observe that these oncogenic variants of the p53 not only suffer a loss in DNA binding, but they also show distinct structural stability, aggregation, and toxicity profiles. The WTp53 and the [R273H]p53 show the least destabilization and aggregation propensity. [R273C]p53 aggregation is disulfide mediated, leading to cross-β, thioflavin-T-positive aggregates, whereas hydrophobic interactions dominate self-assembly in [R273L]p53, leading to a mixture of amyloid and amorphous aggregates. Molecular dynamics simulations indicate different contact maps and secondary structures for the different variants along the course of the simulations. Our study indicates that each of the R273 variants has its own distinct property of stability and self-assembly, the molecular basis of which may lead to different types of cancer pathogenesis in vivo. These studies will aid the design of therapeutic strategies for cancer using residue-specific or process-specific protein aggregation as a target.     

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA-damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment.     

Modelling the interaction between the p53 DNA-binding domain and the p28 peptide fragment of Azurin

Recent experimental data reveal that the peptide fragment of Azurin called p28, constituted by the amino acid residues from 50 to 77 of the whole protein, retains both the Azurin cellular penetration ability and antiproliferative activity. p28 is hypothesized to act by stabilizing the well-known tumour suppressor p53 via a pathway independent from the oncogene Mdm2, which is the main p53 down-regulator, with its anticancer potentiality being probably connected with the binding of its amino acid residues 11 to 18 to p53. However, the p28 mode of action has not been completely elucidated yet, mostly because the details of the p28 interaction with p53 are still unknown. In the present study, computational docking modelling supported by cluster analysis, molecular dynamics simulations and binding free energy calculations have been performed to model the interaction between the DNA-binding domain (DBD) of p53 and the p28 fragment. Since the folding state of p28 when interacting with p53 inside the cell is not known, both the folded and the unfolded structures of this peptide have been taken into consideration. In both the cases, we have found that p28 is able to form with DBD a complex characterized by favourable negative binding free energy, high shape complementarity, and the presence of several hydrogen bonds at the interface. These results suggest that p28 might exert its anticancer action by hampering the binding of ubiquitin ligases to DBD, susceptible to promoting the p53 proteasomal degradation.     

Cellular protein alterations in colorectal cancer: p28, p53 and p65 studies

The expression and intracellular distribution of the p28 protein (MW 28 kD), which is electrophoretically specific for tumour cells, the p53 protein (MW 53 kD), one of the most frequently mutated in cancer, and the oncofoetal p65 protein (MW 65 kD), were investigated in colorectal cancer and normal colonic mucosa. The correlation between the expression of these proteins and the stage of the cancer, was evaluated. Neoplastic and normal tissues were fractionated by differential centrifugation, and protein analysis was performed by means of the Western blot technique in the presence of polyclonal (anti-p28 and anti-p65) or monoclonal (anti-p53) antibodies. Among the colorectal cancer cases examined 69% (11/16), 53% (10/19) and 77% (17/22) were positive for p28, p53 and p65, respectively. Immunoblot analysis revealed that the tumour specific p28 protein expression was mainly evident in the nuclear fraction, while the p53 and p65 proteins accumulated in the cell nuclei and the cytoplasm, although to different extents. The p65 protein appeared to be specifically expressed in the early stages of colorectal cancer, while a high level of p53 protein was typical for more invasive colorectal cancer stages.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.     

Protecting p53 from degradation

Inactivation of the p53 function is a common event in cancer. Approx. 50% of human tumours express mutant p53 and there is evidence that in others, including many childhood tumours, p53 function is impaired in other ways. These defects on p53 function may be due to the alteration of cellular factors that modulate p53 or to the expression of viral oncoproteins. Radiotherapy and many of the chemotherapeutic drugs currently used in cancer treatment are potent activators of p53. However, most of these therapies have a serious drawback; that is, the long-term consequences of their DNA-damaging effects. Understanding the mechanisms regulating p53 stability is crucial for the development of new strategies to activate p53 non-genotoxically. Here we describe the effect of a potent activator of the p53 response, the nuclear export inhibitor leptomycin B, on Mdm2 degradation and we provide evidence for the oligomerization of the p14ARF tumour suppressor and Mdm2 inhibitor in response to oxidative stress.