ADP-ribosylation of p53 tumor suppressor protein: Mutant but not wild-type p53 is modified

Poly(ADP-ribosyl)ation of mutant and wild-type p53 was studied in transformed and nontransformed rat cell lines constitutively expressing the temperature-sensitive p53^135val. It was found that in both cell types at 37.5°C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence studies with affinity purified anti-poly(ADP-ribose) transferase (pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 37.5°C, where p53 adopts wild-type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tighly complexed to pADPRT and becomes modified. At 32.5°C binding to DNA of p53 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein. © 1996 Wiley-Liss, Inc.     

Different recognition of DNA modified by aatitumor cisplatin and its clinically ineffective trans isomer by tumor suppressor protein p53

The p53 gene encodes a nuclear phosphoprotein that is biologically activated in response to genotoxic stresses including treatment with anticancer platinum drugs. The DNA binding activity of p53 protein is crucial for its tumor suppressor function. DNA interactions of active wild-type human p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by antitumor cisplatin and its clinically ineffective trans isomer (transplatin) were investigated by using a gel mobility shift assay. It was found that DNA adducts of cisplatin reduced binding affinity of the consensus DNA sequence to p53, whereas transplatin adducts did not. This result was interpreted to mean that the precise steric fit required for the formation and stability of the tetrameric complex of p53 with the consensus sequence cannot be attained, as a consequence of severe conformational perturbations induced in DNA by cisplatin adducts. The results also demonstrate an increase of the binding affinity of p53 to DNA lacking the consensus sequence and modified by cisplatin but not by transplatin. In addition, only major 1,2-GG intrastrand cross-links of cisplatin are responsible for this enhanced binding affinity of p53. The data base on structures of various DNA adducts of cisplatin and transplatin reveals distinctive structural features of 1,2-intrastrand cross-links of cisplatin, suggesting a unique role for this adduct in the binding of p53 to DNA lacking the consensus sequence. The results support the hypothesis that the mechanism of antitumor activity of cisplatin may also be associated with its efficiency to affect the binding affinity of platinated DNA to active p53 protein.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.     

Activation of the p53 tumor suppressor protein

The p53 tumor suppressor gene plays an important role in preventing cancer development, by arresting or killing potential tumor cells. Mutations within the p53 gene, leading to the loss of p53 activity, are found in about half of all human cancers, while many of the tumors that retain wild type p53 carry mutations in the pathways that allow full activation of p53. In either case, the result is a defect in the ability to induce a p53 response in cells undergoing oncogenic stress. Significant advances have been made recently in our understanding of the molecular pathways through which p53 activity is regulated, bringing with them fresh possibilities for the design of cancer therapies based on reactivation of the p53 response.     

Cell-specific modulation of papovavirus replication by tumor suppressor protein p53

Small DNA tumor viruses like human papillomaviruses, simian virus 40, and adenoviruses modulate the activity of cellular tumor suppressor proteins p53 and/or pRB. These viruses replicate as nuclear multicopy extrachromosomal elements during the S phase of the cell cycle, and it has been suggested that inactivation of p53 and pRb is necessary for directing the cells to the S phase. Mouse polyomavirus (Py), however, modulates only the pRB protein activity without any obvious interference with the action of p53. We show here that Py replication was not suppressed by the p53 protein indeed in all tested different mouse cell lines. In addition, E1- and E2-dependent papillomavirus origin replication was insensitive to the action of p53 in mouse cells. We show that in hamster (Chinese hamster ovary) or human (osteosarcoma 143) cell lines the replication of both Py and papillomavirus origins was efficiently blocked by p53. The block of Py replication in human and hamster cells is not caused by the downregulation of large T-antigen expression. The deletion analysis of the p53 protein shows that the RPA binding, proline-rich regulatory, DNA-binding, and oligomerization domains are necessary for p53 action in both replication systems. These results indicate that in mouse cells the p53 protein could be inactive for the suppression of papovavirus replication.     

肿瘤抑制蛋白p53的活性调控

p53 又称为分子警察或基因的保护神.在面对不同类型和强度的应激时,细胞究竟选择细胞周期停滞、凋亡还是衰老时 p53发挥中心调节作用.作为一种转录调控因子它主要通过对下游的目的基因进行转录调控来发挥功能.p53 结合 DNA 启动子能力也可通过多种方式被调节.这些调节机制主要包括 p53 的亚细胞定位调控、p53 的蛋白稳定性调控和 p53 的翻译后修饰.     

Differential recognition by the tumor suppressor protein p53 of DNA modified by the novel antitumor trinuclear platinum drug BBR3464 and cisplatin

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.     

Interaction of metallothionein with tumor suppressor p53 protein

Previous reports have shown that metallothionein (MT) may modulate p53 activity through zinc exchange. However, little is known on a direct interaction between MT and p53 in cells. The results demonstrate an interaction between MT and p53 can occur in vitro. The complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially associated with Apo-MT. Our data suggest that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells.     

Physical and functional interactions of the tumor suppressor protein p53 and DNA polymerase alpha-primase

The wild-type form of p53 contains an intrinsic 3′–5′-exonuclease activity. As p53 forms a complex with DNA polymerase α-primase (pol-prim) in vivo this finding suggests that p53 might cooperate with pol-prim to stabilize the genetic information of living cells. To test this hypothesis, exonuclease-free DNA pol-prim was expressed alone or together with p53 for purification. Pol-prim formed a complex with p53, which was purified by ion exchange and immunoaffinity chromatography from baculovirus-infected insect cells. The p53-containing pol-prim fractions removed a 3′-unpaired nucleotide with a 1.5–2-fold higher rate than a paired nucleotide, whereas the four subunit pol-prim did not have any exonuclase activity. Therefore, only p53/pol-prim was able to elongate a primer-template that contained a 3′-unpaired primer end in vitro. To achieve this, the 3′–5′-exonuclease activity of p53 excised the unpaired nucleotide at the 3′-end of the primer and created a paired 3′-end, which pol-prim was able to elongate. The exonuclease activity of p53 as well as the elongation of a primer with a mispaired 3′-end was inhibited specifically by the anti-p53 monoclonal antibodies PAb240 and PAb421.     

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.     

Recognition of cisplatin-damaged DNA by p53 protein: critical role of the p53 C-terminal domain

It was shown previously that the p53 protein can recognize DNA modified with antitumor agent cisplatin (cisPt-DNA). Here, we studied p53 binding to the cisPt-DNA using p53 deletion mutants and via modulation of the p53-DNA binding by changes of the protein redox state. Isolated p53 C-terminal domain (CTD) bound to the cisPt-DNA with a significantly higher affinity than to the unmodified DNA. On the other hand, p53 constructs involving the core domain but lacking the C-terminal DNA binding site (CTDBS) exhibited only small binding preference for the cisPt-DNA. Oxidation of cysteine residues within the CD of posttranslationally unmodified full length p53 did not affect its ability to recognize cisPt-DNA. Blocking of the p53 CTDBS by a monoclonal antibody Bp53-10.1 resulted in abolishment of the isolated CTD binding to the cisPt-DNA. Our results demonstrate a crucial role of the basic region of the p53 CTD (aa 363-382) in the cisPt-DNA recognition.     

Refolding and structural characterization of the human p53 tumor suppressor protein

The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level. We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies. To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding. The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces. The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody. Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.     

MDM2--master regulator of the p53 tumor suppressor protein

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 protein binds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion that prevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we present evidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of different species. These highly conserved regions encompass residues 42-94 (CR1), 301-329 (CR2), and 444-483 (CR3) on human MDM2. These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferring ubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potential uses of this knowledge in the fight against cancer.