Development of a sensitive assay to detect reversibly oxidized protein cysteine sulfhydryl groups.

Protein sulfhydryl groups can undergo reversible oxidation reactions in response to reactive oxygen and reactive nitrogen species. Sensitive detection of sulfhydryl group oxidation in specific proteins is required to further our understanding of protein redox changes in biological systems. In general, to detect reversible oxidation reactions the oxidized sulfur atom is reduced to a sulfhydryl group followed by a reaction with a quantifiable agent. Our aim was to develop a sensitive method to detect reversibly oxidized protein sulfhydryl groups in a Western blot format. Conjugation of methoxypolyethylene glycol-maleimide (MAL-PEG) to protein sulfhydryl groups was optimized. Once MAL-PEG forms a covalent bond with the protein, the MAL-PEG-protein conjugate can be detected as a band shift by western analysis. The efficiency of MAL-PEG conjugation to protein was determined with creatine kinase. MAL-PEG conjugated to approximately 100% of the available sulfhydryl groups on creatine kinase within 30 min. Band shift detection sensitivity was measured using the redox-regulated protein p53. MAL-PEG conjugation coupled to western analysis detected a minimum of 0.23 pmol of oxidized p53. The MAL-PEG conjugation method described in this communication can be used to assess the reversible sulfhydryl oxidation status of proteins for which antibodies suitable for western analysis are available.

p53 protein oxidation in cultured cells in response to pyrrolidine dithiocarbamate: a novel method for relating the amount of p53 oxidation in vivo to the regulation of p53-responsive genes.

A novel method was developed to determine the oxidation status of proteins in cultured cells. Methoxy-polyethylene glycol-maleimide MW 2000 (MAL-PEG) was used to covalently tag p53 protein that was oxidized at cysteine residues in cultured cells. Treatment of MCF7 breast cancer cells with pyrrolidine dithiocarbamate (PDTC), a metal chelator, resulted in a minimum of 25% oxidation of p53. The oxidized p53 had an average of one cysteine residue oxidized per p53 protein molecule. The effect of PDTC treatment on downstream components of the p53 signal-transduction pathway was tested. PDTC treatment prevented actinomycin D-mediated up-regulation of two p53 effector gene products, murine double minute clone 2 oncoprotein and p21(WAF1/CIP1) (where WAF1 corresponds to wild-type p53-activated fragment 1 and CIP1 corresponds to cyclin-dependent kinase-interacting protein 1). Actinomycin D treatment led to accumulation of p53 protein in the nucleus. However, when cells were simultaneously treated with PDTC and actinomycin D, p53 accumulated in both the nucleus and the cytoplasm. The data indicate that an average of one cysteine residue per p53 protein molecule is highly sensitive to oxidation and that p53 can be efficiently oxidized by PDTC in cultured cells. PDTC-mediated oxidation of p53 correlates with altered p53 subcellular localization and reduced activation of p53 downstream effector genes. The novel method for detecting protein oxidation detailed in the present study may be used to determine the oxidation status of specific proteins in cells.

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.

Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin.

One mechanism by which chemotherapeutic agents kill tumor cells is by induction of apoptosis. Basic fibroblast growth factor (bFGF/FGF-2) has been reported to inhibit apoptosis in NIH 3T3 cells treated with chemotherapy drugs. We have investigated how bFGF modulates apoptosis induced by cisplatin in NIH 3T3 cells. Treatment with 10 microgram/ml cisplatin for 12 h induced apoptosis in 2 to 13% of the cells at 24 h post-treatment. Preincubation with 10 ng/ml bFGF for 24 h led to cisplatin-induced apoptosis in 20% to 50% of the cells. Preincubation with lower concentrations of bFGF (0.1-1 ng/ml) or simultaneous addition of bFGF and cisplatin had no effect on the amount of apoptosis. Pretreatment with bFGF also significantly decreased the dose-dependent survival of NIH 3T3 cells exposed to cisplatin, as determined by colony formation. Cells treated with 10 ng/ml bFGF showed a distinct morphology, appearing smaller and more refractile, before cisplatin exposure. The enhancement of cisplatin-induced apoptosis and the morphology shift demonstrated the same dose response to bFGF, and both effects were reversible if bFGF was removed from the medium for 24 h before cisplatin treatment. Mitogenic response to bFGF by NIH 3T3 cells saturated at 0.5 ng/ml, as measured by (3)H-thymidine uptake, and this response was blocked by coaddition of suramin, an inhibitor of FGF ligand-receptor interactions. Suramin did not reverse the enhancement of cisplatin-induced apoptosis by bFGF. Therefore, bFGF sensitized NIH 3T3 cells to cisplatin, and this effect might be mediated through a pathway separate from that used for mitogenic signaling.

TP53 tumor suppressor protein in normal human fibroblasts does not respond to 837 MHz microwave exposure.

The TP53 tumor suppressor protein (formerly known as p53) responds to a wide variety of environmental insults. To evaluate the safety of cellular telephones, TP53 responses in human fibroblast cells were studied after exposure to 837 MHz microwaves. Cells were exposed in a temperature-controlled transverse electromagnetic (TEM) chamber to a specific absorption rate (SAR) of 0.9 or 9.0 W/kg at 837 MHz continuous-wave (CW) microwave irradiation for 2 h. The TP53 protein levels were measured by Western blot at 2, 8, 24 and 48 h after treatment. The TP53 protein levels in microwave-treated cells, sham-treated cells, and untreated cells remained unchanged relative to each other at all times tested (Fisher test and Student-Newman-Keuls test, P > 0.05). No morphological alterations were observed in microwave-treated cells compared to sham-treated cells. We conclude that TP53 protein expression levels in cultured human fibroblast cells do not change significantly during a 48-h period after exposure to 837 MHz continuous microwaves for 2 h at SAR levels of 0.9 or 9.0 W/kg.

Pyrrolidine dithiocarbamate prevents p53 activation and promotes p53 cysteine residue oxidation.

Pyrrolidine dithiocarbamate (PDTC) is a thiol compound widely used to study the activation of redox-sensitive transcription factors. Although normally used as an antioxidant, PDTC has been shown to exert pro-oxidant activity on proteins both in vitro and in vivo. Because p53 redox status has been shown to alter its DNA binding capability, we decided to test the effect of PDTC on p53 activation. In this communication, we report that PDTC inhibits the activation of temperature-sensitive murine p53(Val-135) (TSp53) in the transformed rat embryo fibroblast line, A1-5, as well as wild-type human p53 in the normal diploid fibroblast line, WS1neo. In A1-5 cells, PDTC abrogated UV- and temperature shift-induced TSp53 nuclear translocation and p53-mediated transactivation of MDM2. PDTC also blocked UV-induced accumulation of wild-type p53 in WS1neo cells. Continual presence of PDTC was required for its effect as both UV-induced nuclear translocation and accumulation resumed after PDTC removal. We next investigated whether PDTC treatment altered the p53 redox state. We found that PDTC increased p53 cysteine residue oxidation in vivo. This represents the first direct evidence showing that the p53 redox state can be altered in vivo and that increased oxidation correlates with its inability to perform its downstream functions.

The MDM2 gene amplification database.

The p53 tumor suppressor gene is inactivated in human tumors by several distinct mechanisms. The best characterized inactivation mechanisms are: (i) gene mutation; (ii) p53 protein association with viral proteins; (iii) p53 protein association with the MDM2 cellular oncoprotein. The MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines by gene amplification, increased transcript levels and enhanced translation. This communication presents a brief review of the spectrum of MDM2 abnormalities in human tumors and compares the tissue distribution of MDM2 amplification and p53 mutation frequencies. In this study, 3889 samples from tumors or xenografts from 28 tumor types were examined for MDM2 amplification from previously published sources. The overall frequency of MDM2 amplification in these human tumors was 7%. Gene amplification was observed in 19 tumor types, with the highest frequency observed in soft tissue tumors (20%), osteosarcomas (16%) and esophageal carcinomas (13%). Tumors which showed a higher incidence of MDM2 amplification than p53 mutation were soft tissue tumors, testicular germ cell cancers and neuro-blastomas. Data from studies where both MDM2 amplification and p53 mutations were analyzed within the same samples showed that mutations in these two genes do not generally occur within the same tumor. In these studies, 29 out of a total of 33 MDM2 amplification-positive tumors had wild-type p53. We hypothesize that heretofore uncharacterized carcinogens favor MDM2 amplification over p53 mutations in certain tumor types. A database listing the MDM2 gene amplifications is available on the World Wide Web at http://www. infosci.coh.org/mdm2 . Charts of MDM2 amplification frequencies and comparisons with p53 genetic alterations are also available at this Web site.

In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation.

The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G1 arrest and perhaps apoptosis. To learn more about p53's transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53 consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.

Solution conformation of an essential region of the p53 transactivation domain.

BACKGROUND: The peptide segment surrounding residues Leu22 and Trp23 of the p53 transactivation domain plays a critical role in the transactivation activity of p53. This region binds basal transcriptional components such as the TATA-box binding protein associated factors TAFII40 and TAFII60 as well as the mdm-2 and adenovirus type 5 E1B 55 kDa oncoproteins. RESULTS: The structure of residues 14-28 of p53 was studied by nuclear magnetic resonance spectroscopy and found to prefer a two-beta-turn structure stabilized by a hydrophobic cluster consisting of residues known to be important for transactivation and binding to p53-binding proteins. A peptide segment in which Leu22 and Trp23 were replaced by Gln and Ser displays a random structure. CONCLUSIONS: This structural propensity observed in the wild-type p53 peptide is important for understanding the mechanism of transcriptional activation, because very few structural data are available on transactivation domains to date. These results should aid in the design of therapeutics that could competitively inhibit binding of p53 to the oncogene product mdm-2.

Geldanamycin prevents nuclear translocation of mutant p53.

p53 is a tumor suppressor protein that acts in the nucleus to effect cell cycle arrest and apoptosis. In some cells p53 is located in the cytoplasm, perhaps as a means of downregulating its activity. We recently showed that hsp90 forms a complex with the cytoplasmically localized mutant p53 (TSp53vall35) within transformed cells (Sepehrnia et al., J. Biol. Chem. 271, 15084, 1996). The present study was undertaken to determine the p53 conformation bound to hsp90 and the role of hsp90 in p53 nuclear translocation. We show that hsp90 binds both a native and a denatured form of p53 as determined by conformation-specific antibodies. hsp90 does not bind p53 in a spatial-specific manner because it remains bound to p53 when induced to translocate to the nucleus by the protein synthesis inhibitor cycloheximide (CHX). Treatment of transformed cells with geldanamycin (GA), a small molecule that binds hsp90, causes a rapid destabilization of p53 by 50%. Residual p53 that survives GA treatment is incapable of translocating to the nucleus. GA does not destabilize p53 in cells where p53 is genotypically wild type. Although GA appears to dramatically alter the translocating properties of mutant p53 it does not dissociate the p53-hsp90 complex. We suggest that a second chaperone protein, called p23, which we show also binds p53, may play an important role in these GA-mediated effects.

Analysis of the proportion of p53 bound to mdm-2 in cells with defined growth characteristics.

A critical parameter affecting cell growth properties is the relative levels of the p53 tumor suppressor protein and the mdm-2 oncoprotein. Because mdm-2 overexpression is observed in several types of human cancers and its physical association with p53 appears essential for down-regulating p53 activity the proportion of p53 bound to mdm-2 was examined in four types of cells with divergent growth properties: (1) Growth arrested cells (Al) expressing high levels of wild-type p53 activity; (2) Tumorigenic cells (3T3DM) expressing high levels of mdm-2; (3) Immortalized non tumorigenic cells (Swiss3T3 and Balb/c3T3); and (4) Normal murine fibroblasts. In Al cells, greater than 78% of the p53 was not bound to mdm-2, demonstrating that excess free p53 is available for cell cycle arrest. In 3T3DM cells 100% of the p53 was bound to mdm-2 and these cells were unable to support p53-mediated transactivation, a p53 function essential for cell growth inhibition. In Swiss3T3 cells 75% of the p53 was bound to mdm-2. In Balb/c3T3 cells and normal cells no detectable mdm-2 was bound to p53. Since free p53 was detected in several of these cell lines the possibility that mdm-2 is completely titrated by p53 was investigated. However, free mdm-2 was present in all these cells. Phosphorylation of p53 does not appear to control complex formation since the free and the mdm-2-bound form of p53 from Al cells had identical phosphorylation maps. These data suggest that a high proportion of p53 bound to mdm-2 is observed in some cells with a more transformed phenotype and that p53-mdm-2 complex formation is controlled by a posttranslational event other than p53 phosphorylation.

Heat shock protein 84 forms a complex with mutant p53 protein predominantly within a cytoplasmic compartment of the cell.

Cellular DNA damage results in the increased expression and accumulation of the p53 tumor suppressor protein within the nucleus which leads to cell cycle arrest or apoptosis. In some cases, however, wild-type p53 and some mutant forms of p53 reside in the cytoplasm of cancer cells. To understand the mechanism responsible for its cytoplasmic retention, studies were undertaken to determine if unique proteins form a complex with mutant p53 within the cytoplasm of transformed cells. One protein, with an apparent molecular mass of 92 kDa (p92), was observed to form a complex with a temperature-sensitive mutant p53 (TSp53(Val-135)) in the cytoplasm of transformed rat embryo fibroblasts at the non-permissive temperature. p92 copurified with TSp53(Val-135) on a p53-specific immunoaffinity column and a gel filtration column. The protein was purified to homogeneity and identified as hsp84 by partial amino acid sequence analysis. hsp84 is a member of the hsp90 class of proteins. At the non-permissive temperature, TSp53(Val-135) and hsp84 colocalized in the cytoplasm near the nuclear envelope. At the permissive temperature, TSp53(Val-135) resides in the nucleus and expresses a "wild-type like" conformation. Under these conditions hsp84 continued to reside in the cytoplasm and little or no hsp84 formed a complex with p53. The results suggest that hsp84 binds mutant p53 in a spatial and/or conformation dependent manner.

Identification and characterization of multiple mdm-2 proteins and mdm-2-p53 protein complexes.

A protein product of the mdm-2 oncogene (p90) has been recently shown to associate with the protein encoded by the tumor-suppressor gene p53. The mdm-2 gene was originally identified as a gene amplified in a spontaneously transformed Balb/c 3T3 cell line (3T3DM). This report describes the characterization of mdm-2 gene products and their interactions with the p53 protein. Polyclonal and monoclonal antibodies were generated against murine and human mdm-2 protein. These antibodies detected the mdm-2 p90 protein and at least four additional polypeptides (p85, p76, p74, p58-p57) in cultured cells. These additional proteins may arise from different spliced mRNA forms of the mdm-2 gene or post-translational modifications of the mdm-2 protein. The monoclonal antibodies distinguished at least three sets of mdm-2 proteins with distinct combinations of epitopes (p90 and p85; p76 and p74; p58-57). One or two of these proteins forms a complex with the p53 protein (p90, p58). These mdm-2 proteins were found to be overexpressed in 3T3DM cells and a subset of these proteins were complexed with p53. In 3T3DM cells, p90, like p53, had a short half-life of approximately 20 min and was localized to the cell nucleus. In resting cells stimulated with serum p90 levels and p90/p53 complex levels increased in the late G1 phase of the cell cycle. The p90 mdm-2 protein could regulate p53 activity in the late G1 phase of the cell cycle.

Wild-type p53 binds to the TATA-binding protein and represses transcription.

p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.

A cellular phosphoprotein with an apparent molecular mass of 90 kd (p90) that forms a complex with both mutant and wild-type p53 protein has been characterized, purified, and identified. The protein was identified as a product of the murine double minute 2 gene (mdm-2). The mdm-2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. To determine if mdm-2 could modulate p53 transactivation, a p53-responsive element from the muscle creatine kinase gene was employed. A wild-type p53-expressing plasmid enhanced the expression of the p53-responsive element when cotransfected into cells that contain no endogenous p53. When a cosmid expressing mdm-2 was transfected with this p53-expressing plasmid, the transactivation of the p53-responsive element was inhibited. Thus, a product of the mdm-2 oncogene forms a tight complex with the p53 protein, and the mdm-2 oncogene can inhibit p53-mediated transactivation.

The p53 tumour suppressor gene.

The cell cycle is composed of a series of steps which can be negatively or positively regulated by various factors. Chief among the negative regulators is the p53 protein. Alteration or inactivation of p53 by mutation, or by its interactions with oncogene products of DNA tumour viruses, can lead to cancer. These mutations seem to be the most common genetic change in human cancers.

The fidelity of protein synthesis: can mischarging by aspartyl-tRNA(Asp) synthetase lead to the formation of isoaspartyl residues in proteins?

We have tested the hypothesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain beta-carboxyl group is linked to the tRNA(Asp) instead of the main chain alpha-carboxyl group. If this altered Asp-tRNA(Asp) is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or beta-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA(Asp) synthetase from Escherichia coli and [3H]aspartic acid, we have prepared [3H]aspartyl-tRNA(Asp) complexes and directly analyzed the linkage of the [3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the alpha-carboxyl group of aspartate to the tRNA produces [3H]isoasparagine, while the mischarging reaction leads to [3H]asparagine formation after ammonolysis. We have separated [3H]isoasparagine from [3H]asparagine and found an upper limit of 1 asparagine per 10,000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the alpha- and beta-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.