Transcriptome and chromatin landscape changes associated with trastuzumab resistance in HER2+ breast cancer cells.

We set out to uncover transcriptome and chromatin landscape changes that occur in HER2 + breast cancer (BC) cells upon acquiring resistance to trastuzumab. RNA-seq analysis was applied to two independently-derived BC cell lines with acquired resistance to trastuzumab (SKBr3.HerR and BT-474HerR) and their parental drug-sensitive cell lines (SKBr3 and BT-474). Chromatin landscape analysis indicated that the most significant increase in accessibility in resistant cells occurs in PPP1R1B within a segment spanning introns 1b through intron 3. Footprint analysis of this segment revealed that FoxJ3 (within intron 2) and Pou5A1/Sox2 (within inton 3) transcription factor motifs are protected in resistant cells. Overall, 344 shared genes were upregulated in both resistant cell lines relative to their parental counterparts and 453 shared genes were downregulated in both resistant cell lines relative to their parental counterparts. In resistant cells, genes associated with autophagy and mitochondria organization are upregulated and genes associated with ribosome assembly and cell cycle are downregulated relative to parental cells. The five top upregulated genes in drug-resistant breast cancer cells are APOD, AZGP1, ETV5, ALPP, and PPP1R1B. This is the first report of increased chromatin accessibility within PPP1R1B associated with its t-Darpp transcript increase, and points to a possible mechanism for its activation in trastuzumab-resistant cells.

Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction.

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via "computational conformer selection" for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 µM) > bepridil (23 µM) > azelastine (25 µM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

Darpp-32 and t-Darpp protein products of PPP1R1B: Old dogs with new tricks.

The PPP1R1B gene is located on chromosome 17q12 (39,626,208-39,636,626[GRCh38/hg38]), which codes for multiple transcripts and two experimentally-documented proteins Darpp-32 and t-Darpp. Darpp-32 (Dopamine and cAMP Regulated Phosphoprotein), discovered in the early 1980s, is a protein whose phosphorylation is upregulated in response to cAMP in dopamine-responsive tissues in the brain. It's phosphorylation profile modulates its ability to bind and inhibit Protein Phosphatase 1 activity, which, in turn, controls the activity of hundreds of phosphorylated proteins. PPP1R1B knockout mice exhibit subtle learning defects. In 2002, the second protein product of PPP1R1B was discovered in gastric cancers: t-Darpp (truncated Darpp-32). The start codon of t-Darpp is amino acid residue 37 of Darpp-32 and it lacks the domain responsible for modulating Protein Phosphatase 1. Aside from gastric cancers, t-Darpp and/or Darpp-32 is overexpressed in tumor cells from breast, colon, esophagus, lung and prostate tissues. More than one research team has demonstrated that these proteins, through mechanisms that to date remain cloudy, activate AKT, a protein whose phosphorylation leads to cell survival and blocks apoptosis. Furthermore, in Her2 positive breast cancers (an aggressive form of breast cancer), t-Darpp/Darpp-32 overexpression causes resistance to the frequently-administered anti-Her2 drug, trastuzumab (Herceptin), likely through AKT activation. Here we briefly describe how Darpp-32 and t-Darpp were discovered and report on the current state of knowledge of their involvement in cancers. We present a case for the development of an anti-t-Darpp therapeutic agent and outline the unique challenges this endeavor will likely encounter.

t-Darpp Activates IGF-1R Signaling to Regulate Glucose Metabolism in Trastuzumab-Resistant Breast Cancer Cells.

Purpose: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp overexpression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers.Experimental Design: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival. To assess t-Darpp's metabolic effects, we used the Seahorse flux analyzer to measure glucose metabolism in trastuzumab-resistant SK-BR-3 cells (SK.HerR) that have high endogenous t-Darpp levels and SK.tDrp cells that stably overexpress exogenous t-Darpp. To investigate t-Darpp's mechanism of action, we evaluated t-Darpp:IGF-1R complexes by coimmunoprecipitation and proximity ligation assays. We used pathway-specific inhibitors to study the dependence of t-Darpp effects on IGF-1R signaling. We used siRNA knockdown to determine whether glucose reliance in SK.HerR cells was mediated by t-Darpp.Results: In breast tumors, PPP1R1B mRNA levels were inversely correlated with IGF-1R mRNA levels and directly associated with shorter overall survival. t-Darpp overexpression was sufficient to increase glucose metabolism in SK.tDrp cells and essential for the glycolytic phenotype of SK.HerR cells. Recombinant t-Darpp stimulated glucose uptake, glycolysis, and IGF-1R-Akt signaling in SK-BR-3 cells. Finally, t-Darpp stimulated IGF-1R heterodimerization with ErbB receptors and required IGF-1R signaling to confer its metabolic effects.Conclusions: t-Darpp activates IGF-1R signaling through heterodimerization with EGFR and HER2 to stimulate glycolysis and confer trastuzumab resistance. Clin Cancer Res; 24(5); 1216-26. ©2017 AACR.

t-Darpp stimulates protein kinase A activity by forming a complex with its RI regulatory subunit.

t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDpT39A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDpT39A cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp's effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp's activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp-mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.

t-Darpp is an elongated monomer that binds calcium and is phosphorylated by cyclin-dependent kinases 1 and 5.

t-Darpp (truncated isoform of dopamine- and cAMP-regulated phosphoprotein) is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells, t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin-dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 and cyclin-dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated cross-linking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

Clinicopathological and Targeted Exome Gene Features of a Patient with Metastatic Acinic Cell Carcinoma of the Parotid Gland Harboring an ARID2 Nonsense Mutation and CDKN2A/B Deletion.

We describe the presentation, treatment, clinical outcome, and targeted genome analysis of a metastatic salivary acinic cell carcinoma (AciCC). A 71-year-old male presented with a 3 cm right tail of a parotid lesion, first detected as a nodule by the patient seven months earlier. He had a right total parotidectomy with cranial nerve VII resection, right facial nerve resection and grafting, resection of the right conchal cartilage, and right modified radical neck dissection. The primary tumor revealed AciCC with two distinct areas: a well-differentiated component with glandular architecture and a dedifferentiated component with infiltrative growth pattern associated with prominent stromal response, necrosis, perineural invasion, and cellular pleomorphism. Tumor staging was pT4 N0 MX. Immunohistochemistry staining showed pankeratin (+), CD56 (-), and a Ki67 proliferation index of 15%. Upon microscopic inspection, 49 local lymph nodes resected during parotidectomy were negative for cancer cells. Targeted sequencing of the primary tumor revealed deletions of CDKN2A and CDKN2B, a nonsense mutation in ARID2, and single missense mutations of unknown significance in nine other genes. Despite postoperative localized radiation treatment, follow-up whole body PET/CT scan showed lung, soft tissue, bone, and liver metastases. The patient expired 9 months after resection of the primary tumor.

The MDM2 gene family.

MDM2 is an oncoprotein that blocks p53 tumor suppressor-mediated transcriptional transactivation, escorts p53 from the cell nucleus to the cytoplasm, and polyubiquitylates p53. Polyubiquitylated p53 is rapidly degraded in the cytoplasm by the 26S proteasome. MDM2 is abnormally upregulated in several types of cancers, especially those of mesenchymal origin. MDM4 is a homolog of MDM2 that also inhibits p53 by blocking p53-mediated transactivation. MDM4 is required for MDM2-mediated polyubiquitylated of p53 and is abnormally upregulated in several cancer types. MDM2 and MDM4 genes have been detected in all vertebrates to date and only a single gene homolog, named MDM, has been detected in some invertebrates. MDM2, MDM4, and MDM have similar gene structures, suggesting that MDM2 and MDM4 arose through a duplication event more than 440 million years ago. All members of this small MDM2 gene family contain a single really interesting new gene (RING) domain (with the possible exception of lancelet MDM) which places them in the RING-domain superfamily. Similar to MDM2, the vast majority of proteins with RING domains are E3 ubiquitin ligases. Other RING domain E3 ubiquitin ligases that target p53 are COP1, Pirh2, and MSL2. In this report, we present evidence that COP1, Pirh2, and MSL2 evolved independently of MDM2 and MDM4. We also show, through structure homology models of invertebrate MDM RING domains, that MDM2 is more evolutionarily conserved than MDM4.

Identification of FDA-approved drugs that computationally bind to MDM2.

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2--an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction.

The evolution of MDM2 family genes.

MDM2 and MDM4 are proto-oncoproteins that bind to and inhibit members of the p53 protein family, p53, p73 and possibly p63. p53 is a mammalian tumor suppressor and p63 and p73 are critical for development. With the sequencing of genomes from multiple organisms there is mounting evidence for a consensus scenario of p53 gene family evolution. A single p53/p63/p73 gene is in invertebrates and required for maintenance of germline DNA. Gene duplication occurred in an ancestor in common with cartilaginous fishes, giving rise to a separate p53 gene and at least one ancestral p63/p73 gene. In bony vertebrates, all three p53 gene family paralogs, p53, p63, and p73 are distinct genes. This raises the question of how MDM2 and MDM4 genes evolved. We show evidence that MDM2 and MDM4 arose from a gene duplication event prior to the emergence of bony vertebrates more than 440 millionyears ago. Comparative genome studies indicate that invertebrate organisms have only one MDM homolog. In jawed vertebrates, the p53-binding domains of MDM2 and MDM4 proteins evolved at a high rate, approaching the evolution rate of the MDM2-binding domain of p53. However, the MDM2-binding domain of p73 exhibits markedly stronger conservation suggesting novel p53-independent functions. The most conserved domain within all MDM2 family members is the RING domain of the MDM2 ortholog which is responsible for ubiquitination of p53 and heterodimerization with MDM4. We suggest a model where oligomerization is an ancient function of MDM and ubiquitination activity was acquired later near the MDM gene duplication event coinciding with the time of the emergence of p53 as a distinct gene.

Prediction of reversibly oxidized protein cysteine thiols using protein structure properties.

Protein cysteine thiols can be divided into four groups based on their reactivities: those that form permanent structural disulfide bonds, those that coordinate with metals, those that remain in the reduced state, and those that are susceptible to reversible oxidation. Physicochemical parameters of oxidation-susceptible protein thiols were organized into a database named the Balanced Oxidation Susceptible Cysteine Thiol Database (BALOSCTdb). BALOSCTdb contains 161 cysteine thiols that undergo reversible oxidation and 161 cysteine thiols that are not susceptible to oxidation. Each cysteine was represented by a set of 12 parameters, one of which was a label (1/0) to indicate whether its thiol moiety is susceptible to oxidation. A computer program (the C4.5 decision tree classifier re-implemented as the J48 classifier) segregated cysteines into oxidation-susceptible and oxidation-non-susceptible classes. The classifier selected three parameters critical for prediction of thiol oxidation susceptibility: (1) distance to the nearest cysteine sulfur atom, (2) solvent accessibility, and (3) pKa. The classifier was optimized to correctly predict 136 of the 161 cysteine thiols susceptible to oxidation. Leave-one-out cross-validation analysis showed that the percent of correctly classified cysteines was 80.1% and that 16.1% of the oxidation-susceptible cysteine thiols were incorrectly classified. The algorithm developed from these parameters, named the Cysteine Oxidation Prediction Algorithm (COPA), is presented here. COPA prediction of oxidation-susceptible sites can be utilized to locate protein cysteines susceptible to redox-mediated regulation and identify possible enzyme catalytic sites with reactive cysteine thiols.

Capture of p53 by electrodes modified with consensus DNA duplexes and amplified voltammetric detection using ferrocene-capped gold nanoparticle/streptavidin conjugates.

p53, a tumor suppressor protein and a transcription factor, is capable of inhibiting the growth of tumor cells by eliciting either cell-cycle arrest or apoptosis through a cascade of events. p53 binds sites within the promoters of several genes that conform to a sequence commonly defined as the consensus site. In more than 50% of cancer cases, the p53 gene has been found to be mutated and the p53 protein loses its ability to bind the consensus DNA. In this work, double-stranded (ds-) oligonucleotides (ODNs) containing the consensus site are immobilized onto gold electrodes to capture wild-type p53. The cysteine residues on the exterior of the p53 molecule were derivatized for the attachment of gold nanoparticle/streptavidin conjugates capped with multiple ferrocene (Fc) groups. Well-defined voltammetric peaks of high signal intensity were obtained, and p53 concentration as low as 2.2 pM was measured. The peak heights were found to be dependent on the surface density of the consensus ds-ODN, the sequence of the immobilized ODNs, and the p53 concentration. With base pair(s) in the full consensus binding sequence altered, the level of p53 binding was found to decrease sharply, and no p53 binding occurred at electrodes covered with nonconsensus ds-ODNs. The amenability of this method to the analyses of p53 from normal and cancer cell lysates was also demonstrated. Owing to the p53 mutation in the cancer cells, the concentration of the wild-type p53 was found to decrease significantly (by about 50-182 times). The sensitivity and amenability for real sample analysis of the method compared well with enzyme-linked immunosorbant assay (ELISA), and complements ELISA in that wild-type p53, instead of total p53 (wild-type and mutant p53) concentration, is measured. The method described herein is simple and selective and does not require the use of p53 antibodies.

A summer program designed to educate college students for careers in bioinformatics.

A summer program was created for undergraduates and graduate students that teaches bioinformatics concepts, offers skills in professional development, and provides research opportunities in academic and industrial institutions. We estimate that 34 of 38 graduates (89%) are in a career trajectory that will use bioinformatics. Evidence from open-ended research mentor and student survey responses, student exit interview responses, and research mentor exit interview/survey responses identified skills and knowledge from the fields of computer science, biology, and mathematics that are critical for students considering bioinformatics research. Programming knowledge and general computer skills were essential to success on bioinformatics research projects. General mathematics skills obtained through current undergraduate natural sciences programs were adequate for the research projects, although knowledge of probability and statistics should be strengthened. Biology knowledge obtained through the didactic phase of the program and prior undergraduate education was adequate, but advanced or specific knowledge could help students progress on research projects. The curriculum and assessment instruments developed for this program are available for adoption by other bioinformatics programs at http://www.calstatela.edu/SoCalBSI.

WRN exonuclease activity is blocked by DNA termini harboring 3' obstructive groups.

Reactive oxygen species, generated either by cellular respiration or upon exposure to environmental agents such as ionizing radiation (IR), attack DNA to form a variety of oxidized base and sugar modifications. Accumulation of oxidative DNA damage has been associated with age-related disease as well as the aging process. Single-strand breaks harboring oxidative 3' obstructive termini, e.g. 3' phosphates and 3' phosphoglycolates, must be removed prior to DNA repair synthesis or ligation. In addition, 3' tyrosyl-linked protein damage, resulting from therapeutic agents such as camptothecin (CPT), must be processed to initiate repair. Several nucleases participate in DNA repair and the excision of 3' obstructive ends. As the protein defective in the segmental progeroid Werner syndrome (WRN) possesses 3'-5' exonuclease activity, and Werner syndrome cells are hypersensitive to IR and CPT, we examined for WRN exonuclease activity on 3' blocking lesions. Moreover, we compared side-by-side the activity of four prominent human 3'-5' exonucleases (WRN, APE1, TREX1, and p53) on substrates containing 3' phosphates, phosphoglycolates, and tyrosyl residues. Our studies reveal that while WRN degrades 3' hydroxyl containing substrates in a non-processive manner, it does not excise 3' phosphate, phosphoglycolate, or tyrosyl groups. In addition, we found that APE1 was most active at excising 3' blocking termini in comparison to the disease-related exonucleases TREX1, WRN, and p53 under identical physiological reaction conditions, and that TREX1 was the most powerful 3'-5' exonuclease on undamaged oligonucleotide substrates.

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification.

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.

Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization.

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

Purification of recombinant p53 from Sf9 insect cells.

We describe a method for purifying recombinant p53 from baculovirus infected cells in one step by anion exchange chromatography. The p53 is full-length with no flanking sequences and its expression is driven by the baculovirus polyhedron promoter. We also describe how to concentrate the p53 up to 0.9 mg/mL. By gel filtration analysis, we demonstrate that 20% of the p53 forms a tetramer, and 80% forms a monomer. In a DNA binding assay known as the electromobility shift assay, the purified p53/DNA complex forms a single band the gel. This simple procedure should be useful for investigations into the biochemistry of the p53 protein.

Copper uptake is required for pyrrolidine dithiocarbamate-mediated oxidation and protein level increase of p53 in cells.

The p53 tumour-suppressor protein is a transcription factor that activates the expression of genes involved in cell cycle arrest, apoptosis and DNA repair. The p53 protein is vulnerable to oxidation at cysteine thiol groups. The metal-chelating dithiocarbamates, pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate, ethylene(bis)dithiocarbamate and H(2)O(2) were tested for their oxidative effects on p53 in cultured human breast cancer cells. Only PDTC oxidized p53, although all oxidants tested increased the p53 level. Inductively coupled plasma MS analysis indicated that the addition of 60 microM PDTC increased the cellular copper concentration by 4-fold, which was the highest level of copper accumulated amongst all the oxidants tested. Bathocuproinedisulphonic acid, a membrane-impermeable Cu(I) chelator inhibited the PDTC-mediated copper accumulation. Bathocuproinedisulphonic acid as well as the hydroxyl radical scavenger d-mannitol inhibited the PDTC-dependent increase in p53 protein and oxidation. Our results show that a low level of copper accumulation in the range of 25-40 microg/g of cellular protein increases the steady-state levels of p53. At copper accumulation levels higher than 60 microg/g of cellular protein, p53 is oxidized. These results suggest that p53 is vulnerable to free radical-mediated oxidation at cysteine residues.