RIDR-PI-103, ROS-activated prodrug PI3K inhibitor inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor-resistant BRAF-mutant melanoma cells.

Reactive oxygen species (ROS) levels are elevated after acquisition of resistance to v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors including dabrafenib and MEK inhibitors such as trametinib in BRAF-mutant melanoma. To circumvent toxicity to PI-103 (a pan PI3K inhibitor), we utilized a novel ROS-induced drug release (RIDR)-PI-103, with a self-cyclizing moiety linked to PI-103. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to phosphatidylinositol 3,4,5-triphosphate (PIP 3 ). Previous findings demonstrate that trametinib and dabrafenib-resistant (TDR) cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on melanocytes and TDR cells. RIDR-PI-103 exhibited less toxicity compared to PI-103 at 5 µM in melanocytes. RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM. Twenty-four hour treatment with RIDR-PI-103 inhibited p-Akt, p-S6 (Ser240/244) and p-S6 (Ser235/236). We assessed the mechanism of activation of RIDR-PI-103, using glutathione or t-butyl hydrogen peroxide (TBHP) on the TDR cells in the presence or absence of RIDR-PI-103. Addition of the ROS scavenger glutathione to RIDR-PI-103 significantly rescued the cell proliferation in TDR cell lines while addition of the ROS inducer TBHP and RIDR-PI-103 inhibited cell proliferation in WM115 and WM983B TDR cell lines. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor-resistant cells will expand possible treatment options and open avenues for the development of new ROS-based treatment therapies for BRAF-mutant melanoma patients.

Identification of a Noxo1 inhibitor by addition of a polyethylene glycol chain.

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2) which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450, through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. Design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point. The initial compound was then modified by addition of a polyethylene glycol linked biotin. Both inhibitors were found to be non-toxic in human keratinocyte cells. Further in vitro experiments using isothermal calorimetric binding quantification showed the modified biotinylated compound bound Noxo1 peptide with a KD of 2 nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors. Experiments show that ultraviolet light exposure of with the lead compound was able to reduce the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. Further synthesis showed that the polyethylene glycol but not the biotin was essential for inhibition.

Phosphoinositide 3-Kinase (PI3K) Reactive Oxygen Species (ROS)-Activated Prodrug in Combination with Anthracycline Impairs PI3K Signaling, Increases DNA Damage Response and Reduces Breast Cancer Cell Growth.

RIDR-PI-103 is a novel reactive oxygen species (ROS)-induced drug release prodrug with a self-cyclizing moiety linked to a pan-PI3K inhibitor (PI-103). Under high ROS, PI-103 is released in a controlled manner to inhibit PI3K. The efficacy and bioavailability of RIDR-PI-103 in breast cancer remains unexplored. Cell viability of RIDR-PI-103 was assessed on breast cancer cells (MDA-MB-231, MDA-MB-361 and MDA-MB-453), non-tumorigenic MCF10A and fibroblasts. Matrigel colony formation, cell proliferation and migration assays examined the migratory properties of breast cancers upon treatment with RIDR-PI-103 and doxorubicin. Western blots determined the effect of doxorubicin ± RIDR-PI-103 on AKT activation and DNA damage response. Pharmacokinetic (PK) studies using C57BL/6J mice determined systemic exposure (plasma concentrations and overall area under the curve) and T1/2 of RIDR-PI-103. MDA-MB-453, MDA-MB-231 and MDA-MB-361 cells were sensitive to RIDR-PI-103 vs. MCF10A and normal fibroblast. Combination of doxorubicin and RIDR-PI-103 suppressed cancer cell growth and proliferation. Doxorubicin with RIDR-PI-103 inhibited p-AktS473, upregulated p-CHK1/2 and p-P53. PK studies showed that ~200 ng/mL (0.43 µM) RIDR-PI-103 is achievable in mice plasma with an initial dose of 20 mg/kg and a 10 h T1/2. (4) The prodrug RIDR-PI-103 could be a potential therapeutic for treatment of breast cancer patients.

Antifungal Activity of the Lipophilic Antioxidant Ferrostatin-1.

Ferrostatin-1 (Fer-1) is a lipophilic antioxidant that effectively blocks ferroptosis, a distinct non-apoptotic form of cell death caused by lipid peroxidation. During many infections, both pathogens and host cells are subjected to oxidative stress, but the occurrence of ferroptosis had not been investigated. We examined ferroptosis in macrophages infected with the pathogenic yeast Histoplasma capsulatum. Unexpectedly, Fer-1 not only reduced the death of macrophages infected in vitro, but inhibited the growth of H. capsulatum and related species Paracoccidioides lutzii and Blastomyces dermatitidis at concentrations under 10 µm. Other antioxidant ferroptosis inhibitors, including liproxstatin-1, did not prevent fungal growth or reduce macrophage death. Structural analysis revealed a potential similarity of Fer-1 to inhibitors of fungal sterol synthesis, and ergosterol content of H. capsulatum decreased more than twofold after incubation with Fer-1. Strikingly, additional Fer-1 analogues with slight differences from Fer-1 had limited impact on fungal growth. In conclusion, the ferroptosis inhibitor Fer-1 has unexpected antifungal potency distinct from its antiferroptotic activity.

Self-Cyclizing Antioxidants to Prevent DNA Damage Caused by Hydroxyl Radical.

Antioxidant therapy is a promising treatment strategy for protecting DNA from the damage caused by reactive oxygen species (ROS). Here, we report new self-cyclizing antioxidant reagents that are selective for the hydroxyl radical. Our mechanistic investigation revealed that the reagents react with three equivalents of oxidant in a cascade reaction to form a bicyclic final product. Among the reagents synthesized, 1 c showed favorable properties in vitro and in cellular studies. Using As2 O3 , which triggers ROS production, we showed that 1 c prevents formation of the guanine oxidation product 2,2,4-triamino-2H-oxazol-5-one-2'-deoxyribonucleoside and lowers cellular levels of reactive oxygen. The described self-cyclizing antioxidants are efficient, flexible, and tunable reagents with the potential to limit toxic oxidative stress.

Ruthenium Complexes are pH-Activated Metallo Prodrugs (pHAMPs) with Light-Triggered Selective Toxicity Toward Cancer Cells.

Metallo prodrugs that take advantage of the inherent acidity surrounding cancer cells have yet to be developed. We report a new class of pH-activated metallo prodrugs (pHAMPs) that are activated by light- and pH-triggered ligand dissociation. These ruthenium complexes take advantage of a key characteristic of cancer cells and hypoxic solid tumors (acidity) that can be exploited to lessen the side effects of chemotherapy. Five ruthenium complexes of the type [(N,N)2Ru(PL)]2+ were synthesized, fully characterized, and tested for cytotoxicity in cell culture (1A: N,N = 2,2'-bipyridine (bipy) and PL, the photolabile ligand, = 6,6'-dihydroxybipyridine (6,6'-dhbp); 2A: N,N = 1,10-phenanthroline (phen) and PL = 6,6'-dhbp; 3A: N,N = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline (dop) and PL = 6,6'-dhbp; 4A: N,N = bipy and PL = 4,4'-dimethyl-6,6'-dihydroxybipyridine (dmdhbp); 5A: N,N = 1,10-phenanthroline (phen) and PL = 4,4'-dihydroxybipyridine (4,4'-dhbp). The thermodynamic acidity of these complexes was measured in terms of two pKa values for conversion from the acidic form (XA) to the basic form (XB) by removal of two protons. Single-crystal X-ray diffraction data is discussed for 2A, 2B, 3A, 4B, and 5A. All complexes except 5A showed measurable photodissociation with blue light (λ = 450 nm). For complexes 1A-4A and their deprotonated analogues (1B-4B), the protonated form (at pH 5) consistently gave faster rates of photodissociation and larger quantum yields for the photoproduct, [(N,N)2Ru(H2O)2]2+. This shows that low pH can lead to greater rates of photodissociation. Cytotoxicity studies with 1A-5A showed that complex 3A is the most cytotoxic complex of this series with IC50 values as low as 4 µM (with blue light) versus two breast cancer cell lines. Complex 3A is also selectively cytotoxic, with sevenfold higher toxicity toward cancerous versus normal breast cells. Phototoxicity indices with 3A were as high as 120, which shows that dark toxicity is avoided. The key difference between complex 3A and the other complexes tested appears to be higher uptake of the complex as measured by inductively coupled plasma mass spectrometry, and a more hydrophobic complex as compared to 1A, which may enhance uptake. These complexes demonstrate proof of concept for dual activation by both low pH and blue light, thus establishing that a pHAMP approach can be used for selective targeting of cancer cells.

Excessive Reactive Oxygen Species and Exotic DNA Lesions as an Exploitable Liability.

Although the terms "excessive reactive oxygen species (ROS)" and "oxidative stress" are widely used, the implications of oxidative stress are often misunderstood. ROS are not a single species but a variety of compounds, each with unique biochemical properties and abilities to react with biomolecules. ROS cause activation of growth signals through thiol oxidation and may lead to DNA damage at elevated levels. In this review, we first discuss a conceptual framework for the interplay of ROS and antioxidants. This review then describes ROS signaling using FLT3-mediated growth signaling as an example. We then focus on ROS-mediated DNA damage. High concentrations of ROS result in various DNA lesions, including 8-oxo-7,8-dihydro-guanine, oxazolone, DNA-protein cross-links, and hydantoins, that have unique biological impacts. Here we delve into the biochemistry of nine well-characterized DNA lesions. Within each lesion, the types of repair mechanisms, the mutations induced, and their effects on transcription and replication are discussed. Finally, this review will discuss biochemically inspired implications for cancer therapy. Several teams have put forward designs to harness the excessive ROS and the burdened DNA repair systems of tumor cells for treating cancer. We discuss inhibition of the antioxidant system, the targeting of DNA repair, and ROS-activated prodrugs.

A Redox-Active, Compact Molecule for Cross-Linking Amyloidogenic Peptides into Nontoxic, Off-Pathway Aggregates: In Vitro and In Vivo Efficacy and Molecular Mechanisms.

Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-ß (Aß) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand-peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer's disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aß forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aß with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

A ROS-Activatable Agent Elicits Homologous Recombination DNA Repair and Synergizes with Pathway Compounds.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8±0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells.

Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5'-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9±1µM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5'-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.

Developing ICP-MS/MS for the detection and determination of synthetic DNA-protein crosslink models via phosphorus and sulfur detection.

Various endogenous and exogenous agents drive the un-directed formation of covalent bonds between proteins and DNA. These complex molecules are of great biological relevance, as can derive in mutations, but are difficult to study because of their heterogeneous chemical properties. New analytical approaches with sufficient detection capabilities to detect and quantify these compounds can help to standardize study models based on synthesized standards. The use of atomic spectrometry can provide quantitative information on the DNA-protein cross-link reaction yield along with basic stoichiometry of the products, based on internal elemental tags, sulfur from Cys and Met amino acids, and phosphorus from the DNA. A new instrumental approach to remove isobaric and polyatomic interferences from (31)P(+) and (32)S(+) was developed recently, with state-of-the-art for interference removal that captures (31)P(+) in Q1; it reacts with O2 in an octopole collision-reaction cell generating (47)PO(+), therefore allowing detection in Q3 without (31)NOH(+)/(48)Ca/(47)Ti interferences. Similarly, (32)S(+) is reacted to (48)SO(+), eliminating the polyatomic interferences at m/z = 32. In conjunction with the high resolving power of high-performance liquid chromatography (HPLC), this newer technology was applied by to the product purification of a DNA-protein cross link model and some preliminary structural studies.

Investigation of fluorinated and bifunctionalized 3-phenylchroman-4-one (isoflavanone) aromatase inhibitors.

Fluorinated isoflavanones and bifunctionalized isoflavanones were synthesized through a one-step gold(I)-catalyzed annulation reaction. These compounds were evaluated for their in vitro inhibitory activities against aromatase in a fluorescence-based enzymatic assay. Selected compounds were tested for their anti-proliferative effects on human breast cancer cell line MCF-7. Compounds 6-methoxy-3-(pyridin-3-yl)chroman-4-one (3c) and 6-fluoro-3-(pyridin-3-yl)chroman-4-one (3e) were identified as the most potent aromatase inhibitors with IC50 values of 2.5 µM and 0.8 µM. Therefore, these compounds have great potential for the development of pharmaceutical agents against breast cancer.

Novel ROS-activated agents utilize a tethered amine to selectively target acute myeloid leukemia.

This study explores the possible use of reactive oxygen-activated DNA modifying agents against acute myeloid leukemia (AML). A key amine on the lead agent was investigated via cytotoxicity assays and was found necessary for potency. The two best compounds were screened via the NCI-60 cell panel. These two compounds had potency between 200 and 800nM against many of the leukemia cancer cell types. Subsequent experiments explored activity against a transformed AML model that mimics the molecular signatures identified in primary AML patient samples. A lead compound had an IC50 of 760nM against this AML cell line as well as a therapeutic index of 7.7±3 between the transformed AML model cell line and non-cancerous human CD34+ blood stem/progenitor cells (UCB). The selectivity was much greater than the mainstays of AML treatment: doxorubicin and cytarabine. This manuscript demonstrates that this novel type of agent may be useful against AML.

Biologically relevant oxidants cause bound proteins to readily oxidatively cross-link at Guanine.

Oxidative DNA-protein cross-links have received less attention than other types of DNA damage and remain as one of the least understood types of oxidative lesion. A model system using ribonuclease A and a 27-nucleotide DNA was used to determine the propensity of oxidative cross-linking to occur in the presence of oxidants. Cross-link formation was examined using four different oxidation systems that generate singlet oxygen, superoxide, and metal-based Fenton reactions. It is shown that oxidative cross-linking occurs in yields ranging from 14% to a maximal yield of 61% in all oxidative systems when equivalent concentrations of DNA and protein are present. Because singlet oxygen is the most efficient oxidation system in generating DNA-protein cross-links, it was chosen for further analyses. Cross-linking occurred with single-stranded DNA binding protein and not with bovine serum albumin. Addition of salt lowered nonspecific binding affinity and lowered cross-link yield by up to 59%. The yield of cross-linking increased with increased ratios of protein compared with DNA. Cross-linking was highly dependent on the number of guanines in a DNA sequence. Loss of guanine content on the 27-nucleotide DNA led to nearly complete loss in cross-linking, while primer extension studies showed cross-links to predominantly occur at guanine base on a 100-nucleotide DNA. The chemical species generated were examined using two peptides derived from the ribonuclease A sequence, N-acetyl-AAAKF and N-acetyl-AYKTT, which were cross-linked to 2'-deoxyguanosine. The cross-link products were spiroiminodihydantoin, guanidinohydantoin, and tyrosyl-based adducts. Formation of tyrosine-based adducts may be competitive with the more well-studied lysine-based cross-links. We conclude that oxidative cross-links may be present at high levels in cells since the propensity to oxidatively cross-link is high and so much of the genomic DNA is coated with protein.

Novel oxidatively activated agents modify DNA and are enhanced by ercc1 silencing.

Agents that chemically modify DNA form a backbone of many cancer treatments. A key problem for DNA-modifying agents is lack of specificity. To address this issue, we designed novel molecular scaffolds, termed An-Hq and An-Hq(2), which are activated by a hallmark of some cancers: elevated concentrations of reactive oxygen species. Elevated reactive oxygen species are linked to oncogenesis and are found to increase in several aggressive cancers. The agents are quinones that, upon oxidation, form highly electrophilic species. In vitro studies identified the mode of addition to DNA. The aniline portion of An-Hq serves to enhance nucleophilic addition to the ethyl phenyl ether instead of forming common Michael additions. Structural characterization showed that the agents add to 2'-deoxyguanosine at the N2,N3-positions. The product formed is a bulky hydroxy-N2,3-benzetheno-2'-deoxyguanosine adduct. In addition, the oxidatively activated agents added to 2'-deoxyadenosine and 2'-deoxycytidine but not thymidine or 2'-deoxyinosine. These findings are confirmed by primer extension analysis of a 392 base pair DNA. The full-length primer extension product was reduced by 69.0 ± 0.6% upon oxidative activation of An-Hq(2) as compared to controls. Little sequence dependence was observed with 76% of guanine, adenine, and cytosine residues showing an increase in extension stops between 2- and 4-fold above controls. Benzetheno-nucleobase addition to double-stranded DNA was confirmed by LC/MS of a self-complementary oligonucletide. Experiments were carried out to confirm in vivo DNA damage. Because of the lesion identified in vitro, we reasoned that nucleotide excision repair should be involved in reversing the effects of these oxidatively activated agents and enhance toxicity in Drosophila melanogaster. Using an RNAi-based approach, Ercc1 was silenced, and survival was monitored after injection of an agent. As expected, bulky cross-linking DNA-modifying agents, cisplatin and chlorambucil, showed statistically significant enhanced toxicity in Drosophila with silenced Ercc1. In addition, 5-fluorouracil, which does not produce bulky lesions, showed no selective toxicity. An-Hq and An-Hq(2) showed statistically significant toxicity in Drosophila with silenced Ercc1. Examination of cytotoxicity shows renal carcinoma cell lines as a target of these agents with a median IC(50) of 1.8 µM. Taken together, these data show that the designed oxidatively activated agents form distinct, bulky DNA modifications that prove difficult for cancer cells possessing an elevated reactive oxygen species phenotype to overcome. The modification produced is relatively unique among anticancer agents.

Suppression of Breast Cancer by Small Molecules That Block the Prolactin Receptor.

Prolactin (PRL) is a protein hormone which in humans is secreted by pituitary lactotrophs as well as by many normal and malignant non-pituitary sites. Many lines of evidence demonstrate that both circulating and locally produced PRL increase breast cancer (BC) growth and metastases and confer chemoresistance. Our objective was to identify and then characterize small molecules that block the tumorigenic actions of PRL in BC. We employed three cell-based assays in high throughput screening (HTS) of 51,000 small molecules and identified two small molecule inhibitors (SMIs), named SMI-1 and SMI-6. Both compounds bound to the extracellular domain (ECD) of the PRL receptor (PRLR) at 1-3 micromolar affinity and abrogated PRL-induced breast cancer cell (BCC) invasion and malignant lymphocyte proliferation. SMI-6 effectively reduced the viability of multiple BCC types, had much lower activity against various non-malignant cells, displayed high selectivity, and showed no apparent in vitro or in vivo toxicity. In athymic nude mice, SMI-6 rapidly and dramatically suppressed the growth of PRL-expressing BC xenografts. This report represents a pre-clinical phase of developing novel anti-cancer agents with the potential to become effective therapeutics in breast cancer patients.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX.

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / - 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5' primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

Arsenic-induced protein phosphorylation changes in HeLa cells.

Arsenic is well documented as a chemotherapeutic agent capable of inducing cell death while at the same time is considered a human carcinogen and an environmental contaminant. Although arsenic toxicity is well known and has formed an impressive literature over the time, little is known about how its effects are exerted at the proteome level. Protein phosphorylation is an important post-translational modification involved in the regulation of cell signaling and likely is altered by arsenic treatment. Despite the importance of phosphorylation for many regulatory processes in cells, the identification and characterization of phosphorylation, as effected by arsenic through mass spectrometric detection, are not fully studied. Here, we identify phosphorylated proteins, which are related to post-translational modifications after phenylarsine oxide (PAO) inoculation to HeLa cells. PAO was chosen because of its high cytotoxicity, measured earlier in these labs. In this study, size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) is used to establish several molecular weight fractions with phosphorylated proteins by monitoring (31)P signal vs. time via ICP-MS. SEC-ICP-MS fractions are collected and then separated by the nano-LC-CHIP/ITMS system for peptide determination. Spectrum Mill and MASCOT protein database search engines are used for protein identification. Several phosphorylation sites and proteins related to post-translational modifications are also identified.

A role for DNA-mediated charge transport in regulating p53: Oxidation of the DNA-bound protein from a distance.

Charge transport (CT) through the DNA base pairs provides a means to promote redox reactions at a remote site and potentially to effect signaling between molecules bound to DNA. Here we describe the oxidation of a cell-cycle regulatory protein, p53, from a distance through DNA-mediated CT. A consensus p53 binding site as well as three DNA promoters regulated by p53 were synthesized containing a tethered DNA photooxidant, anthraquinone. Photoinduced oxidation of the protein occurs from a distance; introduction of an intervening CA mismatch, which inhibits DNA-mediated CT, prevents oxidation of p53. DNA-mediated oxidation is shown to promote dissociation of p53 from only some promoters, and this sequence-selectivity in oxidative dissociation correlates with the biological regulation of p53. Under severe oxidative stress, effected here through oxidation at long range, p53 dissociates from a promoter that activates DNA repair as well as the promoter for the negative regulator of p53, Mdm2, but not from a promoter activating cell-cycle arrest. Mass spectrometry results are consistent with disulfide bond formation in p53 upon DNA-mediated oxidation. Furthermore, DNA-bound p53 oxidation is shown in vivo by up-regulation of p53 and subsequent irradiation in the presence of a rhodium photooxidant to give a new p53 adduct that can be reversed with thiol treatment. This DNA-mediated oxidation of p53 parallels that seen by treating cells with hydrogen peroxide. These results indicate a unique mechanism using DNA-mediated CT chemistry by which p53 activity on different promoters may be controlled globally under conditions of oxidative stress.

Common mitochondrial DNA mutations generated through DNA-mediated charge transport.

Mutation sites that arise in human mitochondrial DNA as a result of oxidation by a rhodium photooxidant have been identified. HeLa cells were incubated with [Rh(phi)(2)bpy]Cl(3) (phi is 9,10-phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell and bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)(2)bpy](3+) can promote the oxidation of guanine from a distance through DNA-mediated charge transport. After two rounds of photolysis and growth of cells incubated with the rhodium complex, DNA mutations in a portion of the mitochondrial genome were assessed via manual sequencing. The mutational pattern is consistent with dG to dT transversions in the repetitive guanine tracts. Significantly, the mutational pattern found overlaps oxidative damage hot spots seen previously. These mutations are found within conserved sequence block II, a critical regulatory element involved in DNA replication, and these have been identified as sites of low oxidation potential to which oxidative damage is funneled. On the basis of this mutational analysis and its correspondence to sites of long-range oxidative damage, we infer a critical role for DNA charge transport in generating these mutations and, thus, in regulating mitochondrial DNA replication under oxidative stress.