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Danyelle M Townsend

Publications and source records attributed to Danyelle M Townsend.

18 recordsLinked to original sources
PubMed2006

"Skittish" Abca2 knockout mice display tremor, hyperactivity, and abnormal myelin ultrastructure in the central nervous system.

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein most highly expressed in the central and peripheral nervous system tissues and macrophages. Previous studies indicated its role in cholesterol/steroid (estramustine, estradiol, and progesterone) trafficking/sequestration, oxidative stress response, and Alzheimer's disease. Developmental studies have shown its expression during macrophage and oligodendrocyte differentiation, processes requiring membrane growth. To determine the in vivo function(s) of this transporter, we generated a knockout mouse from a gene-targeted disruption of the murine ABCA2 gene. Knockout males and females are viable and fertile. However, a non-Mendelian inheritance pattern was shown among male progeny of heterozygous crosses. Compared to wild-type and heterozygous littermates, knockout mice displayed a tremor without ataxia, hyperactivity, and reduced body weight; the latter two phenotypes were more marked in females than in males. This sexual disparity suggests a role for ABCA2 in hormone-dependent neurological and/or developmental pathways. Myelin sheath thickness in the spinal cords of knockout mice was greatly increased compared to that in wild-type mice, while a significant reduction in myelin membrane periodicity (compaction) was observed in both spinal cords and cerebra of knockout mice. Loss of ABCA2 function in vivo resulted in abnormal myelin compaction in spinal cord and cerebrum, an ultrastructural defect that we propose to be the cause of the phenotypic tremor.

ATP-Binding Cassette Transporters↗
PubMed2006

A novel role for human sulfiredoxin in the reversal of glutathionylation.

Modification of protein cysteine residues by disulfide formation with glutathione (glutathionylation) is a reversible posttranslational modification of critical importance in controlling cell signaling events following oxidative and/or nitrosative stress. Here, we show that human sulfiredoxin, a small redox protein conserved in eukaryotes, can act as a novel regulator of the redox-activated thiol switch in cells by catalyzing deglutathionylation of a number of distinct proteins in response to oxidative and/or nitrosative stress. Actin and protein tyrosine phosphatase 1B were identified in vitro as targets of sulfiredoxin 1 (Srx1)-dependent deglutathionylation and confirmed in vivo by two-dimensional gel electrophoresis analysis. In addition, we show that Srx1-dependent deglutathionylation is functionally relevant through restoration of phosphatase activity. Human sulfiredoxin contains one cysteine residue (Cys(99)) that is conserved in all family members. Mutation of the cysteine residue inhibits deglutathionylation but did not affect its capacity to bind intracellular proteins. Furthermore, sulfiredoxin is not an acceptor molecule for the GS(-) moiety during the reaction process. Using two-dimensional gel electrophoresis, we identified multiple protein targets in vivo that are deglutathionylated by sulfiredoxin following oxidative and/or nitrosative stress. This novel deglutathionylation function of sulfiredoxin suggests it has a central role in redox control with potential implications in cell signaling.

4-Aminobenzoic Acid↗
PubMed2006

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

4-Aminobenzoic Acid↗
PubMed2005

A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins.

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

4-Aminobenzoic Acid↗
PubMed2005

Evaluation of lipophilins as determinants of tumor cell response to estramustine.

Estramustine administered orally as estramustine phosphate (EMP) remains a major tool in hormone refractory prostate cancer chemotherapy. The presence of estramustine binding protein, prostatin, in prostate tissue may be a determinant of response to treatment. Lipophilins are secretory proteins with homology to prostatin. Reverse transcription-polymerase chain reaction was performed to estimate expression patterns of lipophilins A to C in human biopsies and cell lines resistant to estramustine. Although lipophilin A was not expressed in prostate tissue, both lipophilins B and C were expressed in normal and tumor prostate without significant differences. For lipophilin C, a somatic mutation (T to C transition at positions 409 and 412) was found in human tumor samples and absent in normal prostate tissue. No consistent response to EMP was observed in enhanced green fluorescent protein (EGFP)-tagged lipophilin C-transfected PC3 cells compared with parental controls. Among these EGFP-lipophilin C clones, no direct correlation between response to EMP treatment (IC50 values) and EGFP expression was observed (p = 0.73). Lipophilin C mRNA levels did not vary significantly between wild-type and estramustine-resistant cells in prostate (DU145 and PC3) and ovarian (SKOV3) cancer cell lines. Overall, these results suggest that lipophilins are not specific determinants of estramustine efficacy.

Antineoplastic Agents, Hormonal↗
PubMed2005

Sulfiredoxin: a potential therapeutic agent?

The importance of antioxidants in maintaining homeostasis has long been accepted and includes antioxidant proteins such as, peroxiredoxin (Prx), superoxide dismutase and glutathione S transferases. Sulfiredoxin (Srx) is a recently identified antioxidant protein with a role in signaling through catalytic reduction of oxidative modifications. It was first characterized for its regulation of Prx(s) through reduction of the conserved cysteine from sulfinic to sulfenic acid, thereby impacting the role of Prx in regulation of downstream transcription factors and kinase signaling pathways. Furthermore, the reduction of sulfinic to sulfenic acid prevents further oxidation of the conserved cysteine residue to sulfonic acid, the end result of which is degradation. Srx also has a role in the reduction of glutathionylation a post-translational, oxidative modification that occurs on numerous proteins and has been implicated in a wide variety of pathologies, including Parkinson's disease. The regulation of glutathionylation/deglutathionylation (or thiol switch) has been likened to phosphorylation/dephosphorylation, another post-translational modification involved in the regulation of signaling pathways. Unlike, the reduction of Prx over-oxidation, Srx-dependent deglutathionylation appears to be non-specific. Deglutathionylation of multiple proteins has been observed both in vitro and in vivo in response to oxidative and/or nitrosative stress. This review discusses Srx as a novel antioxidant, and focuses on its potential role in the regulation of glutathionylation/deglutathionylation pathways, that have been implicated in a growing number of disease states.

Animals↗
PubMed2005

Glutathione S-transferases as regulators of kinase pathways and anticancer drug targets.

Anticancer drug development using the platform of glutathione (GSH), glutathione S-transferases (GST) and pathways that maintain thiol homeostasis has recently produced a number of lead compounds. GSTpi is a prevalent protein in many solid tumors and is overexpressed in cancers resistant to drugs. It has proved to be a viable target for pro-drug activation with at least one candidate in late-stage clinical development. In addition, GSTpi possesses noncatalytic ligand-binding properties important in the direct regulation of kinase pathways. This has led to the development and testing of agents that bind to GSTpi and interfere with protein-protein interactions, with the phase II clinical testing of one such drug. Attachment of glutathione to acceptor cysteine residues (glutathionylation) is a posttranslational modification that can alter the structure and function of proteins. Two agents in preclinical development (PABA/NO, releasing nitric oxide on GST activation, and NOV-002, a pharmacologically stabilized pharmaceutical form of GSSG) can lead to glutathionylation of a number of cellular proteins. The biological significance of these modifications is linked with the mechanism of action of these drugs. In the short term, glutathione-based systems should continue to provide viable targets and a platform for the development of novel cancer drugs.

4-Aminobenzoic Acid↗
PubMed2004

Sulfur containing amino acids and human disease.

Sulfur containing amino acids contribute substantially to the maintenance and integrity of cellular systems by influencing cellular redox state and cellular capacity to detoxify toxic compounds, free radicals and reactive oxygen species. Methionine and cysteine are the two primary sulfur-containing amino acids in mammals. Methionine is an essential amino acid, obtained by dietary intake while cysteine is non-essential and a metabolite of methionine metabolism. Each of these amino acids contributes significantly to the cellular pool of organic sulfur and generally to sulfur homeostasis as well as playing a significant role in regulation of one carbon metabolism. Genetic defects in the enzymes regulating sulfur pools produce a variety of human pathologies, including homo- and cystinuria, homo- and cysteinemia, and neural tube defects. In addition, thiol imbalance has been associated with multiple disorders, including vascular disease, Alzheimer's, HIV and cancer. Possible treatments to restore the thiol balance are also discussed.

Animals↗
PubMed2004

Organosulfur compounds from alliaceae in the prevention of human pathologies.

A strong association between elevated plasma low-density-lipoprotein (LDL) and the development of cardiovascular diseases (CVD) has been established. Oxidation of LDL (Ox-LDL) promotes vascular dysfunction, enhances the production and release of inflammatory mediators such as reactive oxygen species and contribute to the initiation and progression of atherosclerosis. In addition, Ox-LDL enhances the production and release of tumor necrosis factor (TNF-alpha), interleukin (IL)-6, arachidonic acid metabolites and nitric oxide (NO) that are responsible for various human pathologies including cancer. Organosulfur compounds (OSC) from alliaceae modulate the glutathione (GSH) redox cycle and inhibits NFkappa-B activation in human T cells. Furthermore, OSC bioactivities include antioxidant, antibacterial, anticarcinogenic, antiatherogenic, immunostimulatory, and liver protection potential.

Allyl Compounds↗
PubMed2004

Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug.

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

3T3 Cells↗
PubMed2003

The role of glutathione-S-transferase in anti-cancer drug resistance.

Glutathione-S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs are divided into two distinct super-family members: the membrane-bound microsomal and cytosolic family members. Microsomal GSTs are structurally distinct from the cytosolic in that they homo- and heterotrimerize rather than dimerize to form a single active site. Microsomal GSTs play a key role in the endogenous metabolism of leukotrienes and prostaglandins. Human cytosolic GSTs are highly polymorphic and can be divided into six classes: alpha, mu, omega, pi, theta, and zeta. The pi and mu classes of GSTs play a regulatory role in the mitogen-activated protein (MAP) kinase pathway that participates in cellular survival and death signals via protein : protein interactions with c-Jun N-terminal kinase 1 (JNK1) and ASK1 (apoptosis signal-regulating kinase). JNK and ASK1 are activated in response to cellular stress. GSTs have been implicated in the development of resistance toward chemotherapy agents. It is plausible that GSTs serve two distinct roles in the development of drug resistance via direct detoxification as well as acting as an inhibitor of the MAP kinase pathway. The link between GSTs and the MAP kinase pathway provides a rationale as to why in many cases the drugs used to select for resistance are neither subject to conjugation with GSH, nor substrates for GSTs. GSTs have emerged as a promising therapeutic target because specific isozymes are overexpressed in a wide variety of tumors and may play a role in the etiology of other diseases, including neurodegenerative diseases, multiple sclerosis, and asthma. Some of the therapeutic strategies so far employed are described in this review.

Antineoplastic Agents↗
PubMed2003

Metabolism of Cisplatin to a nephrotoxin in proximal tubule cells.

Cisplatin, a commonly used chemotherapeutic agent, is nephrotoxic. The mechanism by which cisplatin selectively kills the proximal tubule cells was heretofore unknown. Recent studies in mice and rats have shown that the nephrotoxicity of cisplatin can be blocked by acivicin or (aminooxy)acetic acid, the same enzyme inhibitors that block the metabolic activation of a series of nephrotoxic halogenated alkenes. In this study, it was hypothesized that cisplatin is activated in the kidney to a toxic metabolite through the same pathway that has been shown to activate the halogenated alkenes. This activation begins with the formation of a glutathione-conjugate that is metabolized to a cysteinyl-glycine-conjugate, to a cysteine-conjugate, and finally to a reactive thiol. In this study, a protocol was developed in which confluent monolayers of LLC-PK(1) cells were exposed to clinically relevant concentrations of cisplatin or cisplatin-conjugate for 3 h. Cell viability was assayed at 72 h. The role of gamma-glutamyl transpeptidase (GGT) and cysteine-S-conjugate beta-lyase in the metabolism of each of the cisplatin-conjugates was investigated. Pre-incubation of cisplatin with glutathione, cysteinyl-glycine, or N-acetyl-cysteine to allow for the spontaneous formation of cisplatin-conjugates increased the toxicity of cisplatin toward LLC-PK(1) cells. Inhibition of GGT activity showed that GGT was necessary only for the toxicity of the cisplatin-glutathione-conjugate. Inhibition of cysteine-S-conjugate beta-lyase reduced the toxicity of each of the cisplatin-conjugates. These data demonstrate that metabolism of cisplatin in proximal tubule cells is required for its nephrotoxicity. The elucidation of this pathway provides new targets for the inhibition of cisplatin nephrotoxicity.

Animals↗
PubMed2003

High pressure liquid chromatography and mass spectrometry characterization of the nephrotoxic biotransformation products of Cisplatin.

Previous studies have shown that cisplatin requires metabolic activation to become nephrotoxic. The activation is proposed to be via the metabolism of a glutathione-platinum conjugate to a cysteinyl-glycine-platinum conjugate, which is further processed to a cysteine conjugate. Preincubating cisplatin with glutathione (GSH), cysteinyl-glycine, or N-acetylcysteine (NAC) results in a transient increase in the toxicity of cisplatin toward renal proximal tubular cells. In this study, the preincubation solutions were analyzed by high pressure liquid chromatography (HPLC), atomic absorption spectrometry, and mass spectrometry (MS) to characterize the formation and structure of the platinum conjugates. HPLC analysis of the cisplatin-GSH, cisplatin-cysteinyl-glycine, and cisplatin-NAC preincubation solutions revealed two new platinum-containing peaks in each of the solutions. MS-MS analysis of the peaks revealed a diplatinum- and a monoplatinum conjugate in each of the solutions. Analysis of the composition and toxicity of the solutions with time showed that the transient increase in toxicity correlated with the formation of the monoplatinum conjugate whereas prolonged preincubation decreased toxicity and correlated with the formation of the diplatinum conjugate. The monoplatinum-monoglutathione conjugate is a substrate for gamma-glutamyl transpeptidase, an enzyme that is essential for the nephrotoxicity of cisplatin. The monoplatinum-mono-NAC conjugate can be deacetylated to a cysteine conjugate, which is a substrate for pyroxidol phosphate (PLP)-dependent cysteine S-conjugate beta-lyase. This PLP-dependent enzyme is proposed to catalyze the final step in the metabolic activation of cisplatin. Identification of the structure and toxicity of these conjugates further elucidates the metabolism of cisplatin to a nephrotoxin.

Animals↗
PubMed2002

Inhibition of gamma-glutamyl transpeptidase or cysteine S-conjugate beta-lyase activity blocks the nephrotoxicity of cisplatin in mice.

Cisplatin is nephrotoxic. The mechanism underlying this organ-specific toxicity is unknown. We hypothesize that cisplatin is metabolized via a gamma-glutamyl transpeptidase (GGT) and cysteine S-conjugate beta-lyase-dependent pathway that has been shown to activate several haloalkenes to nephrotoxins. To test this hypothesis, we inhibited GGT and cysteine S-conjugate beta-lyase in C57BL/6 mice and analyzed the effect of the inhibitors on the nephrotoxicity of cisplatin. GGT was inhibited by pretreating the mice with acivicin. Cysteine S-conjugate beta-lyase was inhibited by aminooxyacetic acid (AOAA). Male C57BL/6 mice were treated with 15 mg/kg cisplatin (i.p.) and sacrificed on day 5. Half the mice treated with cisplatin alone died before sacrifice. The cisplatin-treated mice sacrificed at 5 days had significantly elevated levels of blood urea nitrogen (BUN). Histologic analysis revealed severe damage to the renal proximal tubules. Pretreatment with acivicin or AOAA protected the mice from the nephrotoxicity of cisplatin. None of the pretreated animals died before sacrifice. BUN levels and quantitative histologic analysis of the kidneys confirmed the protective effect of acivicin and AOAA. Platinum levels in the kidneys were not altered by acivicin or AOAA, indicating that neither affected the uptake of cisplatin into the kidney. Likewise, cisplatin-induced weight loss was not altered by acivicin or AOAA, suggesting that weight loss and nephrotoxicity are via distinct mechanisms. These data support the hypothesis that the nephrotoxicity of cisplatin is due to the metabolism of a platinum-glutathione conjugate by GGT and cysteine S-conjugate beta-lyase to a potent nephrotoxin.

Aminooxyacetic Acid↗
PubMed2002

Efficacy of a glutathione S-transferase pi-activated prodrug in platinum-resistant ovarian cancer cells.

Human ovarian carcinoma cells (C70 and C200) made resistant to cisplatin from A2780 cells demonstrated an approximately 20-fold resistance to the drug. These same cell lines showed no collateral resistance (as compared with the wild-type) to a novel glutathione S-transferase pi-activated prodrug [gamma-glutamyl-alpha-amino-beta[2-ethyl-N,N,N',N'-tetrakis (2-chloroethyl) phosphorodiamidate]-sulfonyl-propionyl-(R)-(-) phenylglycine; TLK286]. Previous results have shown a direct correlation between levels of GST pi expression and cytotoxicity for TLK286 (L. A. Rosario et al., Mol. Pharmacol., 58: 167-174, 2000.). However, protein levels of the isozyme were identical in wild-type C70 and C200 cell lines. In analyzing the DNA repair capacity of C70 and C200, an altered expression of the DNA-dependent protein kinase (DNA-PK) complex (catalytic subunit DNA-PKcs, and the heterodimers Ku70 and Ku80) was found. In C70 and C200 cells, DNA-PKcs was overexpressed at both the transcript and protein levels, whereas amounts of Ku70 and Ku80 were higher only at the level of protein expression. TLK286 in either its parent or activated form inhibited the catalytic kinase activity of purified DNA-PK with an IC50 value of approximately 1 microM. Coimmunoprecipitation of Ku70 after TLK286 treatment of purified DNA-PK and C70 cells showed a drug-induced destabilization of the protein-protein interaction between the catalytic subunit and the Ku heterodimer. Overall, these results implicate inhibition of DNA-PK as a component of TLK286 cytotoxicity and provide a rationale for its use in the clinical management of cisplatin-resistant ovarian cancer.

Antigens, Nuclear↗
PubMedDate not supplied

The importance of glutathione in human disease.

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment and the tripeptide is a co-factor for many cytoplasmic enzymes and may also act as an important post-translational modification in a number of cellular proteins. The cysteine thiol acts as a nucleophile in reactions with both exogenous and endogenous electrophilic species. As a consequence, reactive oxygen species (ROS) are frequently targeted by GSH in both spontaneous and catalytic reactions. Since ROS have defined roles in cell signaling events as well as in human disease pathologies, an imbalance in expression of GSH and associated enzymes has been implicated in a variety of circumstances. Cause and effect links between GSH metabolism and diseases such as cancer, neurodegenerative diseases, cystic fibrosis (CF), HIV, and aging have been shown. Polymorphic expression of enzymes involved in GSH homeostasis influences susceptibility and progression of these conditions. This review provides an overview of the biological importance of GSH at the level of the cell and organism.

Aging↗
PubMedDate not supplied

Characterization of the ATPase activity of human ATP-binding cassette transporter-2 (ABCA2).

BACKGROUND: ABCA2 is a member of the ATP binding cassette transporter family with functional roles in cholesterol homeostasis and drug resistance. MATERIALS AND METHODS: In order to characterize its ATPase activity, we transfected HEK293 cells with an ABCA2 mammalian expression system and isolated ABCA2-enriched membranes. RESULTS: We found no measurable ATPase activity of ABCA2 in isolated membranes, except in the presence of the methyl-beta-cyclodextrin. However, competitive binding of a pseudo-substrate, 8-azido-[alpha-32P]-ATP, was demonstrated. CHO cells transfected with ABCA2 did not have a higher rate of endogenous ATP hydrolysis when compared to the mock-transfected cells. CONCLUSION: Overall, we conclude that, while ABCA2 may have low levels of ATPase activity that can be substrate-stimulated, it is more likely to have a regulatory role in cell physiology.

3T3 Cells↗