Biomedical subjects

Piotr Zimniak

Publications and source records attributed to Piotr Zimniak.

At least 19 recordsLinked to original sources

PubMed2006

Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.

Aldehydes↗

PubMed2006

Regulation of CD95 (Fas) expression and Fas-mediated apoptotic signaling in HLE B-3 cells by 4-hydroxynonenal.

The Fas (apo/CD95) receptor which belongs to the TNF-alpha family is a transmembrane protein involved in the signaling for apoptosis through the extrinsic pathway. During this study, we have examined a correlation between intracellular levels of 4-HNE and expression of Fas in human lens epithelial (HLE B-3) cells. Our results show that in HLE B-3 cells, Fas is induced by 4-HNE in a concentration- and time-dependent manner, and it is accompanied by the activation of JNK, caspase 3, and the onset of apoptosis. Fas induction and activation of JNK are also observed in various tissues of mGsta4 null mice which have elevated levels of 4-HNE. Conversely, when 4-HNE is depleted in HLE B-3 cells by a transient transfection with hGSTA4, Fas expression is suppressed. However, upon the cessation of hGSTA4 expression in these transiently transfected cells, Fas and 4-HNE return to their basal levels. Fas-deficient transformed HLE B-3 cells stably transfected with hGSTA4 show remarkable resistance to apoptosis. Also, the wild-type HLE B-3 cells in which Fas is partially depleted by siRNA acquire resistance to 4-HNE-induced apoptosis, suggesting an at least partial role of Fas in 4-HNE-induced apoptosis in HLE B-3 cells. We also demonstrate that during 4-HNE-induced apoptosis of HLE B-3 cells, Daxx is induced and it binds to Fas. Together, these results show an important role of 4-HNE in regulation of the expression and functions of Fas.

Aldehydes↗

PubMed2005

The course of CCl4 induced hepatotoxicity is altered in mGSTA4-4 null (-/-) mice.

Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the alpha-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the alpha-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl(4)) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl(4) in GSTA4-4 null (-/-) and wild type (+/+) mice. The results show that administration of a single dose of CCl(4) (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both -/- and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in -/- mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of -/- mice. As expected -/- mice did not show any expression of mGSTA4-4; after CCl(4) a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of -/- mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl(4) hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1-6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.

Aldehydes↗

PubMed2005

Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.

Efficient handling of oxidative stress is critical for the survival of organisms. The orphan nuclear receptor pregnane X receptor (PXR) is important in xenobiotic detoxification through its regulation of phase I and phase II drug-metabolizing/detoxifying enzymes and transporters. In this study we unexpectedly found that the expression of an activated human PXR in transgenic female mice resulted in a heightened sensitivity to paraquat, an oxidative xenobiotic toxicant. Heightened paraquat sensitivity was also seen in wild-type mice treated with the mouse PXR agonist pregnenolone-16alpha-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Depletion of 4-hydroxynonenal in hGSTA4-transfected HLE B-3 cells results in profound changes in gene expression.

Previously, we have shown that overexpression of 4-hydroxy-2-nonenal (HNE)-detoxifying enzyme glutathione S-transferase A4-4 (hGSTA4-4) in human lens epithelial cells (HLE B-3) leads to pro-carcinogenic phenotypic transformation of these cells [R. Sharma, et al. Eur. J. Biochem. 271 (2004) 1960-1701]. We now demonstrate that hGSTA4-transfection also causes a profound change in the expression of genes involved in cell adhesion, cell cycle control, proliferation, cell growth, and apoptosis, which is consistent with phenotypic changes of the transformed cells. The expression of p53, p21, p16, fibronectin 1, laminin gamma1, connexin 43, Fas, integrin alpha6, TGFalpha, and c-jun was down-regulated, while the expression of protein kinase C beta II (PKCbetaII), c-myc, cyclin-dependent kinase 2 (CDK2), and TGFbeta was up-regulated in transfected cells. These results demonstrate that HNE serves as a crucial signaling molecule and, by modulating the expression of genes, can influence cellular functions.

Aldehydes↗

PubMed2005

Mutagenic effects of 4-hydroxynonenal triacetate, a chemically protected form of the lipid peroxidation product 4-hydroxynonenal, as assayed in L5178Y/Tk+/- mouse lymphoma cells.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is cytotoxic and genotoxic at superphysiological concentrations. To characterize the mechanism of action of 4-HNE, we assessed genotoxic damage by 4-HNE and by 4-HNE triacetate [4-HNE(Ac)(3)] using the mouse lymphoma assay that measures the mutant frequency in the Tk gene. As a strong electrophile, 4-HNE reacts readily with nucleophilic centers on cellular components. When added extracellularly, it may react preferentially with proteins in culture medium or on the cell surface and not reach deeper cellular targets such as nuclear DNA. Therefore, 4-HNE(Ac)(3), a protected form of 4-HNE that is metabolically converted to 4-HNE in cells (Neely MD, Amarnath V, Weitlauf C, and Montine TJ, Chem Res Toxicol 15:40-47, 2002), was assayed in addition to 4-HNE. When added in serum-containing medium, 4-HNE was not mutagenic in the mouse lymphoma assay up to 38 muM (cytotoxicity = 13%). In contrast, exposure to 4-HNE(Ac)(3), which mimics intracellular formation of 4-HNE, resulted in dose-dependent induction of mutations. At 17 muM 4-HNE(Ac)(3) (cytotoxicity = 33%), the mutant frequency was 719 x 10(-6) (>7-fold higher than the spontaneous mutant frequency). Loss of heterozygosity analysis in the Tk mutants revealed that the majority of mutations induced by 4-HNE(Ac)(3) resulted from clastogenic events affecting a large segment of the chromosome. The results indicate that, in the presence of serum that approximates physiological conditions, 4-HNE generated intracellularly but not extracellularly is a strong mutagen via a clastogenic action at concentrations that may occur during oxidative stress.

Aldehydes↗

PubMed2005

Molecular pathology of aging and its implications for senescent coronary atherosclerosis.

PURPOSE OF REVIEW: This review highlights common mechanisms of organismal aging and inflammatory coronary atherosclerosis. RECENT FINDINGS: A substantial body of evidence now indicates that aging is largely due to molecular damage inflicted by reactive oxygen species, electrophiles, and other reactive endobiotic and xenobiotic metabolites. Our understanding of genetic pathways regulating longevity began 12 years ago with the discovery that a developmental-arrest program in the nematode Caenorhabditis elegans also has marked effects on adult lifespan. This pathway, closely related to the insulin and insulinlike growth factor-signaling pathways of mammals, modulates longevity and stress resistance in several model organisms. Insulin-like signaling also has an impact on redox signaling, antioxidant defenses, and metabolic generation of oxidative stress. Recently, additional signaling pathways--involving Sirtuins, AMP kinase, Jun N-terminal kinase 1, and other master regulatory proteins--have been implicated in longevity and stress-resistance mechanisms. The inflammatory process involves acute production of reactive oxygen species by specialized cells responding to infection, exposure to toxins or allergens, cell damage, hypoxia, ischemia/reperfusion, and other factors, initiating signaling through several of these pathways. Free radical chain reactions arise from lipid oxidation and generate oxidized low-density lipoprotein, a powerful inflammatory signal and potentiator of atherosclerosis. Oxidized low-density lipoprotein accumulates in atherosclerotic arteries, particularly in rupture-prone regions. Inflammation involving oxidative stress, by way of the production of reactive oxygen species, is a hallmark of coronary atherosclerosis. SUMMARY: Common pathways underlie both organismal aging and tissue-autonomous senescent pathologic processes, such as coronary atherosclerosis. The mechanisms discovered in model organisms may lead to pharmacotherapeutic interventions.

Aging↗

PubMed2005

Lifespan and stress resistance of Caenorhabditis elegans are increased by expression of glutathione transferases capable of metabolizing the lipid peroxidation product 4-hydroxynonenal.

Caenorhabditis elegans expresses a glutathione transferase (GST) belonging to the Pi class, for which we propose the name CeGSTP2-2. CeGSTP2-2 (the product of the gst-10 gene) has the ability to conjugate the lipid peroxidation product 4-hydroxynonenal (4-HNE). Transgenic C. elegans strains were generated in which the 5'-flanking region and promoter of gst-10 were placed upstream of gst-10 and mGsta4 cDNAs, respectively. mGsta4 encodes the murine mGSTA4-4, an enzyme with particularly high catalytic efficiency for 4-HNE. The localization of both transgenes was similar to that of native CeGSTP2-2. The 4-HNE-conjugating activity in worm lysates increased in the order: control<mGsta4 transgenic<gst-10 transgenic; and the amount of 4-HNE-protein adducts decreased in the same order, indicating that the transgenic enzymes were active and effective in limiting electrophilic damage by 4-HNE. Stress resistance and lifespan were measured in transgenic animals (five independent lines each) and were compared with two independent control lines. Resistance to paraquat, heat shock, ultraviolet irradiation and hydrogen peroxide was greater in transgenic strains. Median lifespan of mGsta4 and gst-10 transgenic strains vs. control strains was increased by 13% and 22%, respectively. In addition to the cause-effect relationship between GST expression and lifespan observed in the transgenic lines, correlative evidence was also obtained in a series of congenic lines of C. elegans in which lifespan paralleled the 4-HNE-conjugating activity in whole-animal lysates. We conclude that electrophilic damage by 4-HNE may contribute to organismal aging.

Aldehydes↗

PubMed2005

Lifespan extension in hypomorphic daf-2 mutants of Caenorhabditis elegans is partially mediated by glutathione transferase CeGSTP2-2.

Electrophilic stress caused by lipid peroxidation products such as 4-hydroxynonenal (4-HNE) and/or related compounds may contribute to aging. The major mode of 4-HNE metabolism involves glutathione conjugation catalyzed by specialized glutathione transferases. We have previously shown that glutathione transferase CeGSTP2-2, the product of the Caenorhabditis elegans gst-10 gene, has the ability to conjugate 4-HNE, and that its overexpression extends lifespan of C. elegans. We now demonstrate that the expression level of CeGSTP2-2 correlates highly with lifespan in a series of hypomorphic daf-2 mutants of C. elegans. The overexpression of CeGSTP2-2 in daf-2 is abrogated in daf-16; daf-2 mutants, indicating that expression of the gst-10 gene is modulated by insulin-like growth factor signaling. To determine whether the relationship between CeGSTP2-2 and lifespan is causal, we used RNAi to knock down CeGSTP2-2. Treatment with gst-10-specific dsRNA decreased CeGSTP2-2 protein in wild-type N2 and in daf-2 strains to an approximately equal level. The ability to conjugate 4-HNE was similarly decreased by RNAi, suggesting that the increment of that activity in daf-2 over N2 is due largely to the overexpression of CeGSTP2-2. RNAi-mediated knock-down of CeGSTP2-2 led to an increased susceptibility to 4-HNE, paraquat, and heat shock, and to a shortening of lifespan by 13% in both N2 and daf-2 strains. These results indicate that CeGSTP2-2 significantly contributes to the maintenance of the soma, and that this function is augmented in daf-2 mutants concordantly with other longevity assurance genes, probably via insulin-like growth factor signaling.

Animals↗

PubMed2004

Crystal structure of human glutathione S-transferase A3-3 and mechanistic implications for its high steroid isomerase activity.

The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Delta(5)-androstene-3,17-dione (Delta(5)-AD) to Delta(4)-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Delta(5)-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3.GSH.Delta(5)-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS(-)) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO-Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS(-)), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Delta(5)-AD and thus help the thiolate of GS(-) to initiate the proton transfer and the subsequent stabilization of the reaction intermediate.

Amino Acid Substitution↗

PubMed2004

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide.

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Design, synthesis, and structure-activity relationships of haloenol lactones: site-directed and isozyme-selective glutathione S-transferase inhibitors.

Overexpression of glutathione S-transferase (GST), particularly the GST-pi isozyme, has been proposed to be one of the biochemical mechanisms responsible for drug resistance in cancer chemotherapy, and inhibition of overexpressed GST has been suggested as an approach to combat GST-induced drug resistance. 3-Cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone (1a), a lead compound of site-directed GST-pi inactivator, has been shown to potentiate the cytotoxic effect of cisplatin on tumor cells. As an initial step to develop more potent and more selective haloenol lactone inactivators of GST-pi, we examined the relationship between the chemical structures of haloenol lactone derivatives and their GST inhibitory activity. A total of 16 haloenol lactone derivatives were synthesized to probe the effects of (1) halogen electronegativity, (2) electron density of aromatic rings, (3) molecular size and rigidity, (4) lipophilicity, and (5) aromaticity on the potency of GST-pi inactivation. The inhibitory potency of each compound was determined by time-dependent inhibition tests, and recombinant human GST-pi was used to determine their inhibitory activity. Our structure-activity relationship studies demonstrated that (1) reactivity of the halide leaving group plays a weak role in GST inactivation by the haloenol lactones, (2) aromatic electron density may have some influence on the potency of GST inactivation, (3) high rigidity likely disfavors enzyme inhibition, (4) lipophilicity is inversely proportional to enzyme inactivation, and (5) an unsaturated system may be important for enzyme inhibition. This work facilitated understanding of the interaction of GST-pi with haloenol lactone derivatives as site-directed and isozyme-selective inactivators, possibly potentiating cancer chemotherapy.

Drug Design↗

PubMed2004

Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGsta4 null mouse.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) is a strong electrophile that forms covalent adducts with proteins and, to a lesser extent, nucleic acids and phospholipids. The generation of 4-HNE appears to be an inevitable consequence of aerobic metabolism. The metabolism of 4-HNE is mainly, although not entirely, conjugative, and proceeds via Michael addition of glutathione to the double bond of 4-HNE. This reaction is catalyzed by specialized glutathione S-transferases (GSTs) exemplified by the murine mGSTA4-4. To study the (patho)physiological effects of 4-HNE in an intact organism, we disrupted the mGsta4 gene in the mouse. The resulting mGsta4 null mouse expressed no mGsta4 mRNA and no corresponding protein, had a reduced ability to conjugate 4-HNE, and had an increased steady-state level of this aldehyde in tissues. The residual conjugating activity for 4-HNE (23-64% depending on the tissue) is probably attributable to isoforms of glutathione S-transferases which have low catalytic efficiency for 4-HNE but are more abundant than mGSTA4-4, or are upregulated upon mGsta4 gene disruption. Mice homozygous for the disrupted mGsta4 allele were viable and appeared normal except for lower litter size, higher fat content in bones, and greater susceptibility to bacterial infection. The null mice had a significantly lower survival time than wild-type controls when chronically treated with relatively low doses of paraquat, a finding consistent with a role of mGSTA4-4 in the defense against oxidative stress. The mouse model should be useful for the study of degenerative conditions in which 4-HNE is postulated to be a contributing factor.

Adipose Tissue↗

PubMed2004

Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis.

The role of alpha-class mammalian glutathione S-transferases (GSTs) in the protection of many cell types, including vascular smooth muscle cells, against oxidant damage has been demonstrated, but the role of GSTs in the endothelial cell is not well studied. In order to examine the role of GSTs in the endothelial cell, a stable transfection of mouse pancreatic islet endothelial cells (MS1) with cDNA of mGSTA4-4, mouse isozyme of GSTs with activity in vascular wall, was established. Transfected cells demonstrated significantly higher GSTs enzyme activity and expressed significantly increased resistance to the cytotoxicity of allylamine, acrolein, 4-hydroxy-2-nonenal (4-HNE), and H(2)O(2) (P < 0.05). A significantly higher rate of proliferation and lower baseline level of intracellular malondialdehyde (MDA) and 4-HNE were present when compared to wild-type or vector-transfected MS1 endothelial cells (P < 0.05). Transfection protected MS1 endothelial cells from 4-HNE and H(2)O(2) induced apoptosis by inhibiting phosphorylation of c-Jun N-terminal kinases (p-JNK) and consequent activation of p53 and Bax. In early human fibrous atherosclerotic plaques, immunohistochemical studies demonstrated marked induction of hGSTA4-4 in endothelial cells overlying plaque, and in proliferating plaque vascular smooth muscle cells. Our results indicate that endothelial cell mGSTA4-4 can play a key role in protecting blood vessels against oxidative stress and, thus, is likely to be a critical defense mechanism against oxidants that act as atherogens.

Animals↗

PubMed2004

Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells.

4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.

Aldehydes↗

PubMed2003

Transactivation of the epidermal growth factor receptor mediates cholinergic agonist-induced proliferation of H508 human colon cancer cells.

Some human colon cancer cell lines (e.g., H508 cells) express M3 subtype muscarinic receptors that are activated by cholinergic agonists. The objective of the present study was to determine the cellular mechanisms underlying M3 muscarinic receptor-mediated proliferation of H508 human colon cancer cells. In H508 cells, but not in SNU-C4 cells that do not express muscarinic receptors, acetylcholine stimulated calcium-dependent phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and p90 ribosomal S6 kinase and consequent cell proliferation. Atropine or inhibitors of MAPK phosphorylation blocked these effects. Conversely, the actions of epidermal growth factor (EGF) on H508 cells were neither calcium dependent nor mediated by cholinergic mechanisms. Both acetylcholine- and EGF-induced phosphorylation of p44/42 MAPK was abolished in the presence of EGF receptor (EGFR) inhibitors (AG1478 and PD168393). In Chinese hamster ovary cells transfected with the rat M3 muscarinic receptor, which lack EGFR, acetylcholine-induced MAPK phosphorylation was not altered in the presence of EGFR inhibitors. In H508 cells, protein kinase C inhibitors did not alter acetylcholine- or EGF-induced MAPK phosphorylation. Finally, inhibition of EGFR activation abolished acetylcholine-induced H508 cell proliferation. These data indicate that, in H508 human colon cancer cells, cholinergic ligand interaction with M3 muscarinic receptors results in transactivation of EGFR, thereby stimulating cellular proliferation.

Acetylcholine↗

PubMed2003

Transport of glutathione conjugates and chemotherapeutic drugs by RLIP76 (RALBP1): a novel link between G-protein and tyrosine kinase signaling and drug resistance.

Our studies have shown that RLIP76 (RALBP1), a 76 kDa Ral-binding, Rho/Rac-GAP and Ral effector protein, is a novel multispecific transporter of xenobiotics as well as GS-Es. Like previously characterized ABC transporters, it mediates ATP-dependent transport of structurally unrelated amphiphilic xenobiotics and displays inherent ATPase activity, which is stimulated by its substrate allocrites. It does not have significant sequence homology with ABC transporters and differs from the ABC transporters in several other important aspects, including (i) lack of any close homologs in humans, (ii) lack of a classical Walker domain, (iii) integral membrane association without clearly defined transmembrane domains and (iv) its role as a direct link to Ras/Ral/Rho and EGF-R signaling through its multifunctional nature, including GAP activity, regulation of exocytosis as well as clathrin-coated pit-mediated receptor endocytosis. Its multifunctional nature derives from the presence of multiple motifs, including a Rho/Rac GAP domain, a Ral effector domain binding motif, 2 distinct ATP-binding domains, a H(+)-ATPase domain, PKC and tyrosine kinase phosphorylation sites and the ability to undergo fragmentation into multiple smaller peptides which participate as components of macromolecular functional complexes. One of the physiologic functions of RLIP76 is regulation of intracellular concentration of the electrophilic intermediates of oxidative lipid metabolism by mediating efflux of GS-E formed from oxidative degradation of arachidonic acid, including leukotrienes and the 4HNE-GSH conjugate. RLIP76-mediated transport of amphiphilic chemotherapeutic agents such as anthracyclines and vinca alkaloids as well as GS-E produced during oxidative metabolism places this multifunctional protein in a central role as a resistance mechanism for preventing apoptosis caused by chemotherapeutic agents and a variety of external/internal stressors, including oxidative stress, heat shock and radiation.

ATP-Binding Cassette Transporters↗

PubMed2003

Cells preconditioned with mild, transient UVA irradiation acquire resistance to oxidative stress and UVA-induced apoptosis: role of 4-hydroxynonenal in UVA-mediated signaling for apoptosis.

Because 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213-41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis.

Aldehydes↗