Kathryn Z. Guyton

Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury.

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by HO and explored the impact of altering the pathways to kinase activation on cell survival following HO exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of HO treatment, whereupon rapid inactivation ensued. HO activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, HO increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited HO stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by HO was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by HO. HO-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to HO toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to HO toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by HO and suggest that ERK plays a critical role in cell survival following oxidant injury.

Differential activation of ERK, JNK/SAPK and P3/CSBP/RK map kinase family members during the cellular response to arsenite

Exposure of cells to either proliferative or stressful stimuli elicits a complex response involving one or more distinct phosphorylation cascades culminating in the activation of multiple members of the mitogen-activated protein kinase (MAPK) family, including extracellular signal regulated kinase (ERK), stress-activated c-Jun N-terminal kinase (JNK/SAPK), and p38/RK/CSBP protein kinase. While the pathways transducing mitogenic stimuli to these kinases are relatively well established, the early signalling events leading to their activation in response to stress are poorly understood. In the present study, we examined ERK, JNK/SAPK, and p38 activation in cells treated with the sulfhydryl-reactive agent sodium arsenite. Arsenite treatment potently activated both JNK/SAPK and p38, but only moderately activated ERK. Activation of all three kinases was prevented by the free radical scavenger N-Acetyl-L-cysteine, suggesting that an oxidative signal initiates the responses. Suramin, a growth factor receptor poison, significantly inhibited ERK activation by arsenite, but had little effect on either JNK/SAPK or p38 activity. In contrast, suramin inhibited the activation of all three kinases by short wavelength ultraviolet light (UVC) irradiation. In addition, comparative studies with wild-type PC12 cells and PC12 cells expressing a dominant negative Ras mutant allele indicated that arsenite activates ERK primarily through a Ras-dependent pathway(s), while activation of both JNK/SAPK and p38 occurs through a mechanism relatively independent of Ras. These results suggest that JNK/SAPK and p38 may share common upstream regulators distinct from those involved in ERK activation.

Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence.

Chemoprotective 3H-1,2-dithiole-3-thione induces antioxidant genes in vivo.

Several 1,2-dithiole-3-thiones are potent inhibitors of chemical-induced tumors in multiple tissues. Chemoprotection by 1, 2-dithiole-3-thiones has been associated with induction of detoxication enzymes, although several studies suggest that additional mechanisms may be involved. In this study, we examined the induction of hepatic antioxidant genes in rats treated with 3H-1, 2-dithiole-3-thione (D3T). After a 24 h D3T treatment, a 2.4-fold increase in catalase mRNA was observed, which was accompanied by a 1. 5-fold increase in catalase protein expression and a 2.3-fold increase in catalase activity. D3T also mediated 2.9-, 5.9-, and 3. 7-fold increases in the 1.0, 3.0, and 4.0 kb mRNA species of manganese superoxide dismutase (MnSOD), respectively. The induction of MnSOD mRNA by D3T was coincident with 1.7-fold and 4.6-fold increases in MnSOD protein and enzyme activity, respectively. Induction of gamma-glutamylcysteine synthetase mRNA by D3T was accompanied by an increase in glutathione levels. Nuclear run-on assays provided evidence that D3T enhances the transcription rate from MnSOD, catalase, and gamma-glutamylcysteine synthetase. In support of this view, D3T also activated an MnSOD promoter-reporter construct in transiently transfected HepG2 cells. In light of observations that antioxidant enzyme regulation may be altered during carcinogenesis, induction of these genes could provide a potentially important mechanism of action of chemoprotective 1, 2-dithiole-3-thiones.

Expression of stress response genes GADD153, c-jun, and Heme Oxygenase-1 in H2O2 and O2-resistant fibroblasts

In the present study, the expression of the stress-inducible genes GADD153, c-jun, heme oxygenase-1 (HO-1), and HSP70 was compared among parental hamster diploid fibroblasts (HA-1), and cell lines isolated for resistance to either H2O2 (OC14) or O2 (O2R95). Both OC14 and O2R95 cell lines are known to have significantly augmented cellular antioxidant defenses, including increased glutathione content, as well as enhanced catalase, superoxide dismutase, and glutathione peroxidase activities. Northern analysis indicated that basal expression of HO-1 and c-jun is also elevated in these resistant cell lines. Relative to HA-1 values, basal GADD153 mRNA expression was approximately threefold higher in O2R95, but twofold lower in OC14 cells. HSP70 mRNA expression was comparable among parental and resistant cell lines. Both OC14 and O2R95 cells showed greatly enhanced survival following H2O2 exposure. The H2O2 doses that induced 50% toxicity in parental and resistant cells (3 vs. 30-60 x 10(-13) mol/cell, respectively) differed by more than an order of magnitude. Similarly, GADD153, c-jun, and HO-1 mRNA were elevated in control cells following exposure to doses of H2O2 an order of magnitude lower than is required for gene activation in resistant cell lines. Nonetheless, at equitoxic doses, the level of induction of GADD153 and HO-1 was greater in resistant than in parental cell lines. Taken together, our results suggest that alterations in the basal level of expression of certain stress-responsive genes, including HO-1, c-jun, and GADD153, may contribute to the H2O2-resistant phenotype in these fibroblast cell lines. Further, changes in the regulation of these genes in response to adverse stimuli may provide an additional mechanism for enhanced cell survival following oxidative stress.

Induction of Mitogen-Activated Protein Kinase Phosphatase-1 During Acute Hypertension

Recently, we demonstrated that elevated blood pressure activates mitogen-activated protein (MAP) kinases in rat aorta. Here we provide evidence that the vascular response to acute hypertension also includes induction of MAP kinase phosphatase-1 (MKP-1), which has been shown to function in the dephosphorylation and inactivation of MAP kinases. Restraint or immobilization stress, which leads to a rapid rise in blood pressure, resulted in a rapid and transient induction of MKP-1 mRNA followed by elevated MKP-1 protein expression in rat aorta. That the induction of MKP-1 by restraint was due to the rise in blood pressure was supported by the finding that several different hypertensive agents (phenylephrine, vasopressin, and angiotensin II) were likewise capable of eliciting the response, and sodium nitroprusside, a nonspecific vasodilator agent that prevented the acute rise in blood pressure in response to the hypertensive agents, abrogated MKP-1 mRNA induction. The in vivo effects could not be mimicked by treatment of cultured aortic smooth muscle cells with similar doses of the hypertensive agents. These findings support a role for MKP-1 in the in vivo regulation of MAP kinase activity during hemodynamic stress.

Oxidative Stress, Gene Expression, and the Aging Process

Oxidative stress plays a causative role in the development of age-related degenerative diseases and may be an underlying determinant of the aging process itself. In this review, we summarize the evidence from a variety of experimental systems that supports this hypothesis. We discuss the molecular basis for the control of gene expression by oxidants, with particular focus on recent studies demonstrating the activation of mitogen-activated protein kinase signal transduction by oxidative stress. How alterations in such pathways may contribute to aging is addressed, and specific examples of transcription factors whose activities are altered both by oxidants and with aging are given. We provide a model illustrating the multiple sites in signal transduction pathways sensitive to oxidative stress, which may indicate targets for strategies aiming to retard or reverse age-related phenotypic changes. OXIDATIVE STRESS IN AGING: CURRENT VIEWS AND HYPOTHESES Exposure to reactive oxygen intermediates (ROI), including molecular oxygen, superoxide, hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals, occurs ubiquitously in an aerobic environment. Accordingly, aerobic organisms have widely adapted oxidation-reduction reactions to function in key metabolic and regulatory pathways necessary for normal cell growth. In addition, a number of defense mechanisms to control the level of ROI have evolved as an accessory to these processes. However, when ROI and other oxidizing species exceed the cellular antioxidant capacity, oxidative stress results and oxidative damage to lipids, protein, and DNA ensues. The accumulation of oxidative modifications as a causative factor in aging and degenerative processes was originally proposed by Harman 40 years ago (Harman...

Experimental analysis of exome-scale mutational signature of glycidamide, the reactive metabolite of acrylamide

Acrylamide, a probable human carcinogen, is ubiquitously present in the human environment, with sources including heated starchy foods, coffee and cigarette smoke. Humans are also exposed to acrylamide occupationally. Acrylamide is genotoxic, inducing gene mutations and chromosomal aberrations in various experimental settings. Covalent haemoglobin adducts were reported in acrylamide-exposed humans and DNA adducts in experimental systems. The carcinogenicity of acrylamide has been attributed to the effects of glycidamide, its reactive and mutagenic metabolite capable of inducing rodent tumors at various anatomical sites. In order to characterize the pre-mutagenic DNA lesions and global mutation spectra induced by acrylamide and glycidamide, we combined DNA-adduct and whole-exome sequencing analyses in an established exposure-clonal immortalization system based on mouse embryonic fibroblasts. Sequencing and computational analysis revealed a unique mutational signature of glycidamide, characterized by predominant T:A>A:T transversions, followed by T:A>C:G and C:G>A:T mutations exhibiting specific trinucleotide contexts and significant transcription strand bias. Computational interrogation of human cancer genome sequencing data indicated that a combination of the glycidamide signature and an experimental benzo[a]pyrene signature are nearly equivalent to the COSMIC tobacco-smoking related signature 4 in lung adenocarcinomas and squamous cell carcinomas. We found a more variable relationship between the glycidamide‐ and benzo[a]pyrene-signatures and COSMIC signature 4 in liver cancer, indicating more complex exposures in the liver. Our study demonstrates that the controlled experimental characterization of specific genetic damage associated with glycidamide exposure facilitates identifying corresponding patterns in cancer genome data, thereby underscoring how mutation signature laboratory experimentation contributes to the elucidation of cancer causation. A 40-word summary Innovative experimental approaches identify a novel mutational signature of glycidamide, a metabolite of the probable human carcinogen acrylamide. The results may elucidate the cancer risks associated with exposure to acrylamide, commonly found in tobacco smoke, thermally processed foods and beverages.

Chemopreventive Efficacy of Natural Vitamin D and Synthetic Analogs

The activities of vitamin D and synthetic vitamin D analogs (deltanoids) are primarily mediated through binding to vitamin D receptors (VDRs), members of the steroid/thyroid receptor superfamily. Chemopreventive properties of this class of compounds include in vitro and in vivo antiproliferative, pro-apoptotic, pro-differentiating, and anti-angiogenic activities. Natural vitamin D and a number of deltanoids have shown chemopreventive efficacy in preclinical studies. Although also supported by substantial epidemiologic data, the development of natural vitamin D as a cancer chemopreventive has been hindered by dose-limiting hypercalcemic effects. Unfortunately, these same safety concerns also apply to many deltanoids, including those approved for treatment of secondary hyperparathyroidism in patients with end-stage renal disease. However, several new synthetic deltanoids have recently shown promise in preclinical safety and chemopreventive efficacy studies.