RESUMEN
Substantial epidemiologic data support a role for vitamin D in cancer prevention. However, dose-limiting hypercalcemic effects have proved a major obstacle to the development of natural vitamin D as a cancer chemopreventive. Structure-activity studies have sought to disassociate the toxicities and chemopreventive activities of vitamin D, and a number of synthetic deltanoids (vitamin D analogs) have shown considerable promise in this regard. Several such compounds have chemopreventive efficacy in preclinical studies, as does natural vitamin D. Data supporting further development of agents of this class include in vitro and in vivo evidence of antiproliferative, proapoptotic, prodifferentiating and antiangiogenic activities. Ongoing studies are aimed at further defining the molecular mechanisms through which vitamin D and synthetic deltanoids affect gene expression and cellular fate. Additional efforts are focused on establishing the chemopreventive index (efficacy vs toxicity) of each synthetic deltanoid.
Asunto(s)
Neoplasias/prevención & control , Vitamina D/análogos & derivados , Vitamina D/uso terapéutico , Animales , Humanos , Neoplasias/epidemiologíaRESUMEN
Relevant and feasible surrogate end-points are needed for the evaluation of intervention strategies against cancer and other chronic, life-threatening diseases. Carcinogenesis can be viewed as a process of progressive disorganization. This process is characterized by the accumulation of genotypic lesions and corresponding tissue and cellular abnormalities, including loss of proliferation and apoptosis controls. Potential surrogate end-points for cancer incidence include both phenotypic and genotypic biomarkers of this progression. In the US National Cancer Institute chemoprevention programme, histological modulation of a precancer (intraepithelial neoplasia) has so far been the primary phenotypic surrogate end-point in chemoprevention trials. Additionally, high priority has been given to biomarkers measuring specific and general genotypic changes correlated with the carcinogenesis progression model for the targeted cancer (e.g., progressive genomic instability as measured by loss of heterozygosity or amplification at specific microsatellite loci). Other potential surrogate end-points include proliferation and differentiation indices, specific gene and general chromosome damage, cell growth regulatory molecules, and biochemical activities (e.g., enzyme inhibition). Serum biomarkers thought to be associated with cancer progression (e.g., prostate-specific antigen) are particularly appealing surrogate end-points because of accessibility. Potentially chemopreventive effects of the test agent may also be measured (e.g., tissue and serum estrogen levels in studies of steroid aromatase inhibitors). To establish chemopreventive efficacy, prevention of virtually all biomarker lesions, or of those lesions with particular propensity for progression, may be required. Ideally, the phenotype and genotype of any new or remaining precancers in the target tissue of chemopreventive agent-treated subjects would show less, and certainly no greater, potential for progression than those of placebo-treated subjects.
Asunto(s)
Anticarcinógenos/uso terapéutico , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor , Evaluación de Medicamentos/métodos , Neoplasias/prevención & control , Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Humanos , Modelos Genéticos , Neoplasias/epidemiología , Selección de Paciente , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/epidemiologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Antioxidantes/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Tionas/farmacología , Tiofenos/farmacología , Animales , Catalasa/genética , Catalasa/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/metabolismo , Immunoblotting , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Estrés Oxidativo/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Arsenitos/farmacología , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Cartilla de ADN , Células PC12 , Regiones Promotoras Genéticas , Ratas , Factor de Transcripción CHOP , Transcripción GenéticaRESUMEN
Oxidative stress is thought to play a critical role in aging and the pathogenesis of human disease. Molecular studies of both the physiologic function of oxidants and the deleterious consequences of exposure to oxidative stress have suggested that signal transduction cascades may be targeted by oxidants. Here, we review recent studies from this laboratory examining the molecular basis for the activation of mitogen-activated protein kinases by oxidative stress and the influence of these pathways on cellular fate. We examine the association between constitutive activation of extracellular signal-regulated kinase (ERK) and cancer, and discuss how such mechanisms may contribute to oxidant-induced skin carcinogenesis. We also address the relationship between a decline in activation of this same pathway and the aged phenotype. In this regard, we review evidence that a decrease in activation of ERK by growth factor correlates with a reduced proliferative capacity in the isolated rat hepatocyte model, and we provide new data indicating that the activation of the ERK pathway in response to oxidant stimuli is also decreased with age. Further evidence demonstrates that this alteration is associated with both a reduced mitogenic response and a decline in hepatocyte cell survival in response to oxidative stress. Finally, we provide perspective on how modulations in ERK signaling may interplay with other changes in signal transduction cascades in the aging process.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Estrés Oxidativo , Animales , Activación Enzimática , Humanos , Ratas , Transducción de SeñalRESUMEN
Hepatocellular carcinoma (HCC) is among the most prevalent and deadly cancers worldwide. Elevated incidence of HCC is strongly associated with hepatitis B virus infection and dietary exposure to hepatotoxic contaminants. Vaccination programs against viral hepatitis hold hope for the eventual reduction of HCC incidence, and screening of cirrhotic patients may provide one mechanism for improving prognosis of HCC patients. Additional efforts have focused on preventing or retarding liver cancer development in populations at risk for HCC mortality. This review highlights recent clinical trials aimed at exploring the efficacy and practicality of chemoprevention efforts in these cohorts. Preclinical findings that provide insight into mechanisms of hepatocarcinogenesis and point to potential strategies for intervention also are discussed. Finally, novel molecular mechanisms contributing to the known ability of chemopreventive agents to inhibit liver cancer in experimental models are explored.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Neoplasias Hepáticas/prevención & control , Carcinoma Hepatocelular/virología , Hepatitis B/complicaciones , Humanos , Neoplasias Hepáticas/virología , Factores de RiesgoRESUMEN
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.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hipertensión/enzimología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Enfermedad Aguda , Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Western Blotting , Sistema Cardiovascular/enzimología , Sistema Cardiovascular/fisiopatología , Células Cultivadas , Interpretación Estadística de Datos , Activación Enzimática , Hipertensión/etiología , Hipertensión/fisiopatología , Masculino , Mitógenos/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Nitroprusiato/farmacología , Fenilefrina/farmacología , Proteína Fosfatasa 1 , ARN/análisis , Ratas , Ratas Wistar , Restricción Física , Estrés Fisiológico/complicaciones , Vasoconstrictores/farmacología , Vasodilatadores/farmacología , Vasopresinas/farmacologíaRESUMEN
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.
Asunto(s)
Apoptosis , Carcinoma/patología , Neoplasias Colorrectales/patología , Ciclinas/genética , Prostaglandinas A/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma/genética , División Celular , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica , HumanosRESUMEN
Phenylacetate (PA) and related aromatic fatty acids constitute a novel class of relatively nontoxic antineoplastic agents. These compounds induce tumor cytostasis and growth inhibition and differentiation of cancer cells, but little is known regarding the molecular events mediating these biological effects. Using human breast carcinoma MCF-7 cells as a model, we show here that PA-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and dephosphorylation of the retinoblastoma protein (pRB). The induction of p21WAF1/CIP1 mRNA by PA was independent of the cellular p53 status. To directly assess the contribution of p21Waf1/Cip1 to PA-mediated cytostasis, we compared the effects of PA in parental MCF-7 cells and cells expressing reduced levels of p21Waf1/Cip1 protein (clones AS.3 and AS.4), accomplished through constitutive expression of antisense p21Waf1/Cip1 transcripts. In contrast to parental cells, AS.3 and AS.4 cells did not show reduced pRB phosphorylation following PA treatment, indicating that p21Waf1/Cip1 induction by PA is required for dephosphorylation (inactivation) of pRB, a known mediator of cell cycle control. A prominent role for p21Waf1/Cip1 in mediating PA-induced growth arrest was further supported by the demonstration that embryonal fibroblasts derived from a p21WAF1/CIP1 knockout mouse (p21-/- mouse embryonal fibroblasts) did not growth arrest following PA treatment, whereas PA effectively induced p21WAF1/CIP1 mRNA and growth inhibition of the wild-type mouse embryonal fibroblasts. Taken together, our findings strongly support a role for p21Waf1/Cip1 in the PA-mediated inhibition of cell growth.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Quinasas CDC2-CDC28 , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Fenilacetatos/farmacología , Animales , Elementos sin Sentido (Genética) , Neoplasias de la Mama , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/fisiología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The mitogen-activated protein kinase (MAPK) cascade plays an important role in carcinogenic development. Herein, we show that the skin tumor promoter butylated hydroxytoluene hydroperoxide (BHTOOH) stimulates a rapid and potent (14- to 20-fold) activation of extracellular signal-regulated kinase (ERK) in vivo and in cultured mouse keratinocytes. BHTOOH also moderately (5-fold) activated c-jun-N-terminal kinase, and 38-kDa MAPK-related protein in these same cells. N-acetylcysteine and o-phenanthroline abolished ERK activation by BHTOOH, consistent with a requirement for metal-dependent formation of reactive intermediates. Indeed, 4-CD3-BHTOOH, an analogue that generates less of the metabolite BHT-quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) and fewer tumors in vivo, accordingly exhibited diminished potency for activating ERK. ERK activation by BHTOOH was inhibited by suramin, and by expression of dominant-negative Ras-N-17 in PC12 cells, suggesting overlap between the pathways for BHTOOH and growth factor signaling. Induction of MAPK-dependent genes c-fos and MAPK phosphatase-1 by BHTOOH was also blocked by Ras-N-17 expression. Moreover, expression of Ras-N-17 or kinase-defective MAPK kinase (MEK) diminished cell survival following BHTOOH exposure. Similarly, pretreatment with suramin or the MEK inhibitor PD098059 also potentiated the toxicity of BHTOOH. On the other hand, expression of constitutively active MEK enhanced cell survival. Thus, we demonstrate that the MAPK cascade is critical to the cellular response to BHTOOH. This study suggests a functional role for MAPK activation in tumor promotion stimulated by oxidants and other agents.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Carcinógenos/toxicidad , Quinonas/toxicidad , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/enzimología , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Oxidantes/toxicidad , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Piel/efectos de los fármacos , Piel/enzimología , Estimulación Química , Proteínas ras/fisiologíaRESUMEN
GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter-reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H2O2 and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H2O2 and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H2O2, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H2O2. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Estrés Oxidativo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Arsenitos/farmacología , Secuencia de Bases , Línea Celular Transformada , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oxidantes/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Compuestos de Sulfhidrilo/metabolismo , Factor de Transcripción CHOP , Activación Transcripcional , Rayos UltravioletaRESUMEN
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 H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, H2O2 increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 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 H2O2. H2O2-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 H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to H2O2 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 H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Músculo Liso Vascular/efectos de los fármacos , Células 3T3 , Animales , Aorta/citología , Aorta/efectos de los fármacos , Aorta/fisiología , Western Blotting , Células Cultivadas , Activación Enzimática , Radicales Libres/análisis , Radicales Libres/metabolismo , Expresión Génica/efectos de los fármacos , Genes fos , Genes jun , Células HeLa , Humanos , Cinética , Luciferasas/análisis , Luciferasas/biosíntesis , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Células PC12 , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfotirosina/análisis , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
A number of studies have demonstrated that the proliferative capacity of cells declines with aging. In particular, epidermal growth factor (EGF)-stimulated DNA synthesis is reduced in hepatocytes from aged rats relative to young rats. Growth factor stimulation activates a genetic program in large part regulated by a family of mitogen-activated protein kinases (MAPK) that phosphorylate and thereby activate transcription factors involved in controlling the expression of proliferation-associated genes. In the present study, we compared the activation of the extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1) MAPK in EGF-stimulated hepatocytes derived from young (6-month) and aged (24-month) rats. JNK activity was not appreciably altered by EGF treatment of cells from either age group. In contrast, ERK2 was highly activated by EGF treatment, but the magnitude of activation was significantly lower in hepatocytes of aged animals compared to those of young animals (7-fold versus 20-fold, respectively). The reduced ERK2 activity in response to EGF was associated with decreased c-fos and c-jun mRNA expression and lower levels of AP-1 transcription factor DNA binding activity in the aged hepatocytes. Finally, the basal expression of MAPK phosphatase 1, a MAPK-regulated gene involved in regulating MAPK activity, was higher in aged hepatocytes. Taken together, these findings suggest that an alteration in the balance between MAP kinase-phosphatase activities could contribute to the age-related decline in proliferative capacity.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Factor de Crecimiento Epidérmico/farmacología , Hígado/enzimología , Proteínas Quinasas Activadas por Mitógenos , Fosfoproteínas Fosfatasas , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Células Cultivadas , Fosfatasa 1 de Especificidad Dual , Activación Enzimática , Expresión Génica , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Cinética , Hígado/efectos de los fármacos , Hígado/crecimiento & desarrollo , Masculino , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción AP-1/metabolismoRESUMEN
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.
Asunto(s)
Arsenitos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Compuestos de Sodio/farmacología , Acetilcisteína/farmacología , Animales , Western Blotting , Línea Celular , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Fibroblastos , Depuradores de Radicales Libres/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Células PC12 , Ratas , Suramina/farmacología , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por Mitógenos , Proteínas ras/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/genética , Oxígeno/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Proteínas de Unión al ADN/biosíntesis , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hemo Oxigenasa (Desciclizante)/biosíntesis , Oxidorreductasas/metabolismo , Factor de Transcripción CHOP , Factores de Transcripción/biosíntesis , Activación TranscripcionalRESUMEN
Metabolism of the skin tumor promoter butylated hydroxy-toluene hydroperoxide (2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone; BHTOOH) to reactive intermediates is required for tumor promotion by this compound. In particular, an electrophilic quinone methide is known to mediate both in vivo tumor promotion as well as in vitro cytotoxicity by BHTOOH. In the present study, the role of this reactive intermediate in the induction of ornithine decarboxylase (ODC), a gene strongly associated with tumor promotion, was investigated in cultured keratinocytes. BHTOOH stimulates a time-dependent increase in ODC enzyme activity, paralleled by ODC mRNA induction, suggesting transcriptional regulation of ODC by BHTOOH. Depletion of intracellular glutathione caused a 5-fold potentiation of keratinocyte sensitivity to BHTOOH. Concordantly, ODC induction by BHTOOH could be completely inhibited by soluble thiol compounds. These results suggest that ODC induction is mediated by a thiol-reactive metabolite of BHTOOH. The iron-specific chelator desferal blocked ODC induction by BHTOOH, indicating that formation of this intermediate is iron-dependent. Substitution of the 4-methyl group of BHTOOH with alkyl groups of incrementally larger size is known to reduce accordingly quinone methide production; comparative study of these BHTOOH analogs demonstrated a corresponding loss of potency for ODC induction, indicating that BHT-quinone methide mediates the in vitro induction of ODC by BHTOOH. Finally, kinase inhibitor studies suggested a role for protein kinase C in the induction of ODC by BHTOOH. Taken together, these results provide insight into the cellular mechanisms through which the reactive electrophile BHT-quinone methide can mediate alterations in gene expression, such as occur in tumor promotion in vivo.
Asunto(s)
Hidroxitolueno Butilado/análogos & derivados , Carcinógenos/farmacología , Indolquinonas , Indoles/farmacología , Ornitina Descarboxilasa/biosíntesis , Peróxidos/farmacología , Quinonas/farmacología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/enzimología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Animales , Secuencia de Bases , Hidroxitolueno Butilado/farmacología , Células Cultivadas , Quelantes/farmacología , Sinergismo Farmacológico , Inducción Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Isoquinolinas/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Queratinocitos/fisiología , Ratones , Datos de Secuencia Molecular , Ornitina Descarboxilasa/genética , Inhibidores de la Ornitina Descarboxilasa , Papiloma/inducido químicamente , Papiloma/enzimología , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
An electrophilic quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) functions in the toxicity of butylated hydroxytoluene (BHT) in both rodent liver and lung. BHT-QM has also been demonstrated to mediate tumor promotion in mouse skin by another metabolite of BHT, 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH). In the present study, the role of BHT-QM in the cytotoxicity of BHTOOH was investigated. The toxicity of BHTOOH was potentiated by glutathione depletion and inhibited by thiol compounds, indicating that BHTOOH is activated to a thiol-reactive, toxic intermediate. This activation process was suggested to be iron-dependent by the ability of an Fe(III)-specific chelator to inhibit BHTOOH toxicity. Comparative study of analogs of BHTOOH in which the 4-methyl group was substituted with CD3, ethyl, isopropyl or tert-butyl supported the hypothesis that BHT-QM mediates this toxicological response. The decreased rate of reactivity of quinone methides that occurs as the 4-alkyl group is enlarged was accompanied by a corresponding reduction in toxic potency. The structural requirements for quinone methide toxicity were also explored with a series of BHTOOH analogs substituted at the 2- and 6-positions of the molecule. Reducing the lipophilicity of the 2,6-tert-butyl groups is known to increase quinone methide reactivity with glutathione but does not diminish the rate of quinone methide formation from the hydroperoxide. Interestingly, alteration of only one of the tert-butyl groups did not change the toxic potency, whereas removal or replacement of both tert-butyl groups dramatically reduced the toxicity in control cells but not glutathione-depleted cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hidroxitolueno Butilado/análogos & derivados , Carcinógenos/toxicidad , Cocarcinogénesis , Indolquinonas , Indoles/toxicidad , Peróxidos/toxicidad , Quinonas/toxicidad , Neoplasias Cutáneas/inducido químicamente , Animales , Hidroxitolueno Butilado/toxicidad , Quelantes/farmacología , Colorimetría , Deferoxamina/farmacología , Ratones , Microscopía Electrónica , Relación Estructura-Actividad , Sales de Tetrazolio , Tiazoles , Azul de Tripano , Células Tumorales CultivadasRESUMEN
Cancer in humans and animals is a multistep disease process. In this process, a single cell can develop from an otherwise normal tissue into a malignancy that can eventually destroy the organism. The complex series of cellular and molecular changes that occur through the development of cancers can be mediated by a diversity of endogenous and environmental stimuli. Active oxygen species and other free radicals have long been known to be mutagenic; further, these agents have more recently emerged as mediators of the other phenotypic and genotypic changes that lead from mutation to neoplasia. Free radical production is ubiquitous in all respiring organisms, and is enhanced in many disease states, by carcinogen exposure, and under conditions of stress. Free radicals may therefore contribute widely to cancer development in humans. This review explores the molecular mechanisms through which free radicals can participate in the carcinogenic process.
Asunto(s)
Daño del ADN , Neoplasias/etiología , Oxígeno/metabolismo , Animales , Cobre/metabolismo , Radicales Libres/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Mutación , Oxidación-Reducción , Especies Reactivas de OxígenoRESUMEN
Surprisingly, both of the synthetic 1-(hydroxymethyl)-25-hydroxyvitamin D3 diastereomers (-)-2 and (+)-3 retained the antiproliferative activity of natural calcitriol in murine keratinocytes. Preliminary studies indicated, however, that both of these synthetic diastereomers were less than 0.1% as effective as calcitriol for binding to the 1,25-(OH)2-D3 receptor and that they were less than 0.1% as potent as calcitriol for calbindin-D28K induction in organ-cultured embryonic chick duodenum. 1-(Hydroxymethyl)-25-hydroxyvitamin D3 homologs (-)-2 and (+)-3 were synthesized in a convergent manner by combining enantiomerically pure C,D-ring ketone 12 with highly enantiomerically enriched A-ring phosphine oxides (-)-11a and (+)-11b. These A-ring chirons were prepared starting from thermal [2 + 4] cycloaddition of 3-bromo-2-pyrone and acrolein.
Asunto(s)
Calcitriol/análogos & derivados , Queratinocitos/citología , Animales , Unión Competitiva , Calbindina 1 , Calbindinas , Calcitriol/síntesis química , Calcitriol/metabolismo , Calcitriol/farmacología , División Celular/efectos de los fármacos , Línea Celular , Embrión de Pollo , Duodeno/efectos de los fármacos , Duodeno/embriología , Duodeno/metabolismo , Queratinocitos/efectos de los fármacos , Ratones , Estructura Molecular , Técnicas de Cultivo de Órganos , Ornitina Descarboxilasa/metabolismo , Receptores de Calcitriol , Receptores de Esteroides/metabolismo , Proteína G de Unión al Calcio S100/biosíntesis , Estereoisomerismo , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Free radical derivatives of peroxides, hydroperoxides, and anthrones are thought to mediate tumor promotion by these compounds. Further, the promoting activity of phorbol esters is attributed, in part, to their ability to stimulate the cellular generation of oxygen radicals. A hydroperoxide metabolite of butylated hydroxytoluene, 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHTOOH), has previously been shown to be a tumor promoter in mouse skin. BHTOOH is extensively metabolized by murine keratinocytes to several radical species. The primary radical generated from BHTOOH is a phenoxyl radical that can disproportionate to form butylated hydroxytoluene quinone methide, a reactive electrophile. Since electrophilic species have not been previously postulated to mediate tumor promotion, the present study was undertaken to examine the role of this electrophile in the promoting activity of BHTOOH. The biological activities of two chemical analogs of BHTOOH, 4-trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH, were compared with that of the parent compound. 4-Trideuteromethyl-BHTOOH and 4-tert-butyl-BHTOOH have a reduced ability or inability, respectively, to form a quinone methide; however, like the parent compound, they both generate a phenoxyl radical when incubated with keratinocyte cytosol. The potency of BHTOOH, 4-trideuteromethyl-BHTOOH, and 4-tert-butyl-BHTOOH as inducers of ornithine decarboxylase, a marker of tumor promotion, was commensurate with their capacity for generating butylated hydroxytoluene quinone methide. These initial results were confirmed in a two-stage tumor promotion protocol in female SENCAR mice. Together, these data indicate that a quinone methide is mediating tumor promotion by BHTOOH, providing direct evidence that an electrophilic intermediate can elicit this stage of carcinogenesis.