RESUMEN
Menopausal estrogen replacement therapy is thought to be responsible for the recent decline in colorectal cancer (CRC) incidence among women. In the C57BL/6J-Min/+ mouse, an animal model of CRC, 17beta-estradiol (E(2)) prevents tumor formation in ovariectomized females. We examined human CRC intestinal cell lines to determine whether particular E(2) metabolites produced anti-tumor effects. Treatment of CRC cells with 2-methoxyestradiol (2-MeOE(2)) increased expression of p53 and p21(WAF1/CIP1) proteins and induced apoptosis, but did not produce changes in expression of estrogen receptor (ER)alpha or ERbeta. The finding that 2-MeOE(2) induces p53-mediated colon cell apoptosis in vitro supports a role for 2-MeOE(2) as an endogenous mediator of intestinal tumor suppression.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Colorrectales/patología , Estradiol/análogos & derivados , Estradiol/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , 2-Metoxiestradiol , Western Blotting , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptores de Estrógenos/metabolismo , Células Tumorales Cultivadas/metabolismoRESUMEN
The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.
Asunto(s)
Adenoma/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Uniones Adherentes/química , Transactivadores , Alelos , Animales , Cadherinas/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citosol/metabolismo , Enterocitos/metabolismo , Femenino , Eliminación de Gen , Genes Dominantes , Heterocigoto , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Mutantes , Mutación , Fosforilación , Pruebas de Precipitina , Unión Proteica , Fracciones Subcelulares , Tirosina/metabolismo , beta CateninaRESUMEN
Menopausal hormone replacement therapy has been widely used to alleviate the symptoms of menopause and to decrease the detrimental effects of ovarian hormone loss on bone density and cardiovascular health. Multiple studies of colorectal cancer epidemiology also support a role for hormone replacement therapy in prevention of colorectal cancer. We studied the effect of ovariectomy and estrogen replacement on tumor formation in C57BL/6J-Min/+ (Min/+) mice, animals that bear a germline mutation in murine Apc. These mice develop multiple intestinal tumors that show loss of wild-type Apc protein. After ovariectomy, intestinal adenomas in Min/+ mice increased by 77% (P = 0.0004). Ovariectomized Min/+ mice that were treated with a replacement dose of 17beta-estradiol had the same number of tumors as Min/+ mice that were neither castrated nor treated with estrogen replacement (P = 0.85). Examination of estrogen receptor (ER) levels in intestinal tissue by immunoblot showed changes in relative expression levels of ERalpha and ERbeta, with highest ERalpha and lowest ERbeta expression in the normal-appearing intestine of Min/+ mice, and lowest ERalpha and highest ERbeta expression in the enterocytes of animals that received 17beta-estradiol. These results suggest that endogenous estrogens protect against Apc-associated tumor formation and that tumor prevention by 17beta-estradiol is associated with an increase in ERbeta and a decrease in ERalpha expression in the target tissue.
Asunto(s)
Estrógenos/fisiología , Neoplasias Intestinales/metabolismo , Receptores de Estrógenos/biosíntesis , Proteína de la Poliposis Adenomatosa del Colon , Animales , Proteínas del Citoesqueleto/genética , Enterocitos/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Terapia de Reemplazo de Estrógeno , Femenino , Genes APC/genética , Mutación de Línea Germinal , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/etiología , Neoplasias Intestinales/genética , Ratones , Ratones Endogámicos C57BL , OvariectomíaRESUMEN
Colorectal cancer is sensitive to dietary influences. Epidemiological data linking high intake of fruits and vegetables to decreased cancer risk have prompted the search for specific plant constituents implicated in tumor prevention. This task is difficult because of the complex chemical composition of plant foods and the multifactorial nature of carcinogenesis. Researchers are aided in this effort by the C57BL/6J-Min/+ (Min/+) mouse, an animal bearing a germline defect in Apc that is similar to the initiating genetic event in the majority of human colorectal cancers. In this study, we treated Min/+ mice with (+)-catechin, a phenolic antioxidant abundant in certain fruits. Administration of (+)-catechin in an AIN-76A diet at doses of 0.1 and 1% decreased the intestinal tumor number by 75 and 71%, respectively. Mechanistic studies linked this effect to (+)-catechin-induced changes in integrin-mediated intestinal cell-survival signaling, including structural alteration of the actin cytoskeleton and decreased focal adhesion kinase (FAK) tyrosine phosphorylation. Immunoblot analysis of small intestine scrapings from Min/+ mice and Apc+/+ wild-type C57BL/6J littermates together with excised Min/+ adenomas showed increased expression of phosphorylated FAK in the macroscopically normal enterocytes of untreated Min/+ mice and adenomas. Confirming the relevance of this signaling pathway, treatment of Min/+ mice with (+)-catechin reduced the expression of phosphorylated FAK to a level similar to the wild-type littermate controls. Thus, the natural abundance and favorable bioavailability of (+)-catechin make it a promising addition to the list of potential colorectal cancer chemopreventive agents.
Asunto(s)
Anticarcinógenos/farmacología , Catequina/farmacología , Neoplasias Intestinales/prevención & control , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Células 3T3 , Animales , División Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Enterocitos/efectos de los fármacos , Enterocitos/enzimología , Activación Enzimática , Femenino , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrinas/fisiología , Neoplasias Intestinales/patología , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estereoisomerismo , Células Tumorales Cultivadas/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Epidemiological studies consistently indicate that consumption of fruits and vegetables lowers cancer risk in humans and suggest that certain dietary constituents may be effective in preventing colon cancer. Plant-derived phenolic compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis in vivo. In this study, we investigated the efficacy of several plant-derived phenolics, including caffeic acid phenethyl ester (CAPE), curcumin, quercetin and rutin, for the prevention of tumors in C57BL/6J-Min/+ (Min/+) mice. These animals bear a germline mutation in the Apc gene and spontaneously develop numerous intestinal adenomas by 15 weeks of age. At a dietary level of 0.15%, CAPE decreased tumor formation in Min/+ mice by 63%. Curcumin induced a similar tumor inhibition. Quercetin and rutin, however, both failed to alter tumor formation at dietary levels of 2%. Examination of intestinal tissue from the treated animals showed that tumor prevention by CAPE and curcumin was associated with increased enterocyte apoptosis and proliferation. CAPE and curcumin also decreased expression of the oncoprotein beta-catenin in the enterocytes of the Min/+ mouse, an observation previously associated with an antitumor effect. These data place the plant phenolics CAPE and curcumin among a growing list of anti-inflammatory agents that suppress Apc-associated intestinal carcinogenesis.
Asunto(s)
Poliposis Adenomatosa del Colon/patología , Neoplasias Intestinales/prevención & control , Fenoles/farmacología , Plantas/química , Transactivadores , Poliposis Adenomatosa del Colon/metabolismo , Animales , División Celular/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Enterocitos/citología , Enterocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , beta CateninaRESUMEN
Epidemiological studies of colorectal cancer incidence suggest that the development of this disease can be modulated by dietary factors. Among the micronutrients showing significant efficacy in tumor prevention are polyphenolic antioxidants found in fruits and vegetables. Epidemiological studies also indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colorectal cancer. Integrin-mediated cell-matrix contact provides critical signaling that regulates cellular proliferation, migration, and apoptosis. A signaling mediator for this system is focal adhesion kinase (FAK). Thus far, FAK has not been identified as a target for the inhibitory action of any chemopreventive drug in vivo or in vitro. However, the loss of integrin-mediated cell-matrix contact can induce apoptosis (anoikis), and effective chemopreventive agents typically increase the rate of enterocyte apoptosis. Therefore, we asked whether the NSAID, sulindac sulfide, and the phenolic antioxidant, caffeic acid phenethyl ester (CAPE), affected FAK expression or tyrosine phosphorylation in human colon carcinoma cells. We show that subapoptotic doses of both sulindac sulfide and CAPE caused a rearrangement of the actin cytoskeleton and consequently the loss of focal adhesion plaques. These drugs also reduced the tyrosine phosphorylation of FAK and an associated factor, p130Cas. Steady-state levels of these proteins, together with other relevant signaling molecules, remained unchanged after treatments. Finally, we show that both CAPE and sulindac reduced cell invasion, a functional assay for the inhibition of signaling downstream of FAK. These data strongly suggest that chemopreventive drugs can regulate FAK activity. In conclusion, these novel studies add modulation of integrin-mediated signaling to the spectrum of activity of NSAIDs and plant phenolics.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/prevención & control , Integrinas/fisiología , Proteínas , Transducción de Señal/efectos de los fármacos , Actinas/efectos de los fármacos , Actinas/metabolismo , Ácidos Cafeicos/farmacología , Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Proteína Sustrato Asociada a CrK , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Sulindac/análogos & derivados , Sulindac/farmacología , Células Tumorales CultivadasRESUMEN
We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Anticarcinógenos/farmacología , Ácidos Cafeicos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Inflamación/genética , Isoenzimas/biosíntesis , Mucosa Bucal/citología , Alcohol Feniletílico/análogos & derivados , Regiones Promotoras Genéticas/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Aire , Animales , Ácidos Araquidónicos/metabolismo , Calcimicina/antagonistas & inhibidores , Calcimicina/farmacología , Carcinoma de Células Escamosas/patología , Carragenina/toxicidad , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Vectores Genéticos/genética , Humanos , Indometacina/farmacología , Inflamación/inducido químicamente , Inflamación/metabolismo , Ionóforos/antagonistas & inhibidores , Ionóforos/farmacología , Isoenzimas/genética , Masculino , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana , Nucleopoliedrovirus/genética , Alcohol Feniletílico/farmacología , Fosfolípidos/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes de Fusión/biosíntesis , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level. In contrast, binding of factors to this probe was inhibited after either in vivo treatment of cells with CAPE or in vitro addition of this compound to the nuclear extract. In view of the clear stimulation by CAPE of gene expression mediated by hARE, possible explanations of this result are discussed.
Asunto(s)
Antioxidantes/farmacología , Ácidos Cafeicos/farmacología , Alcohol Feniletílico/análogos & derivados , Quinona Reductasas/genética , ADN/metabolismo , Humanos , Alcohol Feniletílico/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismoRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) with sterically hindered fjord region diol epoxides are interesting with respect to their potency as carcinogens, interactions with DNA and mutagenic specificities. Unlike the bay region PAH derivative, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydroxybenzo[a]pyrene (BPDE), reactive metabolites of two fjord region PAH, trans-3,4-dihydroxy-anti-1, 2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene [(+/-)-anti-BcPHDE] and trans-11,12-dihydroxy-syn-BgCDE], react with DNA to yield high levels of adenine adducts. We previously found that forward mutations induced by (+/-)-anti-BcPHDE in the dihydrofolate reductase (dhfr) gene of Chinese hamster ovary (CHO) cells preferentially targeted mRNA splice acceptor sites. (+/-)-anti-BcPHDE and (+/-)-syn-BgCDE are structurally similar; they differ only by the presence of an additional benzene ring. Thus we used (+/-)-syn-BgCDE to learn if the mutational target bias reflects aspects of the mutagen structure or its capacity to efficiently modify deoxyadenosine (dA) in vivo. dhfr(-) mutants were induced after treatment of hemizygous UA21 cells with a 0.75 microM dose of (+/-)-syn-BgCDE. Cell survival after carcinogen exposure was 40%. The induced mutation frequency was 9 x 10(-6), nearly 10-fold higher than the spontaneous one, but approximately 19-fold lower than formerly observed using (+/-)-anti-BcPHDE. In the 26 confirmed null dhfr(-) mutants 27 mutations were identified by DNA sequencing. The types of (+/-)-syn-BgCDE-induced mutations were very similar to those formerly induced by (+/-)-anti-BcPHDE. Consistent with the binding specificity, both chemicals induced transversion base substitution at purines (R-->T). The most prevalent type of mutation was A-->T, which represented 59% of the induced changes, compared with 42% for (+/-)-anti-BcPHDE. (+/-)-syn-BgCDE mutated mostly novel targets in the dhfr gene, sites not found mutated with any of the several other mutagens we have used in former studies. Whereas the 25 kg dhfr gene contains six coding exons, the majority (16/27) of (+/-)-syn-BgCDE-induced mutations were located in a single one (exon 4). A random distribution of mutations affecting splice acceptor sites (22%) was induced by (+/-)-syn-BgCDE. Hence, preferential mutation of these sites by (+/-)-anti-BcPHDE may reflect aspects of chromatin structure in vivo which make these sequences better targets for modification. Alternatively, the sequence context of these sites may dictate an adduct conformation which is poorer for damage recognition and/or efficient repair.
Asunto(s)
Crisenos/toxicidad , Mutágenos/toxicidad , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Cricetinae , Aductos de ADN/química , Sensibilidad y EspecificidadRESUMEN
Efficient replication of HIV-1 requires integration of a DNA copy of the viral genome into a chromosome of the host cell. Integration is catalyzed by the viral integrase, and we have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE, 2), and curcumin confer inhibitory activity against HIV-1 integrase. We now extend these findings by performing a comprehensive structure-activity relationship using CAPE analogues. Approximately 30 compounds have been prepared as HIV integrase inhibitors based on the structural lead provided by CAPE, which has previously been shown to exhibit an IC50 value of 7 microM in our integration assay. These analogues were designed to examine specific features of the parent CAPE structure which may be important for activity. Among the features examined for their effects on inhibitory potency were ring substitution, side chain length and composition, and phenyl ring conformational orientation. In an assay which measured the combined effect of two sequential steps, dinucleotide cleavage and strand transfer, several analogues have IC50 values for 3'-processing and strand transfer lower than those of CAPE. Inhibition of strand transfer was assayed using both blunt-ended and "precleaved" DNA substrates. Disintegration using an integrase mutant lacking the N-terminal zinc finger and C-terminal DNA-binding domains was also inhibited by these analogues, suggesting that the binding site for these compounds resides in the central catalytic core. Several CAPE analogues were also tested for selective activity against transformed cells. Taken together, these results suggest that the development of novel antiviral agents for the treatment of acquired immune deficiency syndrome can be based upon inhibition of HIV-1 integrase.
Asunto(s)
Antivirales/síntesis química , Ácidos Cafeicos/química , ADN Nucleotidiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Alcohol Feniletílico/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Ácidos Cafeicos/farmacología , Línea Celular Transformada , ADN/química , ADN/metabolismo , VIH/efectos de los fármacos , Humanos , Hidroxilación , Integrasas , Espectroscopía de Resonancia Magnética , Conformación Molecular , Datos de Secuencia Molecular , Estructura Molecular , Alcohol Feniletílico/química , Alcohol Feniletílico/farmacología , Ratas , Relación Estructura-Actividad , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE-treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Cafeicos/farmacología , Transformación Celular Neoplásica/patología , Alcohol Feniletílico/análogos & derivados , Acetilcisteína/farmacología , Animales , Catalasa/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular , Daño del ADN/efectos de los fármacos , Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Alcohol Feniletílico/farmacología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Ratas , Ratas Endogámicas F344RESUMEN
This study presents the physical characterization of mutants induced in mammalian cells by high linear energy transfer alpha-particle radiation that simulates exposure to radon daughters. Alpha-Particles from accelerated 4He at 150 keV/microns were used to induce 20 Chinese hamster ovary mutants that are deficient in dihydrofolate reductase (DHFR) activity. Parental cells were the hemizygous UA21 line. Cell survival decreased exponentially in response to radiation dose from 0.25 to 1.75 Gy. Mutants were obtained at 1.0 (17/20) and 1.25 Gy (3/20); treatments at 1.50 Gy failed to yield mutants. The induced frequency of mutation was 2.3 x 10(-6) at the 1.0 Gy dose, approximately 18-fold greater than the spontaneous mutation rate at this locus. DNA of the 20 confirmed null mutants were examined for alterations in the 25 kb DHFR gene by Southern blotting using a mixed probe that scans a continuous 34 kb of sequence. Deletions were the most prevalent induced change (18/20). Of the two point mutants, DNA sequencing showed that one carries a T:A-->G:C base substitution that changed Val135 to Gly in exon 5; carcinogen-induced reversion to a DHFR+ phenotype at a frequency of 3 x 10(-6) confirmed that the other also carried a single base change. The distribution of deletion break sites in the DHFR locus was non-random. In half of the mutants deletion break sites were clustered within a single 9.4 kb DHFR intron. Fine mapping within the gene of 14 mutants by Southern blotting localized 10 distinct break sites to small restriction fragments (< 2 kb). Results of this mapping indicated that three unequivocally independent mutants apparently arose by the same deletion and that others shared single break sites. Deletion sizes in these mutants were determined by Southern analysis using six cosmids and two plasmid probes that together span approximately 500 kb of sequence in the region of the DHFR locus. Probing blots with the cosmids defined a maximal deletion size in 15/17 mutants and confirmed that deletions extended < 150 kb in 11, a qualitatively different result from that previously obtained after a similar analysis of gamma-ray-induced DHFR- mutants. Since deletions were non-randomly distributed, typically less than replicon size and spared regions containing matrix attachment sites, the results suggest a model whereby alpha-particles induce double-strand breaks in accessible chromatin loops.
Asunto(s)
Partículas alfa , Deleción Cromosómica , Radón/toxicidad , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Cricetinae , Mutación PuntualRESUMEN
Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural determinations of (deoxyguanosin-8-yl)-aminofluorene; the carcinogen is in the B-DNA minor groove and its adopts a syn conformation mispaired with an anti A. The implications of this conformational distortion in DNA structure for damage recognition by cellular repair enzymes are discussed.
Asunto(s)
Carcinógenos/toxicidad , Reparación del ADN , Desoxiguanosina/análogos & derivados , Imidazoles/metabolismo , Imidazoles/toxicidad , Mutágenos/toxicidad , Piridinas/toxicidad , Tetrahidrofolato Deshidrogenasa/genética , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , ADN/metabolismo , Desoxiguanosina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Mutagenicidad , Conformación de Ácido NucleicoRESUMEN
Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.
Asunto(s)
Exones/efectos de los fármacos , Fluorenos/toxicidad , Mutación Puntual , Tetrahidrofolato Deshidrogenasa/deficiencia , Acetoxiacetilaminofluoreno/toxicidad , Animales , Secuencia de Bases , Células CHO , Cricetinae , Exones/genética , Datos de Secuencia MolecularRESUMEN
Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.
Asunto(s)
Empalme del ARN , Tetrahidrofolato Deshidrogenasa/genética , Empalme Alternativo , Animales , Secuencia de Bases , Células CHO , Secuencia de Consenso , Cricetinae , Exones , Genes Supresores , Intrones , Datos de Secuencia Molecular , Mutación , ARN Mensajero/genéticaRESUMEN
We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line of repair-deficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations.
Asunto(s)
Reparación del ADN , Fenantrenos/química , Tetrahidrofolato Deshidrogenasa/genética , Animales , Células CHO , Carcinógenos/química , Cricetinae , ADN/química , Daño del ADN , GenesRESUMEN
We previously showed that the preferred mutation induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy- 1,2,3,4-tetrahydrobenzo[c]phenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple purine target was present on both strands and all R----T transversion mutations yielded amino acid substitutions that were compatible with dihydrofolate reductase enzyme activity. From the size of the targets, a 2:1 ratio of mutations at the purines on the nontranscribed strand was expected if the DNA strands were mutationally equivalent. We isolated a total of 66 BcPHDE-induced revertants of two mutants that carry point mutations at either the 5' or the 3' end of the gene. All reversions at the 5' end arose by substitution on the nontranscribed strand; those at the 3' end showed a strand bias that favored this strand by 7:1. For both mutants, R----T transversions accounted for 88% of all the induced base changes. Thus, in this system, mutational strand bias is independent of the selection for phenotype. The results are consistent with the model of preferential repair of the transcribed strand as proposed by others. The involvement of RNA polymerase in the selective repair recruitment is discussed.
Asunto(s)
Benzo(a)Antracenos , Daño del ADN , Mutagénesis , Fenantrenos/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Animales , Secuencia de Bases , Carcinógenos/química , Línea Celular , Cricetinae , Cricetulus , Reparación del ADN , Femenino , Técnicas In Vitro , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes/efectos de los fármacos , Mutación , Fenantrenos/farmacología , Empalme del ARN , Tetrahidrofolato Deshidrogenasa/genética , Animales , Secuencia de Bases , Línea Celular , Cricetinae , Cricetulus , Inducción Enzimática , Femenino , Datos de Secuencia Molecular , Mutágenos/farmacología , Sondas de Oligonucleótidos , OvarioAsunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Tetrahidrofolato Deshidrogenasa/genética , Animales , Línea Celular , Cricetinae , Cricetulus , Inducción Enzimática , Femenino , Genes/efectos de los fármacos , Genes/efectos de la radiación , Mutágenos/farmacología , Ovario , Biosíntesis de Proteínas , Empalme del ARNRESUMEN
We previously treated Chinese hamster ovary (CHO) cells with benzo[a]pyrene diol epoxide (BPDE) and mutants at the dihydrofolate reductase (dhfr) locus were isolated. On the basis of Southern blotting and RNA heteroduplex mapping experiments, 14 of the 15 mutants were presumed to carry point mutations. Two restriction fragment length polymorphism mutants were cloned and sequenced; one carried a point mutation, the other a -1 frameshift mutation. Using polymerase chain reaction techniques and direct sequencing of amplified mutant DNA, we have now determined the induced changes in the remaining 12 cell lines. All changes occurred at guanine bases; all target guanines except one were on the non-transcribed coding strand. Most mutants (79%) contained base substitutions; the rest (3/14) carried frameshift mutations. Of the point mutations, all but one (91%) were GC----TA transversions either in the dhfr coding sequence or at splice sites. The single exception was a GC----AT transition. Of the frameshift mutations, two were deletions of a GC pair and the other was an insertion of an AT pair. Four different mutations (29%) were clustered in a 3 bp region in exon 4. Tandem guanine bases adjacent to adenine were favored sites for mutation occurring in 9/14 cases (64%). These results are consistent with data previously obtained by others in the supF shuttle vector system and the CHO aprt gene.