RESUMEN
In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs.
Asunto(s)
Mucosa Intestinal/metabolismo , Intestinos/citología , Neuropéptidos/metabolismo , Smegmamorpha/anatomía & histología , Smegmamorpha/metabolismo , Animales , Inmunohistoquímica , Intestinos/anatomía & histología , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/metabolismoRESUMEN
There is convincing evidence that cellular prooxidant states--that is, increased concentrations of active oxygen and organic peroxides and radicals--can promote initiated cells to neoplastic growth. Prooxidant states can be caused by different classes of agents, including hyperbaric oxygen, radiation, xenobiotic metabolites and Fenton-type reagents, modulators of the cytochrome P-450 electron-transport chain, peroxisome proliferators, inhibitors of the antioxidant defense, and membrane-active agents. Many of these agents are promoters or complete carcinogens. They cause chromosomal damage by indirect action, but the role of this damage in carcinogenesis remains unclear. Prooxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense and low molecular weight scavenger molecules, and many antioxidants are antipromoters and anticarcinogens. Finally, prooxidant states may modulate the expression of a family of prooxidant genes, which are related to cell growth and differentiation, by inducing alterations in DNA structure or by epigenetic mechanisms, for example, by polyadenosine diphosphate-ribosylation of chromosomal proteins.
Asunto(s)
Carcinógenos , Transformación Celular Neoplásica , Neoplasias/inducido químicamente , Oxígeno/metabolismo , Animales , Antioxidantes/farmacología , Carcinógenos/metabolismo , Carcinógenos/farmacología , Cationes/metabolismo , Diferenciación Celular , División Celular , Línea Celular , Membrana Celular/fisiología , Aberraciones Cromosómicas , Cromosomas/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/metabolismo , Transporte de Electrón , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Hidróxidos/metabolismo , Radical Hidroxilo , Peróxidos Lipídicos/metabolismo , Microcuerpos/metabolismo , Mutación , Oxidación-Reducción , Oxígeno/fisiología , Poli Adenosina Difosfato Ribosa/metabolismo , Oxígeno Singlete , Compuestos de Sulfhidrilo/fisiología , Superóxidos/metabolismo , Rayos UltravioletaRESUMEN
Fifty-eight percent of hepatocellular carcinomas (HCCs) from Qidong, China, contain an AGG to AGT mutation at codon 249 of the p53 tumor suppressor gene, a mutation that is rarely seen in HCCs from Western countries. The population of Qidong is exposed to high levels of aflatoxin B1 (AFB1), a fungal toxin that has been shown to induce the same mutation in cultured human HCC cells. To investigate the role of AFB1 and of these p53 mutations in hepatocarcinogenesis, normal liver samples from the United States, Thailand, and Qidong (where AFB1 exposures are negligible, low and high, respectively) were examined for p53 mutations. The frequency of the AGG to AGT mutation at codon 249 paralleled the level of AFB1 exposure, which supports the hypothesis that this toxin has a causative--and probably early--role in hepatocarcinogenesis.
Asunto(s)
Aflatoxina B1/efectos adversos , Carcinoma Hepatocelular/genética , Genes p53 , Neoplasias Hepáticas/genética , Hígado/química , Mutación Puntual , Carcinoma Hepatocelular/inducido químicamente , China , Codón/genética , Humanos , Neoplasias Hepáticas/inducido químicamente , Tailandia , Estados UnidosRESUMEN
Oxidative stress may play an important role in the carcinogenic action of UVB light (290 to 320 nm). UVB light induces the growth-related immediate-early gene c-fos in JB6 mouse epidermal cells, but at the same time it causes structural damage to DNA, in particular DNA strand breakage. We have studied the effect of the modulation of the frequencies of DNA breaks on the transcriptional induction of c-fos by changing the cellular antioxidant defense or by inhibiting break repair. Reduction of UVB-induced DNA breakage in a stable transfectant with an increased complement of glutathione peroxidase enhanced the induction of c-fos. In contrast, c-fos induction was diminished in stable transfectants with Cu,Zn-superoxide dismutase. Increasing the stationary concentration of UVB-induced DNA breaks by inhibition of repair in the presence of the adenosine diphosphoribose (ADPR)-transferase inhibitor 3-amino-benzamide suppressed the induction of c-fos. We conclude that DNA breaks which are induced by UVB via oxidative processes interfere with the transcriptional induction of c-fos. DNA breaks appear to exert a long-range effect on chromatin conformation which is incompatible with efficient transcription. This notion is supported by the observation that inhibition of break rejoining by 3-amino-benzamide suppressed the UVB induction of the endogenous c-fos gene and of a stably integrated construct containing the c-fos regulatory sequences linked to a reporter gene. In contrast, the induction of the same construct was not inhibited when it remained extrachromosomal in transient transfection experiments.
Asunto(s)
Daño del ADN , ADN/efectos de la radiación , Genes fos/efectos de la radiación , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Rayos Ultravioleta , Animales , Catalasa/metabolismo , Células Clonales , Epidermis , Expresión Génica/efectos de la radiación , Glutatión Peroxidasa/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-fos/efectos de la radiación , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de la radiación , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa/metabolismo , Transcripción Genética/efectos de la radiación , TransfecciónRESUMEN
BACKGROUND: The abundance and the conservation of the repeated element (rep) genes in Ichnoviruses genomes suggest that this gene family plays an important role in viral cycles. In the Ichnovirus associated with the wasp Hyposoter didymator, named HdIV, 10 rep genes were identified to date. In this work, we report a relative quantitative transcription study of these HdIV rep genes in several tissues of the lepidopteran host Spodoptera frugiperda as well as in the H. didymator wasps. RESULTS: The data obtained in this work indicate that, in the early phases of infection (24 hours), HdIV rep genes each display different levels of transcripts in parasitized 2nd instar or HdIV-injected last instar S. frugiperda larvae. Only one, rep1, is significantly transcribed in female wasps. Transcript levels of the HdIV rep genes were found as not correlated to their copy number in HdIV genome. Our results also show that HdIV rep genes display different tissue specificity, and that they are primarily transcribed in S. frugiperda fat body and cuticular epithelium. CONCLUSION: This work is the first quantitative analysis of transcription of the ichnovirus rep gene family, and the first investigation on a correlation between transcript levels and gene copy numbers in Ichnoviruses. Our data indicate that, despite similar gene copy numbers, not all the members of this gene family are significantly transcribed 24 hours after infection in lepidopteran larvae. Additionally, our data show that, as opposed to other described HdIV genes, rep genes are little transcribed in hemocytes, thus suggesting that they are not directly associated with the disruption of the immune response but rather involved in other physiological alterations of the infected lepidopteran larva.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de Insectos/metabolismo , Polydnaviridae/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Spodoptera/virología , Proteínas Virales/genética , Avispas/virología , Secuencia de Aminoácidos , Animales , Dosificación de Gen , Genes Virales , Virus de Insectos/genética , Virus de Insectos/fisiología , Larva/virología , Datos de Secuencia Molecular , Familia de Multigenes , Polydnaviridae/metabolismo , Polydnaviridae/fisiología , Transcripción Genética , Proteínas Virales/metabolismoRESUMEN
The mechanism of in vitro transformation of the mouse embryo fibroblast C3H/10T 1/2 clone 8 by aflatoxin B1 (AFB1) was studied in confluent holding (CH) experiments. Confluent cultures of C3H/10T 1/2 cells were treated with AFB1 for 16 hours, and the DNA adduct composition and concentration were determined by chromatographic procedures after 0, 8, 16, and 40 hours of CH when the cells were replated at low density for the expression of their colony-forming ability and the formation of transformed foci. Total adduct concentration and the concentration of the major primary adduct 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua) decreased continuously during CH due to spontaneous decomposition and probably also due to enzymatic repair processes. In contrast, the more chemically stable secondary product 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-triamino-Py) accumulated in the DNA and reached its maximum concentration after 16 hours of CH. While the loss of total AFB1-DNA adducts during CH was reflected in recovery of viability, the potential to form transformed foci reached a maximum after 16 hours of CH and then decreased with continued CH below the initial value. Therefore, no simple relationship exists between the concentration of the total adducts AFB1-N7-Gua and AFB1-triamino-Py at the time of release from CH and the potential to form transformed foci. However, DNA lesions or abnormal DNA configurations formed during CH as a consequence of the cellular processing of AFB1-DNA adducts may play a role in the transformation process.
Asunto(s)
Aflatoxinas/metabolismo , Carcinógenos/metabolismo , Transformación Celular Neoplásica , ADN/metabolismo , Aflatoxina B1 , Animales , Radioisótopos de Carbono , Células Cultivadas , Cromatografía Líquida de Alta Presión , ADN/aislamiento & purificación , Replicación del ADN , Embrión de Mamíferos , Cinética , Ratones , Ratones Endogámicos C3H , TritioRESUMEN
We have studied changes in intracellular localization and phosphorylating activity of protein kinase C (PKC) in mouse epidermal JB6 cells treated with oxidants. Exposure to hydrogen peroxide, reagent grade or generated enzymatically by glucose/glucose oxidase, at concentrations known to result in elevated intracellular free Ca2+ resulted in an increase in binding of [3H]phorbol dibutyrate to intact cells. Ca2+ chelation, either intracellularly by quin 2 or extracellularly by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, abolished the increase in radioligand binding. In contrast to H2O2, superoxide generated extracellularly by xanthine/xanthine oxidase or intracellularly by menadione was inactive. Scatchard plot analysis revealed that the enhancement in binding resulted from both increased receptor affinity and increased maximal binding capacity. Treatment of cells with superoxide, generated extracellularly by xanthine/xanthine oxidase or intracellularly by menadione, diminished the [3H]phorbol dibutyrate-binding capacity of the cytosol fractions prepared at low Ca2+ concentration. This decrease was not accompanied by a compensatory increase in the binding to membrane components. In contrast to superoxide, reagent H2O2, H2O2 produced by glucose/glucose oxidase, and the Ca2+ ionophore A23187 had no significant effect on the [3H]phorbol dibutyrate-binding capacities of either cellular fraction. Exposure of cells to low concentrations of extra- or intracellular superoxide resulted in an increase in the Ca2+- and phospholipid-dependent phosphorylating activity of cytosolic extracts towards adenosine diphosphoribose transferase which has been reported to be a specific substrate for PKC. The increase in phosphorylation could be diminished by the extracellular addition of copper-zinc-containing superoxide dismutase but not catalase suggesting that superoxide rather than H2O2 represents the active oxygen species in this reaction. The observation that reagent H2O2 or glucose/glucose oxidase failed to increase the phosphorylating activity of cytosolic preparations supports this conclusion. Treatment of cells or cytosolic extracts with the sulfhydryl reagent diamide stimulated the Ca2+/phospholipid-dependent phosphorylating activity toward adenosine diphosphoribose transferase. In a reconstituted system containing purified PKC, diamide induced a 25-30% increase in phospholipid-dependent phosphorylation of H1 whereas no change in activity was observed with the reducing agent dithiothreitol. It is concluded that H2O2 but not superoxide induces an increase in the phorbol ester binding, presumably to PKC, of intact JB6 cells. On the other hand
Asunto(s)
Oxidación-Reducción , Proteína Quinasa C/metabolismo , Animales , Calcimicina/farmacología , Calcio/metabolismo , Compartimento Celular/efectos de los fármacos , Línea Celular , Diamida/farmacología , Activación Enzimática/efectos de los fármacos , Glucosa Oxidasa/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Fosforilación , Xantina Oxidasa/metabolismoRESUMEN
The excision and persistence of covalent DNA adducts formed by N-acetoxy-2-acetylaminofluorene (AAAF) were studied in human skin fibroblasts. The changes in adduct concentration as a function of posttreatment incubation were measured in purified nucleosomal core DNA and in total nuclear DNA, and from these data the changes in adduct concentration in nucleosomal linker DNA were calculated. Immediately after N-acetoxy-2-acetylaminofluorene treatment which introduced 20 to 38 mumol adducts per mol DNA-P, the adduct concentration was 4 to 5 times higher in linker DNA than in core DNA. Adduct removal was rapid during the first 8 hr of incubation and occurred 3.5 to 4 times more efficiently from linker DNA than from core DNA. After 24 hr incubation, adduct removal continued only at a very low rate, leaving a substantial fraction of adducts unexcised. This fraction of persistent adducts had a value of 0.5 and was independent of the initial adduct concentration in the range of 12 to 115 mumol adducts per mol DNA-P. Approximately 65% of the persisting adducts were located in nucleosomal cores. The initial differences in adduct concentration between linker DNA and core DNA diminished during posttreatment incubation. This was entirely due to preferential early adduct excision from linker DNA. No evidence was obtained for repair-induced long-range nucleosomal movement in normal fibroblasts or constitutive movement in the absence of excision repair in xeroderma pigmentosum fibroblasts. Nucleosomal movement would tend to diminish the concentration differences between linker DNA and core DNA.
Asunto(s)
2-Acetilaminofluoreno , Acetoxiacetilaminofluoreno , Cromatina/metabolismo , Reparación del ADN , ADN/metabolismo , 2-Acetilaminofluoreno/análogos & derivados , Células Cultivadas , Fibroblastos , Humanos , Nucleosomas/metabolismo , Xerodermia Pigmentosa/metabolismoRESUMEN
Point mutations in ras protooncogenes and in the p53 tumor suppressor gene are common in many forms of human cancer. The identification of carcinogens which are responsible for their induction in humans is of great interest because it may suggest measures for disease prevention. Furthermore, the load of somatic mutations in cancer-related genes in premalignant tissues may become a useful parameter for risk assessment. For the measurement of such mutations, highly sensitive genotypic mutation systems are required which avoid the selection and clonal expansion of cells on the basis of a mutated phenotype. We have developed the restriction fragment length polymorphism/polymerase chain reaction method for genotypic mutation analysis and applied it to the study of the mutability of hot-spot codons in c-H-ras1 and p53 genes with human carcinogens. In particular, we studied the mutability of codons 247-250 of p53 with the mycotoxin aflatoxin B1 (AFB1) in human hepatocytes. AFB1 preferentially induced the transversion of guanosine to thymidine in the third position of codon 249, generating the same mutation which is found in a large fraction of hepatocellular carcinomas from regions of the world with AFB1-contaminated food. Our results are in support of AFB1 as an etiological factor for hepatocellular carcinoma in AFB1-contaminated areas. In an ongoing study we are comparing the load of mutations in hot-spot codon 12 of c-H-ras1 in urinary bladder carcinoma and in normal tissue, by restriction fragment length polymorphism/polymerase chain reaction. We observed moderately elevated abundances of guanosine to thymidine transversions in the middle position of codon 12 in tumor DNA. These results may reflect a mutator phenotype of the tumor tissue or they could be the consequence of the heterogeneity of the biopsies which were analyzed.
Asunto(s)
Aflatoxina B1/toxicidad , Genes p53 , Genes ras , Mutagénesis , Neoplasias/genética , Polimorfismo de Longitud del Fragmento de Restricción , Proto-Oncogenes , Secuencia de Bases , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica , Codón , Cartilla de ADN , Etilnitrosourea/toxicidad , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
In light of recent studies implicating low catalase activities in the pathogenesis of the cancer-prone disease xeroderma pigmentosum (XP) we have measured catalase activity, protein levels, and mRNA concentrations in six XP fibroblast strains and three normal controls. Only one XP strain of complementation group A (XP1223) possessed significantly lower catalase by all three criteria. The other five XP strains (two XP variants, two strains of complementation group D, and one strain of complementation group C) possessed catalase levels which fell into the range of the interindividual variations of normal controls. We further assessed the total enzymatic antioxidant defense status by measuring the levels of copper, zinc, and manganese superoxide dismutase and glutathione peroxidase. None of these enzymes showed significant deviations from controls in XP cells. Our results do not support the notion that a deficient enzymatic antioxidant defense facilitates the establishment of a prooxidant state in XP upon exposure to near-UV. However, they do not argue against the participation of active oxygen in near-UV-induced carcinogenesis in XP.
Asunto(s)
Antioxidantes/análisis , Catalasa/análisis , Xerodermia Pigmentosa/enzimología , Fibroblastos/enzimología , Glutatión Peroxidasa/análisis , Humanos , ARN Mensajero/análisis , Superóxido Dismutasa/análisisRESUMEN
We have compared the mechanisms of the transcriptional induction of c-fos in mouse epidermal cells JB6 (clone 30) by an extracellular burst of active oxygen of the type produced by inflammatory phagocytes to induction by serum and phorbol ester. All three inducers elicit a characteristic immediate early response of c-fos which is inhibited by the protein kinase inhibitor H7 but enhanced by the protein synthesis inhibitor cycloheximide. Experiments with stable transfectants containing fos 5' upstream regulatory sequences linked to an HSV-tk-chloram-phenicol-acetyl-transferase reporter construct indicate that the joint dyad symmetry element-AP-1 motifs exert the most potent enhancer effect in response to active oxygen as well as serum. It is concluded that the different signal transduction pathways used by these inducers converge to the same 5' regulatory sequences of c-fos. In contrast to these common features only active oxygen induction of c-fos required the poly-ADP-ribosylation of chromosomal proteins. The inhibitors of ADP-ribose transferase benzamide and 3-amino-benzamide suppressed the elongation of the c-fos message and the de novo synthesis of nuclear factors, among them c-Fos and c-Jun, which bind to the fos-AP-1 motif in vitro only following stimulation with active oxygen. No active oxygen-induced change was observed in the protein complex which binds to an oligonucleotide containing the SIF and dyad symmetry element motifs in vitro. The presence of Fos and Jun proteins was detected in this complex. Only active oxygen, but not serum or phorbol ester, induces DNA breakage. We propose that poly-ADP-ribosylation is required because it participates in the repair of DNA breaks which interfere with transcription. We observed that Fos protein is weakly poly-ADP-ribosylated in response to active oxygen, but the functional role of this modification remains unclear.
Asunto(s)
Genes fos/efectos de los fármacos , Proteínas Oncogénicas v-fos/metabolismo , Oxígeno/farmacología , Transcripción Genética/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Benzamidas/farmacología , Línea Celular , Cicloheximida/farmacología , Reparación del ADN/efectos de los fármacos , Isoquinolinas/farmacología , Datos de Secuencia Molecular , Proteína Oncogénica p65(gag-jun)/metabolismo , Proteínas Oncogénicas v-fos/genética , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas , Transducción de Señal/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
The processing of covalent aflatoxin B1 (AFB1)-DNA adducts was investigated in confluent normal fibroblasts (NF) and xeroderma pigmentosum skin fibroblasts of Complementation Group A (XPA) following treatment with rat liver microsome-activated AFB1 for 30 min. Following rapid DNA isolation at slightly acidic pH by a new filter technique, more than 90% of the adducts corresponded to 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-AFB1 (AFB1-N7-Gua) according to the analysis of acid hydrolysates by high-pressure liquid chromatography. The changes in adduct concentration and composition were compared between DNA isolated immediately following AFB1 treatment and incubated at neutrality in vitro and DNA in situ in the cell isolated after different lengths of incubation. The following conclusions were reached from the observed differences in the kinetics of adduct removal from free DNA and DNA in situ in NF and XPA: (a) AFB1-N7-Gua is removed spontaneously and enzymatically in NF but probably only spontaneously in XPA. This result suggests that these lesions are removed via nucleotide excision repair in NF; (b) the putative 2,3-dihydro-2(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 is formed in a secondary reaction from AFB1-N7-Gua in vitro and in situ in the cell. It accumulates more rapidly and to a greater extent in XPA than in NF and persists in both cells types over prolonged periods. The reaction of AFB1-N7-Gua to 2,3-dihydro-2-(N5-formyl-2',5'6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatox in B1 represents the transformation of a repairable lesion to a nonrepairable, persistent lesion.
Asunto(s)
Aflatoxinas/metabolismo , Reparación del ADN , ADN/metabolismo , Aflatoxina B1 , Biotransformación , Células Cultivadas , Humanos , Cinética , Microsomas Hepáticos/metabolismoRESUMEN
Blood monocytes from 45 selected patients with lung cancer and 30 healthy controls were incubated with [G-3H]-benzo(a)pyrene for 30 h, and the formation of covalently bound DNA adducts was determined. The lung cancer patients were either relatively young (below 46 yr), nonsmokers, or had at least one first degree relative with lung cancer. Therefore, they might be considered cancer prone. The DNA adducts were significantly elevated in 22 patients with early age cancer (4.34 fmol/micrograms of DNA; P less than 0.04). In 12 familial cases, the slight elevation (2.77 fmol/micrograms of DNA) was not statistically significant in comparison to healthy controls. Benzo(a)pyrene:DNA adduct levels did not differ significantly between smokers and nonsmokers. Eight of 9 lung cancer patients with DNA adducts equal or above 4.5 fmol/micrograms of DNA but only 16 of 36 with adducts below this value had either oat cell or squamous cell cancer (P less than 0.05). The observed enhanced formation of covalent DNA adducts in blood monocytes exposed to a carcinogenic polycyclic hydrocarbon may be genetically determined and could play a role in the development of lung cancer at an early age.
Asunto(s)
Benzo(a)pireno/metabolismo , ADN/metabolismo , Neoplasias Pulmonares/metabolismo , Monocitos/metabolismo , Adulto , Femenino , Humanos , Neoplasias Pulmonares/etiología , Masculino , Persona de Mediana Edad , FumarRESUMEN
The UVB (290-320 nm) portion of the solar spectrum possesses the highest activity for the induction of skin cancer and has the capacity to stimulate epidermal proliferation. We report that UVB is a transcriptional inducer of the c-fos protooncogene in mouse JB6 epidermal cells. Induction is biphasic with an immediate early peak at 30-60 min and a second broader peak 8 h following irradiation. The immediate early phase is suppressed by inhibitors of nuclear adenosine diphosphoribose transferase. For UVB induction, the formation of full-length messages is less efficient than of early, short messages, while both types of messages are produced at similar rates following serum stimulation. Experiments with stable transfectants with reporter constructs linked to 5'-upstream sequences of c-fos indicate that UVB and serum stimulation both require the sequences from -345 to -285 which contain the joint DSE-AP-1 enhancer motifs for efficient induction. Mobility shift data reveal that the complement of c-Fos and c-Jun proteins which bind to the fos-AP-1 octanucleotide decrease immediately following irradiation. Increased binding of Fos and Jun is observed 8-24 h later. UVB did not cause an observable change in the nuclear proteins which bind to the dyad symmetry element oligonucleotide in vitro. Fos protein was detected among the binding proteins. We propose that the two phases of UVB-induced c-fos expression occur by quite different mechanisms. The immediate early phase is inhibited by adenosine diphosphoribose transferase inhibitors because poly-ADP ribosylation of chromosomal proteins is required for the resealing of UVB-induced DNA strand breaks which otherwise retard message elongation. The production of an autocrine factor may be responsible for the late phase of c-fos induction.
Asunto(s)
Epidermis/fisiología , Epidermis/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes fos/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Secuencia de Bases , Benzamidas/farmacología , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Daño del ADN , Regulación hacia Abajo/efectos de la radiación , Células Epidérmicas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes fos/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Proteína Oncogénica p65(gag-jun)/genética , Proteína Oncogénica p65(gag-jun)/metabolismo , Proteínas Oncogénicas v-fos/genética , Proteínas Oncogénicas v-fos/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Transcripción Genética/efectos de la radiaciónRESUMEN
The formation of single-strand breaks by near-ultraviolet light at 313 nm and by aerobic gamma-rays was compared for skin fibroblast monolayer cultures from 4 normal donors (NF) and 8 patients with Bloom's syndrome (BS) by the alkaline elution method. In 6 of 8 BS strains, the number of breaks induced by near-ultraviolet light, 2.25 kJ/sq m, at 0 degrees was comparable to NF, while elevated breakage was observed in BS strains HG 369 and HG 916. Breakage frequencies were increased substantially in 6 of 8 BS strains relative to NF when the near-ultraviolet light exposure was at 37 degrees. BS strain GM 2520 represents an exception since normal breakage frequencies were induced both at 0 degrees and 37 degrees. Aerobic gamma-rays (75 R) induced comparable numbers of single-strand breaks in BS and NF strains at 0 degrees. The breakage frequencies were reduced an average of 17% in NF when the same dose was given at 30 degrees followed by 6 min incubation. Under the same conditions, the breakage frequencies were on the average reduced by 42% relative to 0 degrees in the BS strains, indicating that they possess normal or possibly slightly increased capacities for the rejoining of gamma-ray-induced breaks.
Asunto(s)
ADN/efectos de la radiación , Telangiectasia/genética , Rayos Ultravioleta , Aerobiosis , Células Cultivadas , Reparación del ADN , Enanismo , Rayos gamma , HumanosRESUMEN
Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.
Asunto(s)
Transformación Celular Neoplásica , Genes ras , Queratinocitos/patología , Northern Blotting , División Celular , Línea Celular , Humanos , Trasplante de Neoplasias , Fenotipo , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas p21(ras) , TransfecciónRESUMEN
Human papillomaviruses (HPV) are associated with papillomatosis of the larynx, trachea, and bronchi in decreasing order of frequency, and these papillomatosis lesions may become malignant. When the patients are not selected for a history of papillomatosis, the frequency of HPV in bronchogenic carcinoma tissue is 1-5%. In order to develop a model for investigating the role of HPV in human bronchogenic carcinogenesis, normal human bronchial epithelial cells were transfected with cloned full-length HPV16 or HPV18. Two HPV18-transformed cell lines (BEP1 and BEP2) and one HPV16-transformed cell line (BEP3) were established. These nontumorigenic epithelial cell lines have: (a) attained over 100 population doublings in vitro; (b) mutually exclusive human marker chromosomes; (c) HPV DNA in forms that are consistent with chromosomal integration by Southern analysis; (d) HPV E6, E7, and E6* mRNA transcripts by Northern and reverse transcriptase-polymerase chain reaction analysis; and (e) diminished confluence-induced squamous differentiation. These cell lines should be useful for studying mechanisms involved in proliferation, differentiation, and neoplastic transformation of human bronchial epithelial cells.
Asunto(s)
Bronquios/microbiología , Línea Celular , Transformación Celular Viral/genética , Papillomaviridae , Secuencia de Bases , Northern Blotting , Southern Blotting , Mapeo Cromosómico , Dermatoglifia del ADN , Epitelio , Humanos , Queratinas/biosíntesis , Datos de Secuencia Molecular , Papillomaviridae/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Transcripción Genética , TransfecciónRESUMEN
Base pair mutations in the p53 tumor suppressor gene are among the most frequently observed genetic changes in human malignancies. Several mutational hotspots have been identified and tumor- and tissue-specific differences have been observed. We studied the mutability of hotspot codon 248, CGG, in human fibroblasts in response to the alkylating agent N-ethyl-N-nitrosourea (ENU) by MspI RFLP/PCR analysis. Alkylating agents are implicated as etiological agents in carcinogenesis in the gut and other tissues in the human. ENU induced preferentially G to A transitions in the non-transcribed strand. The predominant mutation involving the G-residue of the CpG dinucleotide was observed with an absolute frequency of 4 x 10(-7). The corresponding C to T transition in the first position of codon 248 was observed at several fold lower frequency suggesting more efficient excision repair of the transcribed strand. The G to A transition in the third position of codon 248 occurred at low frequency. Our results are compatible with a role for aliphatic alkylating agents in human tumors with codon 248 mutations since almost all mutations reported in this codon are transitions in the CpG-dinucleotide.
Asunto(s)
Codón , Etilnitrosourea/toxicidad , Genes p53/efectos de los fármacos , Mutagénesis , Secuencia de Bases , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Oxidants are suspected to represent important human carcinogens. They are mutagenic and may participate in the activation of proto-oncogenes and the inactivation of tumor suppressor genes. We have studied the capacity of hydrogen peroxide plus ferric chloride (FeCl3) to induce base pair changes in the hotspot codons 248 and 249 of the p53 tumor suppressor gene in human fibroblasts. In codon 248 (CGG) H2O2/FeCl3 only induced the transversion of G to C in the second position and the transition of G to A in the third position. No evidence was obtained for spontaneous or oxidant-induced deamination of 5-methylcytosine in the CpG dinucleotide of codon 248 since neither C to T transitions in the first position nor G to A transitions in the middle position were observed. H2O2/FeCl3 efficiently induced G to T transversions at both G-residues of codon 249 (AGG) and C to A transversions at the first position of codon 250 (CCC). It is evident that H2O2/FeCl3 possesses essentially the same mutagenic specificity for codons 249 and 250 of p53 as bulky carcinogens such as aflatoxin B1, benzo(a)pyrene or heterocyclic amines. In particular, it is not possible to eliminate oxidants from the list of candidate carcinogens which may be responsible for the high incidence of p53 codon 249 AGT mutations in hepatocellular carcinoma from certain areas of the world.
Asunto(s)
Codón , Genes p53 , Peróxido de Hidrógeno/toxicidad , Mutagénesis , Células Cultivadas , Cloruros , Citosina/metabolismo , Compuestos Férricos/farmacología , Radicales Libres , Humanos , MetilaciónRESUMEN
Monomeric products of the 5,6-dihydroxy-dihydrothymine type are produced in the DNA by both ultraviolet and ionizing radiations. The capacity of nuclear preparations from normal and Xeroderma pigmentosum (XP) cells (Complementation groups A, B, C and D) to excise such products from ultraviolet or gamma-irradiated T7DNA was comparable and was independent of radiation induced strand breaks.