RESUMEN

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

Asunto(s)

ADN/química , Marcación de Gen/métodos , Genes jun , Conformación de Ácido Nucleico , Nucleótidos de Purina/química , Cationes Bivalentes/química , Cationes Bivalentes/efectos de la radiación , ADN/efectos de la radiación , Genes jun/efectos de la radiación , Calor , Humanos , Magnesio/química , Magnesio/efectos de la radiación , Conformación de Ácido Nucleico/efectos de la radiación , Desnaturalización de Ácido Nucleico/efectos de la radiación , Nucleótidos de Purina/efectos de la radiación , Nucleótidos de Pirimidina/química , Nucleótidos de Pirimidina/efectos de la radiación , Sodio/química , Sodio/efectos de la radiación , Rayos Ultravioleta

RESUMEN

PURPOSE: A class of naturally occurring isoforms of tocopherol (tocols) was shown to have varying degrees of protection when administered before radiation exposure. We recently demonstrated that α-tocopherol succinate (TS) is a potential radiation prophylactic agent. Our objective in this study was to further investigate the mechanism of action of TS in mice exposed to (60)Co γ-radiation. METHODS AND MATERIALS: We evaluated the effects of TS on expression of antioxidant enzymes and oncogenes by quantitative RT-PCR in bone marrow cells of (60)Co γ-irradiated mice. Further, we tested the ability of TS to rescue and repopulate hematopoietic stem cells by analyzing bone marrow cellularity and spleen colony forming unit in spleen of TS-injected and irradiated mice. RESULTS: Our results demonstrate that TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice at different time points. TS also increased colony forming unit-spleen numbers and bone marrow cellularity in irradiated mice. CONCLUSIONS: Results provide additional support for the observed radioprotective efficacy of TS and insight into mechanisms.

Asunto(s)

Antioxidantes/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Protectores contra Radiación/farmacología , alfa-Tocoferol/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de la radiación , Radioisótopos de Cobalto/farmacología , Ensayo de Unidades Formadoras de Colonias/métodos , Cartilla de ADN/genética , Genes jun/efectos de los fármacos , Genes jun/efectos de la radiación , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/efectos de los fármacos , Glutatión Reductasa/metabolismo , Glutatión Transferasa/efectos de los fármacos , Glutatión Transferasa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , Células Madre Hematopoyéticas/efectos de la radiación , Masculino , Ratones , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/efectos de la radiación , Esternón/citología , Esternón/efectos de los fármacos , Esternón/efectos de la radiación , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/metabolismo

RESUMEN

OBJECTIVE: To analyze the upstream region of radiation-induced junB gene. METHODS: Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization. RESULTS: CAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation. CONCLUSIONS: 590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.

Asunto(s)

Cloranfenicol O-Acetiltransferasa/genética , Regulación de la Expresión Génica/efectos de la radiación , Genes jun/efectos de la radiación , Rayos X , Actinas/análisis , Actinas/metabolismo , Animales , Northern Blotting , Células Cultivadas , Genes Reporteros , Genes jun/genética , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Plásmidos/análisis , Plásmidos/genética , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Factores de Tiempo

RESUMEN

Potential targets for chemoprevention of nonmelanoma skin cancer include UV-induced nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activation in keratinocytes. Inhibition of both these ultraviolet light B (UVB)-induced transcription factors has been shown with the dominant-negative c-jun mutant, TAM67; however, its mechanism of action has not yet been determined. Here we demonstrated that transient transfection of a mouse keratinocyte cell line (308) with a dominant-negative phosphorylation mutant of c-Jun before exposure to 250 J/m(2) UVB inhibits transactivation mediated by both AP-1 and NF-kappaB transcription factors to levels below those of UVB exposed controls. Through the utilization of immunoprecipitation techniques, protein-protein interactions between NF-kappaB family members IkappaBalpha, IkappaBbeta, p50, and p65 (Rel-A) were identified with an Xpress tagged dominant-negative c-Jun (TAM67) protein. Expression of the leucine zipper domain of the TAM67 protein inhibited UVB-induced NF-kappaB transactivation but not AP-1 transactivation. Expression of the bZIP domain of the TAM67 protein was able to inhibit transactivation mediated by both transcription factors. These data demonstrate that TAM67 is able to inhibit two significant UVB-induced molecular targets AP-1 and NF-kappaB, and that the inhibition of these two transcription factor families is potentially due to protein-protein interactions between different regions of the dominant-negative c-Jun protein.

Asunto(s)

Genes jun/efectos de la radiación , FN-kappa B/genética , Proteínas Nucleares/efectos de la radiación , Proteínas de Unión al ARN/efectos de la radiación , Factor de Transcripción AP-1/genética , Factores de Transcripción/efectos de la radiación , Activación Transcripcional/efectos de la radiación , Rayos Ultravioleta , Animales , Línea Celular , Núcleo Celular/fisiología , Núcleo Celular/efectos de la radiación , Queratinocitos , Ratones , FN-kappa B/efectos de la radiación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/efectos de la radiación , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/efectos de la radiación , Factor de Transcripción AP-1/efectos de la radiación

RESUMEN

Expression of immediate early response genes such as c-fos, c-jun, and c-myc in response to 1-500 microT resultant (r) 60 Hz elliptically polarized (EP) magnetic fields (MFs), typical of environmental MFs polarization under overhead power lines, was analyzed in both at transcriptional and translational levels using human glioblastoma (T98G) cells. Pseudo synchronized T98G cells at G1 phase were exposed to EP-MFs (1, 20, 100, and 500 microTr) for up to 3 h, but produced no statistical difference (P>0.05) in the levels of expression ratio at both the transcriptional and translational levels at 30 min for c-fos and c-jun and at 180 min for c-myc after serum stimulation. In addition, exposure of T98G cells to linearly (vertical and horizontal) and/or circularly polarized MFs (500 microTr) produced no significant change (P>0.05) in the expression ratio at both transcriptional and post-transcriptional levels. Thus, there was no evidence that linearly or rotating polarized MFs enhanced early response gene expression in these studies. These results suggest that environmental MFs at 1-500 microT flux density are unlikely to induce carcinogenesis through a mechanism involving altered expression of the immediate early response genes.

Asunto(s)

Neoplasias Encefálicas/genética , Campos Electromagnéticos , Regulación de la Expresión Génica/efectos de la radiación , Glioblastoma/genética , ARN Ribosómico/metabolismo , Transcripción Genética/efectos de la radiación , Northern Blotting , Genes fos/genética , Genes fos/efectos de la radiación , Genes jun/genética , Genes jun/efectos de la radiación , Genes myc/genética , Genes myc/efectos de la radiación , Humanos , Procesamiento Postranscripcional del ARN/efectos de la radiación , ARN Ribosómico/genética , Células Tumorales Cultivadas

RESUMEN

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.

Asunto(s)

Dermatitis Fototóxica/prevención & control , Piel/efectos de los fármacos , Piel/efectos de la radiación , Té/química , Administración Oral , Administración Tópica , Adulto , Animales , Línea Celular , Receptores ErbB/metabolismo , Receptores ErbB/efectos de la radiación , Femenino , Genes fos/efectos de los fármacos , Genes fos/efectos de la radiación , Genes jun/efectos de los fármacos , Genes jun/efectos de la radiación , Genes p53/efectos de los fármacos , Genes p53/efectos de la radiación , Humanos , Ratones , Ratones Pelados , Piel/lesiones , Rayos Ultravioleta/efectos adversos

RESUMEN

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Asunto(s)

Proto-Oncogenes/efectos de la radiación , Ondas de Radio/efectos adversos , Teléfono , Factores de Transcripción/metabolismo , Animales , Ciclo Celular , Línea Celular , Medios de Cultivo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Expresión Génica/efectos de la radiación , Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Genes myc/efectos de la radiación , Ratones , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/etiología , Estrés Fisiológico/genética , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-2

RESUMEN

The known nucleotide excision repair (NER) defects of xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells can be exploited to analyze mechanisms of repair of UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide (nt.) resolution. The two gene products of the CS complementation groups (CSA and CSB) have been implicated in the preferential repair of the transcribed strand of human genes. We had previously described very efficient repair of CPDs at sequences near the transcription initiation site of the human JUN gene in normal fibroblasts. Here, we have analyzed repair in a CSA fibroblast strain. CSA cells exhibited rapid repair near the transcription initiation site (positions -45 to +15) but were deficient in repair of sequences on the transcribed strand beginning around nt. +20. There was also no strand-selective repair of sequences further downstream of the start site (+260 to +450). The results suggest that the transcription-repair coupling factor (TRCF) CSA is required for efficient repair only during the elongation stages of RNA polymerase II transcription. We also discuss possible mechanisms of differential repair observed near the transcription initiation site in XP and CS cells and conclude that these in vivo repair data support some recent models obtained from nucleotide excision repair experiments in vitro.

Asunto(s)

Reparación del ADN/fisiología , Extensión de la Cadena Peptídica de Translación/genética , Proteínas/fisiología , ARN Polimerasa II/genética , Transcripción Genética/fisiología , Línea Celular , Síndrome de Cockayne/genética , Codón Iniciador , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Enzimas Reparadoras del ADN , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Genes jun/efectos de la radiación , Humanos , Factores de Transcripción , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Xerodermia Pigmentosa/genética

RESUMEN

Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of p53 mRNA and protein. However, the p53 expressed by the wild-type L1210 cells was found to be a mutant form of p53 (relative to normal mouse fibroblast p53 sequence) having a point mutation in the DNA binding domain of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT cells had previously been shown to lack the expression of p53 but to respond to cycloheximide (CHX) treatment by superinduction of p53 mRNA. The mRNA for p53 induced by CHX had the same sequence as the p53 from normal mouse fibroblasts. Although the Y8 cells had no constitutive levels of p53 mRNA or protein, the Y8 cells expressed constitutive levels of WAF1 mRNA and protein. Gadd45 mRNA was also present in the Y8 cells. Subjecting the WT or Y8 cells to ionizing radiation did not result in a G0/G1 cell cycle block; the cells blocked in G2/M. The Y8 cells were much more sensitive to the irradiation treatment than the WT cells, resulting in marked increases in apoptosis in the Y8 cells. Although radiation treatment induced p53 mRNA, but no p53 protein, in the Y8 cells, WAF1 mRNA was induced in the Y8 cells. These data indicate that there are p53-independent pathway(s) that may still involve WAF1 and Gadd45 with respect to cell cycle control and apoptosis.

Asunto(s)

Leucemia L1210/genética , Leucemia L1210/radioterapia , Mutación , Animales , Desoxiadenosinas/farmacología , Resistencia a Antineoplásicos , Genes jun/efectos de la radiación , Genes myc/efectos de la radiación , Leucemia L1210/tratamiento farmacológico , Ratones , FN-kappa B/metabolismo , FN-kappa B/efectos de la radiación , Radiación Ionizante , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.

Asunto(s)

Carcinoma de Células Escamosas/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Proto-Oncogenes/genética , Neoplasias Cutáneas/genética , Rayos Ultravioleta , Animales , Carcinoma de Células Escamosas/etiología , Genes fos/genética , Genes fos/efectos de la radiación , Genes jun/genética , Genes jun/efectos de la radiación , Genes myc/genética , Genes myc/efectos de la radiación , Genes ras/genética , Genes ras/efectos de la radiación , Ratones , Ratones Pelados , Proto-Oncogenes/efectos de la radiación , Piel/efectos de la radiación , Neoplasias Cutáneas/etiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiación

RESUMEN

Rat PC12 pheochromocytoma cells have been treated with nerve growth factor and then exposed to athermal levels of a packet-modulated radiofrequency field at 836.55 MHz. This signal was produced by a prototype time-domain multiple-access (TDMA) transmitter that conforms to the North American digital cellular telephone standard. Three slot average power densities were used: 0.09, 0.9, and 9 mW/cm2. Exposures were for 20, 40, and 60 min and included an intermittent exposure regimen (20 min on/20 min off), resulting in total incubation times of 20, 60, and 100 min, respectively. Concurrent controls were sham exposed. After extracting total cellular RNA, Northern blot analysis was used to assess the expression of the immediate early genes, c-fos and c-jun, in all cell populations. No change in c-fos transcript levels were detected after 20 min exposure at each field intensity (20 min was the only time period at which c-fos message could be detected consistently). Transcript levels for c-jun were altered only after 20 min exposure to 9 mW/cm2 (average 38% decrease).

Asunto(s)

Campos Electromagnéticos , Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Factores de Crecimiento Nervioso/farmacología , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Ondas de Radio , Neoplasias de las Glándulas Suprarrenales , Animales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Células PC12 , Feocromocitoma , Ratas , Transcripción Genética/efectos de los fármacos , Transcripción Genética/efectos de la radiación

RESUMEN

The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST-P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST-P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10-25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST-P mRNA expression, with a 70% increase at 250 microM concentration. Alterations in possible trans-acting factors were examined to clarify the mechanism of repression by UV irradiation. c-Jun mRNA was induced 3.5-fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c-Fos mRNA was increased 10-fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c-Jun and c-Fos mRNA differed from that of GST-P mRNA. The level of mRNA for silencer factor-B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST-P gene did not suppress CAT activity. Although basal expression of the GST-P gene was mainly dependent on GPE I, altered expression of c-jun, c-fos and other genes coding for factors possibly trans-acting on GPE I did not appear to be responsible for the decreased GST-P mRNA levels.

Asunto(s)

Glutatión Transferasa/biosíntesis , Isoenzimas/biosíntesis , Queratinocitos/enzimología , Queratinocitos/efectos de la radiación , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Glutatión Transferasa/clasificación , Glutatión Transferasa/genética , Peróxido de Hidrógeno/farmacología , Isoenzimas/genética , Queratinocitos/metabolismo , ARN Mensajero/efectos de la radiación , Ratas , Transactivadores/metabolismo , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta

RESUMEN

The purpose of this study was to determine the role of radiation-induced expression of c-jun and c-fos in radiation-induced apoptosis of cells of the Jurkat T-cell line. Doses of 10-20 Gy caused a massive number of cells to undergo apoptosis within the first 24 h. This was accompanied by extensive increases in c-jun mRNA levels and moderate increases in c-fos levels, both occurring at the time of onset of internucleosomal DNA fragmentation. Increased c-jun and c-fos expression was maximum at 8 h after irradiation with a 10-fold increase in c-jun and a 2-fold increase in c-fos mRNA levels. In comparison, stimulation of the Jurkat cells with PMA resulted in rapid induction of c-jun and c-fos within 1 h. The late induction of c-jun and c-fos was not preceded by induction of tumor necrosis factor-alpha (TNF-alpha) or the bifunctional repair endonuclease and nuclear redox factor Ref-1; rather a slow decrease in Ref-1 mRNA levels was found over the first 24 h. Our results showed that radiation-induced c-jun and c-fos expression is a late response in Jurkat cells, and is most likely a secondary effect not necessary for radiation-induced apoptosis. Furthermore, apoptosis was induced by the RNA synthesis inhibitor actinomycin D, which does not induce c-jun or c-fos expression. This demonstrates that massive late induction of c-jun and c-fos is not a general requirement for apoptosis in Jurkat cells.

Asunto(s)

Apoptosis/efectos de la radiación , Liasas de Carbono-Oxígeno , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Apoptosis/efectos de los fármacos , Northern Blotting , Línea Celular , Supervivencia Celular , Reparación del ADN , ADN de Neoplasias/aislamiento & purificación , ADN de Neoplasias/efectos de la radiación , Dactinomicina/farmacología , Relación Dosis-Respuesta en la Radiación , Electroforesis en Gel de Agar , Rayos gamma , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Proteínas Nucleares/biosíntesis , Linfocitos T , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Asunto(s)

Ataxia Telangiectasia/genética , Expresión Génica/efectos de la radiación , Genes jun/efectos de la radiación , Transcripción Genética/efectos de la radiación , Ataxia Telangiectasia/patología , Cloranfenicol O-Acetiltransferasa/genética , Fibroblastos/efectos de la radiación , Fase G1/genética , Fase G1/efectos de la radiación , Genes jun/fisiología , Humanos , Transfección

RESUMEN

Radiotherapy administrated to patients with head and neck malignancies and prior to bone marrow transplantation often results in severe xerostomia. We evaluated the expression of early response proto-oncogenes (c-fos and jun B), tissue specific genes (proline rich protein [PRP] and kallikrein), and proteolysis linked utiquitin gene following exposure to 15 Gy irradiation alone or in combination with beta-adrenergic stimulation of the rat submandibular glands. Head and neck irradiation resulted not only in dysfunction and tissue loss of the salivary glands but also in a systemic effect expressed as profound body weight loss. Irradiation alone was found to induce expression of the jun B but not the c-fos proto-oncogenes. The combination of irradiation and beta-adrenergic stimulation by isoproterenol induced earlier expression of jun B and profound expression of the c-fos proto-oncogene in comparison to irradiation alone. In contrast, the kallikrein and ubiquitin genes were expressed constitutively and were not affected by irradiation alone or in combination with beta-adrenergic stimulation. In addition, irradiation had no effect on submandibular gland mRNA translation. We observed that the expression of the genes whose regulation is associated with DNA damage (i.e. jun B and c-fos) was enhanced by irradiation alone or in combination with isoproterenol administration. In contrast, the expression of genes associated with the routine functional integrity of the cell (i.e. kallikrein, ubiquitin, and PRP) was unaffected. These findings, in addition to delayed gland dysfunction, leads us to believe that the irradiation induced injury to the submandibular glands is to be attributed to reproductive stem cell death which may be partly obliterated in the clinical setting by better understanding.

Asunto(s)

Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Glándula Submandibular/efectos de la radiación , Agonistas Adrenérgicos beta/farmacología , Animales , Isoproterenol/farmacología , Calicreínas/metabolismo , Calicreínas/efectos de la radiación , Masculino , Péptidos/metabolismo , Péptidos/efectos de la radiación , Dominios Proteicos Ricos en Prolina , Dosis de Radiación , Ratas , Ratas Wistar , Saliva/metabolismo , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/efectos de la radiación

RESUMEN

Ultraviolet light (UV) and different DNA-damaging agents are known to induce AP-l-transcription-factor activity. Whereas UV induction appears to be triggered by events at the cell membrane, the mechanism of AP-l activation by alkylating or platinating agents is not known. We have here examined the effect of cisplatin on AP-l activity in RPMI-8322 melanoma cells. Cisplatin was found to induce binding of nuclear proteins to TRE elements from the c-jun and collagenase-gene promoters, and was also found to induce activation of a c-jun-promoter reporter construct. Compared with stimulation by UV, cisplatin stimulation of c-jun-promoter activity was found to be less sensitive to a dominant negative mutant of Raf-I protein kinase. Furthermore, whereas UV treatment resulted in strong MAP-kinase activation, cisplatin treatment resulted only in a weak and transient increase. These data suggest that the Raf-MAPK pathway is of minor importance for the induction of c-jun-promoter activity by cisplatin. Finally, we report that cisplatin induction of c-jun in RPMI-8322 cells was blocked by herbimycin A, an inhibitor of Src-family tyrosine kinases. In contrast, UV induction of c-jun was not blocked by herbimycin A. In conclusion, our data strongly suggest that UV and cisplatin induction of c-jun mRNA in RPMI-8322 melanoma cells occur by distinct mechanisms.

Asunto(s)

Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes jun/efectos de los fármacos , ARN Mensajero/biosíntesis , Rayos Ultravioleta , Benzoquinonas , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Genes jun/efectos de la radiación , Células HeLa , Humanos , Lactamas Macrocíclicas , Melanoma/genética , Melanoma/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Proteínas Proto-Oncogénicas c-raf , Quinonas/farmacología , Rifabutina/análogos & derivados , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-1/efectos de la radiación , Células Tumorales Cultivadas

RESUMEN

We describe a new form of DNA repair heterogeneity along the genome. The repair rate of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured at single nucleotide resolution along the promoter and transcribed sequences of the human JUN gene in UV-irradiated diploid fibroblasts. The promoter of this gene contains an array of sequence-specific transcription factors located between nucleotides -200 and -50 relative to the major transcription start site. These sequences are repaired slowly; at many sites >50% of the CPDs are left unrepaired after 24 h. However, repair rates are 10-fold faster near the transcription initiation site. This very fast repair is seen on both DNA strands between nucleotides -40 and +100 where at most positions >90% of the dimers are repaired within 4 h. There is a general gradient of repair efficiency of the transcribed DNA strand with faster repair within the 5'-end and diminished repair towards the 3'-end of the gene. The fast repair rates seen near the transcription initiation site may be explained by increased local concentrations of DNA repair factors that are associated with general transcription factors (e.g. TFIIH) functioning in transcription initiation. This domain-specific DNA repair may aid in maintaining transcription initiation of essential genes after DNA damage.

Asunto(s)

Reparación del ADN/genética , Genes jun , Secuencia de Bases , Células Cultivadas , ADN/química , ADN/genética , ADN/efectos de la radiación , Cartilla de ADN/genética , Genes jun/efectos de la radiación , Humanos , Cinética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/efectos de la radiación , Dímeros de Pirimidina/química , Transcripción Genética , Rayos Ultravioleta

RESUMEN

Exposure of mammalian cells to ionizing radiation results in the induction of the immediate early genes, c-jun and Egr-1, which encode transcription factors implicated in cell growth as well as the cellular response to oxidative stress. We studied the role of these immediate early genes in cell cycle kinetics and cell survival following x-irradiation of clones containing inducible dominant negatives to c-jun and Egr-1. The dominant negative constructs to c-jun (delta 9) and Egr-1 (WT/Egr) prevented x-ray induction of transcription through the AP-1 and Egr binding sites, respectively. Twenty percent of confluent, serum-deprived SQ20B human tumor cells, normal fibroblasts, and fibroblasts from patients with ataxia telangiectasia entered S phase within 5 h of irradiation. Clones containing inducible delta 9 and WT/Egr dominant negative constructs demonstrated attenuation of the percentage of cells exiting G1 phase and reduced survival following irradiation. These data indicate that the dominant negatives to the stress-inducible immediate early genes Egr-1 and c-jun prevent the onset of S phase and reduce the survival of human cells exposed to ionizing radiation.

Asunto(s)

Supervivencia Celular/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN de Neoplasias/biosíntesis , Genes Inmediatos-Precoces/efectos de la radiación , Genes jun/efectos de la radiación , Proteínas Inmediatas-Precoces , Proto-Oncogenes/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular , Clonación Molecular , ADN de Neoplasias/efectos de la radiación , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Cinética , Neoplasias Laríngeas , Sulfatos/farmacología , Factores de Tiempo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Rayos X , Compuestos de Zinc/farmacología , Sulfato de Zinc

RESUMEN

The effects of ionizing radiation on c-fos, c-jun and jun-B mRNA levels were determined in cultures of rat perinatal type 1 astrocytes and two rat brain tumour cell lines, 175A and 9L. In astrocyte cultures X-ray doses as low as 1 Gy induced the expression of c-fos and jun-B but had essentially no effect on c-jun. The maximum increase in expression was found 1 h after irradiation, which then rapidly returned to control levels. These findings suggest that astrocytes may play a role in mediating the radiation response of the central nervous system via X-ray-induced changes in gene expression. In contrast, doses of up to 20 Gy had no effect on c-fos, c-jun and jun-B mRNA levels in the two brain tumour cell lines. In addition, whereas 12-O-tetradecanoylphorbol-13-acetate induced the expression of these genes in astrocytes, it had little or no effect on fos or jun expression in 9L or 175A cells. These results suggest that the signal transduction pathways mediating radiation-induced gene expression may be different in normal astrocytes and brain tumour cells.

Asunto(s)

Genes fos/efectos de la radiación , Genes jun/efectos de la radiación , Animales , Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/efectos de la radiación , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , ARN Mensajero/análisis , Dosis de Radiación , Radiación Ionizante , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas

RESUMEN

The relevance of tyrosine phosphorylation and c-jun protooncogene expression to radiation sensitization was investigated in B16 melanoma. These cells are sensitized by bromodeoxyuridine (BrdU), a thymidine analog, showing extensive DNA fragmentation reminiscent of apoptosis, after UV radiation. UV-irradiated unsensitized cells did not reveal DNA fragmentation but showed increased expression of c-jun and greater protein tyrosine phosphorylation in response to sodium vanadate, an inhibitor of tyrosine phosphatases. However, these responses were inhibited in UV-irradiated BrdU-treated cells. Our data suggest that the bromodeoxyuridine-induced sensitization to radiation can lead to DNA fragmentation and cell death, partly because of a defective tyrosine kinase signalling and an impaired c-jun expression, both of which appear important for cell survival in response to UV radiation.

Asunto(s)

Bromodesoxiuridina/farmacología , Muerte Celular/efectos de la radiación , Daño del ADN , ADN de Neoplasias/efectos de la radiación , Genes jun/efectos de la radiación , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Fármacos Sensibilizantes a Radiaciones/farmacología , Transducción de Señal/efectos de la radiación , Tirosina/análogos & derivados , Rayos Ultravioleta , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , ADN de Neoplasias/efectos de los fármacos , Éteres Cíclicos/farmacología , Genes jun/efectos de los fármacos , Immunoblotting , Melanoma Experimental , Ratones , Ácido Ocadaico , Fosforilación , Fosfotirosina , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Tirosina/metabolismo , Vanadatos/farmacología