RESUMEN
Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.
Asunto(s)
Proteína Quinasa C/genética , Receptores Opioides kappa/genética , Transducción de Señal/efectos de los fármacos , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero/farmacología , Animales , Reacción de Prevención , Quinasas de Receptores Acoplados a Proteína-G , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Fosfoproteínas , Fosforilación , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/efectos de la radiación , Inhibidores de Proteínas Quinasas , Proteómica , Receptor Cannabinoide CB1/efectos de los fármacos , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/efectos de la radiación , Serina-Treonina Quinasas TOR/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
Asunto(s)
Diglicéridos/metabolismo , Diglicéridos/efectos de la radiación , Procesos Fotoquímicos/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Rayos Ultravioleta , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efectos de la radiación , Diglicéridos/química , Ratones , Fenómenos Ópticos , Proteína Quinasa C/química , Transducción de Señal/efectos de la radiaciónRESUMEN
12-O-Tetradecanoylphorbol-13-acetate (TPA) is a non-genotoxic tumor promoter that dysregulates the protein kinase C (PKC) pathway and causes variable cellular responses to DNA damage in different experimental models. In the present study, we pretreated human lymphoblastoid TK6 cells (wild-type p53) for 72 h with TPA, and five other PKC-activating tumor promoters, to determine how sustained exposure to these chemicals modulates key DNA damage response (DDR) endpoints induced by UVC-irradiation. Here we show that pre-treatment with PKC-activating tumor promoters augmented the sensitivity of TK6 cells to UVC-irradiation characterized by a synergistic increase in apoptosis compared to that induced by either stress alone. In addition, high residual levels of the DNA damage repair signal γH2AX was observed in tumor promoter treated cells indicating a delayed DDR recovery. NH32 (p53-null, isogenic to TK6) cells were resistant to the synergistic effects on apoptosis implicating p53 as a central mediator of the DDR modulating effects. In addition, analysis of p53 target genes in TPA-pre-treated TK6 cells revealed a significant modulation of UVC-induced gene expression that supported a shift toward a pro-apoptotic phenotype. Therefore, sustained exposure to tumor promoting agents modulates the UVC-induced DDR in TK6 cells, which may represent important synergistic interactions that occur during tumor promotion.
Asunto(s)
Carcinógenos/metabolismo , Daño del ADN , Proteína Quinasa C/metabolismo , Rayos Ultravioleta , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Carcinógenos/efectos de la radiación , Carcinógenos/toxicidad , Línea Celular , Línea Celular Tumoral , Activación Enzimática/efectos de la radiación , Genes p53/efectos de los fármacos , Histonas/metabolismo , Humanos , Fosforilación/efectos de la radiación , Proteína Quinasa C/efectos de la radiación , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Acetato de Tetradecanoilforbol/toxicidad , Transcripción Genética/genética , Transcripción Genética/efectos de la radiaciónRESUMEN
Recently, there have been several reports referring to detrimental effects due to radio frequency electromagnetic fields (RF-EMF) exposure. Special attention was given to investigate the effect of mobile phone exposure on the rat brain. Since the integrative mechanism of the entire body lies in the brain, it is suggestive to analyze its biochemical aspects. For this, 35-day old Wistar rats were exposed to a mobile phone for 2 h per day for a duration of 45 days where specific absorption rate (SAR) was 0.9 W/Kg. Animals were divided in two groups: sham exposed (n = 6) and exposed group (n = 6). Our observations indicate a significant decrease (P < 0.05) in the level of glutathione peroxidase, superoxide dismutase, and an increase in catalase activity. Moreover, protein kinase shows a significant decrease in exposed group (P < 0.05) of hippocampus and whole brain. Also, a significant decrease (P < 0.05) in the level of pineal melatonin and a significant increase (P < 0.05) in creatine kinase and caspase 3 was observed in exposed group of whole brain as compared with sham exposed. Finally, a significant increase in the level of ROS (reactive oxygen species) (P < 0.05) was also recorded. The study concludes that a reduction or an increase in antioxidative enzyme activities, protein kinase C, melatonin, caspase 3, and creatine kinase are related to overproduction of reactive oxygen species (ROS) in animals under mobile phone radiation exposure. Our findings on these biomarkers are clear indications of possible health implications.
Asunto(s)
Encéfalo/efectos de la radiación , Teléfono Celular , Microondas , Animales , Antioxidantes/metabolismo , Antioxidantes/efectos de la radiación , Encéfalo/enzimología , Encéfalo/metabolismo , Catalasa/metabolismo , Catalasa/efectos de la radiación , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/efectos de la radiación , Masculino , Melatonina/metabolismo , Melatonina/efectos de la radiación , Oxidación-Reducción/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/efectos de la radiación , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/efectos de la radiación , Factores de TiempoRESUMEN
Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of ß-galactosidase by using an eosin-labeled anti-ß-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.
Asunto(s)
Eosina Amarillenta-(YS)/farmacología , Fármacos Fotosensibilizantes/farmacología , beta-Galactosidasa/antagonistas & inhibidores , Aurora Quinasa B , Aurora Quinasas , Colorantes Fluorescentes/farmacología , Células HeLa , Humanos , Rayos Láser , Ligandos , Luz , Proteína Quinasa C/efectos de la radiación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Oxígeno Singlete , beta-Galactosidasa/inmunología , beta-Galactosidasa/efectos de la radiaciónRESUMEN
Cellular lesions (e.g. DSBs) are induced into DNA upon exposure to radiation, with DSB complexity increasing with radiation ionization density. Using M059K and M059J human glioblastoma cells (proficient and deficient in DNA-PKcs activity, respectively), we investigated the repair of DNA damage, including DSBs, induced by high- and low-LET radiation [gamma rays, alpha particles and high-charge and energy (HZE) ions]. In the absence of DNA-PKcs activity, less DSB repair and increased recruitment of RAD51 was seen at 24 h. After exposure to (56)Fe heavy ions, the number of cells with RAD51 tracks was less than the number of cells with gamma-H2AX at 24 h with both cell lines. Using alpha particles, comparable numbers of cells with visible gamma-H2AX and RAD51 were seen at 24 h in both cell lines. M059J cells irradiated with alpha particles accumulated in S phase, with a greater number of cyclin A and RAD51 co-stained cells seen at 24 h compared with M059K cells, where an S-phase block is absent. It is proposed that DNA-PKcs plays a role in the repair of some frank DSBs, which are longer-lived in NHEJ-deficient cells, and some non-DSB clustered damage sites that are converted into DSBs at replication as the cell cycles through to S phase.
Asunto(s)
Proteína Quinasa Activada por ADN/efectos de la radiación , Rayos gamma/efectos adversos , Radioisótopos de Hierro/farmacología , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/aislamiento & purificación , Proteína Quinasa Activada por ADN/metabolismo , Electroforesis en Gel de Campo Pulsado , Fibroblastos/enzimología , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Glioblastoma , Humanos , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Dosis de RadiaciónRESUMEN
Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-beta inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5 microM) in combination with irradiation (2-8 Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7 microM) and irradiation (10 Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25 Gy increased PKC-alpha activity by 2.5-fold compared to untreated controls, whereas PKC-epsilon and -betaII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75 mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.
Asunto(s)
Neoplasias de la Mama/enzimología , Inhibidores Enzimáticos/farmacología , Rayos gamma , Indoles/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Citosol/química , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Fosforilación/efectos de los fármacos , Proteína Quinasa C/efectos de la radiación , Proteínas Represoras/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Studies over the last several years have revealed the existence of a biological phenomenon known as "bystander effect", wherein cells that are not exposed to radiation elicit a similar response to that of irradiated cells. Understanding the mechanism(s) underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for cancer radiotherapy. Evaluation of signaling pathways in bystander cells may provide an insight to understand the molecular mechanisms(s) responsible for this complex phenomenon. With this objective, the time course kinetics of intracellular distribution of protein kinase C (PKC isoforms PKC-betaII, PKC-alpha/beta, PKC-theta) was investigated in total and subcellular (cytosolic and nuclear) fractions of human lung fibroblast (MRC-5) cells. MRC-5 cells were either irradiated or treated with the irradiated conditioned medium collected 1h after 1 or 10 Gy of gamma-irradiation. The radiation dose selected was in the range of therapeutic usage of radiation for the human cancer treatment. Unexpectedly, bystander cells showed higher activation of protein kinase C isoforms as compared to irradiated and sham-treated control cells. Protein kinase C isoforms were more enriched in the nuclear fraction than the cytosolic fraction proteins. Induction of PKC isoforms in bystander cells are due to post-translational modifications as shown by the non-phosphorylated protein kinase C level in both irradiated and bystander cells did not differ from the sham-treated control cells. The specific activation of protein kinase C isoforms in bystander cells as demonstrated for the first time in this study may help to identify the effect of therapeutically used radiation exposure for the tumor destructions along with its implications for adjacent non-irradiated cells and organs.
Asunto(s)
Efecto Espectador , Fibroblastos/enzimología , Isoenzimas/metabolismo , Isoenzimas/efectos de la radiación , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-alfa/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , Núcleo Celular/metabolismo , Medios de Cultivo Condicionados , Citosol/metabolismo , Relación Dosis-Respuesta en la Radiación , Activación Enzimática/efectos de la radiación , Fibroblastos/ultraestructura , Humanos , Pulmón/citología , Proteína Quinasa C beta , Proteína Quinasa C-theta , Procesamiento Proteico-Postraduccional , Radiación Ionizante , Transducción de SeñalRESUMEN
Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.
Asunto(s)
Señalización del Calcio/efectos de la radiación , Calcio/metabolismo , Campos Electromagnéticos/efectos adversos , Hipocampo/efectos de la radiación , Receptores de N-Metil-D-Aspartato/efectos de la radiación , Animales , Unión Competitiva/fisiología , Unión Competitiva/efectos de la radiación , Calcineurina , Señalización del Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de la radiación , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de la radiación , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Trastornos de la Memoria/etiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiología , Transmisión Sináptica/efectos de la radiaciónRESUMEN
There is growing concern by the public regarding the potential human health hazard due to exposure to microwave frequencies. 2.45 GHz radiation widespread use in industry, research, and medicine, and leakage into the environment is possible. In order to quantitate this, experiments were performed on developing rat brain. Male Wistar 35-day-old rats (n = 6) were used for this study. Animals were exposed to 2.45 GHz radiation for 2 h/day for a period of 35 days at a power density of 0.344 mW/cm(2) (SAR 0.11 W/kg). The control group was sham irradiated. After 35 days these rats were sacrificed and whole brain tissue was isolated for protein kinase C (PKC) assay. For morphological study the forebrain was isolated from the whole brain and PKC activity was measured using P(32) labeled ATP. Our study reveals a statistically significant (p < 0.05) decrease in PKC activity in hippocampus as compared to the remaining portion of the whole brain and the control group. A similar experiment conducted on hippocampus and the whole brain gave a similar result. Electron microscopic study shows an increase in the glial cell population in the exposed group as compared to the control group. This present study is indicative of a significant change after exposure to the above-mentioned field intensity. This suggests that chronic exposures may affect brain growth and development.
Asunto(s)
Encéfalo/efectos de la radiación , Microondas/efectos adversos , Neuroglía/efectos de la radiación , Proteína Quinasa C/efectos de la radiación , Ondas de Radio/efectos adversos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Masculino , Microscopía Electrónica , Neuroglía/metabolismo , Proteína Quinasa C/metabolismo , Dosis de Radiación , Ratas , Ratas WistarRESUMEN
Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.
Asunto(s)
Apoptosis/efectos de la radiación , Proliferación Celular/efectos de la radiación , Activación Enzimática/efectos de la radiación , Terapia por Luz de Baja Intensidad , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Acetato de Tetradecanoilforbol/farmacología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Modelos Biológicos , Espectrometría de Fluorescencia , Factores de Tiempo , TransfecciónAsunto(s)
Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transducción de Señal , Animales , Antiinflamatorios no Esteroideos/farmacología , Anticarcinógenos/farmacología , Apoptosis/efectos de la radiación , Arsénico/toxicidad , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Diseño de Fármacos , Histonas/metabolismo , Histonas/efectos de la radiación , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Neoplasias/patología , Fosforilación/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Rayos UltravioletaRESUMEN
In this study, we show that one single dose of gamma-irradiation at birth induces an inhibition of the cerebellar calcium dependent nitric oxide synthase (NOS) activity, probably correlated to the motor abnormalities and the disarrangement in the cerebellar cytoarchitecture observed in adult rats. This decrease in calcium dependent NOS activity could be associated with an increased protein kinase C (PKC) activity. PKC inhibition partially restores calcium dependent NOS activity, indicating that PKC activity could be negatively modulating the catalytic activity of calcium dependent NOS. These findings suggest that a decrease in nitric oxide (NO) production and the related increase in PKC activity could be intracellular events that participate in the onset of motor and cerebellar abnormalities induced by postnatal gamma-irradiation at early stages of life.
Asunto(s)
Cerebelo/enzimología , Cerebelo/efectos de la radiación , Rayos gamma , Óxido Nítrico Sintasa/efectos de la radiación , Proteína Quinasa C/efectos de la radiación , Traumatismos Experimentales por Radiación/enzimología , Análisis de Varianza , Animales , Animales Recién Nacidos , Calbindinas , Calcio/metabolismo , Cerebelo/patología , Femenino , Marcha/efectos de la radiación , Masculino , Neuronas/enzimología , Neuronas/patología , Neuronas/efectos de la radiación , Ratas , Ratas Wistar , Proteína G de Unión al Calcio S100/efectos de la radiación , Transducción de Señal/efectos de la radiación , Factores de TiempoRESUMEN
Various kinases, such as tyrosine, protein kinase C (PKC) and MAP kinase, play important role in the cellular response to radiation, but little is known about the specific response in the whole animal. Most studies, except a few, are based on single cells. There is a paucity of data where signaling following whole body irradiation is concerned. In this study a comparison has been made between the activities of these kinases following ex vivo and in vivo irradiation. Tyrosine kinase activity showed no difference in the lymphocytes irradiated ex vivo or in vivo. A significant differential dose-dependent response could be observed in PKC activity. PKC was seen to be activated at the higher dose, i.e., 1 Gy in, in vivo irradiated lymphocytes, whereas in ex vivo irradiated lymphocytes, PKC was seen to be activated at the lower dose, i.e., 0.1 Gy. MAP kinase activity was seen to decrease with an increasing dose in ex vivo irradiated lymphocytes. In vivo MAP kinase activity was seen to increase as the dose increased, with maximum activation at 3 Gy. These kinases are being used to manipulate the tumor response to radiotherapy. Thus it is essential to study the behavior of the above kinases in the whole animal because the difference in response of a single cell to the whole animal may be different.
Asunto(s)
Linfocitos/enzimología , Linfocitos/efectos de la radiación , Fosfotransferasas/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Activación Enzimática/efectos de la radiación , Rayos gamma , Linfocitos/citología , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de la radiación , Fosfotransferasas/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/efectos de la radiación , Dosis de Radiación , Irradiación Corporal TotalRESUMEN
Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase-3 activation, we paid attention to the influence exerted by Protein kinase C delta on transcription factor NF-kappaB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC delta/NF-kappaB pathway activation, probably mediated by novel IKKs and a role for PI-3-kinase in activating PKC delta at Thr 505 by PDK-1 phosphorylation is suggested. In addition, since caspase-3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF-kappaB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF-kappaB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings.
Asunto(s)
Apoptosis/fisiología , Apoptosis/efectos de la radiación , Leucemia/patología , FN-kappa B/fisiología , Caspasa 3 , Caspasas/metabolismo , Caspasas/efectos de la radiación , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Activación Enzimática/efectos de la radiación , Humanos , Proteínas I-kappa B/metabolismo , Proteínas I-kappa B/efectos de la radiación , Etiquetado Corte-Fin in Situ , Células Jurkat/citología , Células Jurkat/efectos de la radiación , Células K562 , Leucemia/metabolismo , Microscopía Fluorescente , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de la radiación , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Radiación IonizanteRESUMEN
Natural phenolic compounds were tested in vitro for their effect on the activity of protein kinase C (PKC) isolated from the liver cytosol and the particulate fraction of unirradiated mice and mice irradiated at 5 Gy. Following irradiation, the PKC activity was found to be increased in both cytosolic and particulate fractions. Curcumin, ellagic acid and quercetin were effective in inhibiting radiation-induced PKC activity. Curcumin and ellagic acid were found to be more inhibitory towards radiation-induced PKC activity, while quercetin was the least effective. Curcumin was found to inhibit the activated cytosolic and particulate PKC at very low concentrations. Activation of PKC is one of the means of conferring radioresistance on a tumour cell. Suppression of PKC activity by phenolics may be one of the means of preventing the development of radioresistance following radiotherapy.
Asunto(s)
Fenoles/farmacología , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/efectos de la radiación , Animales , Curcumina/farmacología , Citosol/enzimología , Citosol/efectos de la radiación , Ácido Elágico/farmacología , Masculino , Ratones , Quercetina/farmacologíaRESUMEN
We used a resonant cavity which delivered a continuous wave exposure at 864.3 MHz at an average specific absorption rate (SAR) of 7 W/kg to determine non-thermal biological effects of microwave exposure. A human mast cell line, HMC-1, was used as the biological target. Cells were given three exposures each of 20-min duration daily for 7 days. The temperature of the cell culture medium during the exposure fell to 26.5 degrees C. Effects were seen on localization of protein kinase C, and expression of three genes of 588 screened. The affected genes included the proto-oncogene c-kit, the transcription factor Nucleoside diphosphate kinase B and the apoptosis-associated gene DAD-1. Stress response genes were variably upregulated. No significant effect on morphology or on F-actin distribution was detected. We conclude that low-power microwave exposure may act on HMC-1 cells by altering gene expression via a mechanism involving activation of protein kinase C, and at temperatures well below those known to induce a heat shock response.
Asunto(s)
Proteínas de Caenorhabditis elegans , Regulación de la Expresión Génica/efectos de la radiación , Microondas , Proteína Quinasa C/efectos de la radiación , Actinas/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis , Línea Celular , Humanos , Cinética , Mastocitos , Nucleósido-Difosfato Quinasa/genética , Proteína Quinasa C/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Represoras/genética , TemperaturaRESUMEN
Since ultraviolet (UV) radiation is a major etiologic factor in the development of human skin cancers, investigating the signal transduction pathways initiated by UV radiation may help with the understanding of the molecular mechanisms of UV-induced carcinogenesis. Our previous studies demonstrated that UV-induced activator protein-1 (AP-1) activation is blocked by dominant negative atypical PKCs (aPKCs). Here we investigated the role of aPKC in UV-induced activation of mitogen activated protein (MAP) kinase family members which are considered to be the mediators of AP-1 activation. We found that UV radiation led to translocation of protein kinase C (PKC) zeta and activation of MAP kinase family members as well as an increase of AP-1-dependent transcription activation at the same dose range. Pretreatment of cells or mouse skin with antisense oligonucleotides of PKCzeta impaired UV-induced activation of AP-1 in JB6 cells as well as in AP-1-luciferase transgenic mice. It also inhibited UV-induced activation of ERKs but not of JNK and p38 kinases in JB6 cells. In contrast, no significant inhibition of AP-1 activation and MAP kinase activation were observed in cells treated with sense oligonucleotides of PKCzeta. Furthermore, overexpression of a dominant negative mutant of PKClambda/iota specifically inhibited activation of extracellular signal-regulated protein kinases (ERKs) but not of c-jun N-terminal kinases (JNKs) nor p38 kinases induced by UV radiation. These results demonstrated that inhibition of aPKC impairs UV-induced AP-1 activation via suppression of ERKs activation but not of JNKs or p38 kinase activation.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Rayos Ultravioleta , Animales , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/efectos de la radiación , Línea Celular , Membrana Celular/enzimología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Inducción Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de la radiación , Femenino , Humanos , Isoenzimas , Proteínas Quinasas JNK Activadas por Mitógenos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oligonucleótidos Antisentido/farmacología , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/genética , Proteína Quinasa C/fisiología , Proteína Quinasa C/efectos de la radiación , Factor de Transcripción AP-1/efectos de la radiación , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Photoaffinity probes PPDA and PPTD, which have diazoacetyl and trifluorodiazopropionyl group at C-13 position in phorbol, respectively, were synthesized. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking.