RESUMO
The treatment for spinal sarcomas is difficult due to inadequate surgical margin and an inability to deliver high dose radiation. Advanced technology of stereotactic radiosurgery (SRS) enabled higher biological effective doses of radiation to be delivered to spinal sarcomas by hypofractionation method. The authors evaluated local control rate following SRS for primary and metastatic spinal sarcomas. Thirty-two spinal sarcomas (10 primary tumors, 22 metastatic tumors) in 27 patients were treated by SRS from November 2002 to September 2009. Patients were assessed for pain status, neurological status and radiological response by regular follow-up. Overall survival and local progression-free survival were calculated and prognostic factors were sought. Median tumor volume was 18.6 ml. Radiation doses to the tumor margins ranged from 16 to 45 Gy in one to three fractions, and the median single session equivalent dose was 21.8 Gy. Follow-up ranged from 4 to 68 months (median, 22 months). Overall median survival was 29 months and no related prognostic factors were identified. During follow-up, pain was controlled in 89.3% (25/28) lesions at 6 months, in 68.2% (15/22) at 1 year, and in 61.5% (8/13) at 2 years. Tumor volume was found to be significantly related to post-SRS pain control rate. Radiological evaluation showed that local control was maintained in 96.7% (29/30) lesions at 6 months, in 78.3% (18/23) at 1 year, and in 76.9% (10/13) at 2 years. Radiation dose and tumor volume were found to be related to radiological control at 24 months following SRS. Nine cases developed recurrence between 2 and 33 months, median local progression-free survival was 23 months. Age was found to be predictive of local progression-free survival (P = 0.009). SRS proved to be an effective modality for the local control of primary and metastatic spinal sarcomas, and age was significantly related to local recurrence.
Assuntos
Radiocirurgia/métodos , Sarcoma/cirurgia , Neoplasias da Coluna Vertebral/cirurgia , Adolescente , Adulto , Fatores Etários , Idoso , Criança , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Medição da Dor , Dosagem Radioterapêutica , Estudos Retrospectivos , Sarcoma/mortalidade , Neoplasias da Coluna Vertebral/mortalidade , Adulto JovemRESUMO
BACKGROUND: this study was to estimate the predicted effect-site concentration of propofol administered by a target-controlled infusion (TCI) for maintenance of anesthesia based on the bispectral (BIS) index as a measure of hypnosis in laparoscopic surgery. METHOD: one-hundred and sixty unpremedicated patients undergoing gynecologic laparoscopy were assigned randomly to receive one of the target effect-site concentrations of propofol 2.0, 2.5, 3.0, 3.5 and 4.0 microg/ml during TCI with propofol and sufentanil. The dose-response relationship of propofol for the maintenance of adequate anesthesia based on BIS, movement and hemodynamic response was investigated using a fixed effect-site concentration of sufentanil (0.2 ng/ml). The BIS values, hemodynamic variables, time course during emergence and intraoperative awareness were also assessed. RESULTS: the predicted effect-site propofol concentrations for adequate anesthesia at the skin incision in 50% (EC(50) ) and 95% (EC(95) ) of patients undergoing gynecologic laparoscopy were 2.2 and 3.7 microg/ml, respectively. The predicted propofol EC(50) and EC(95) to maintain adequate anesthesia in these patients were 2.6 microg/ml (95% CI 2.3-2.7 microg/ml) and 3.6 microg/ml (95% CI 3.3-4.0 microg/ml), respectively. The BIS values, effect-site concentration of propofol, hemodynamic data and time course during emergence and post-operative adverse events were comparable in each group. There were no reports of intraoperative awareness in the post-anesthetic care unit. CONCLUSION: based on the anesthetic depth assessed by the clinical signs and BIS monitoring, the predicted effect-site propofol concentrations for the maintenance of anesthesia in patients undergoing gynecologic laparoscopy were similar in those administered adequate anesthesia at the skin incision during TCI.
Assuntos
Anestesia Intravenosa , Anestésicos Intravenosos/administração & dosagem , Procedimentos Cirúrgicos em Ginecologia , Laparoscopia , Propofol/administração & dosagem , Sufentanil/administração & dosagem , Adolescente , Adulto , Anestesia Intravenosa/efeitos adversos , Anestésicos Intravenosos/efeitos adversos , Pressão Sanguínea/efeitos dos fármacos , Monitores de Consciência , Relação Dose-Resposta a Droga , Eletroencefalografia , Feminino , Previsões , Hemodinâmica/efeitos dos fármacos , Humanos , Infusões Intravenosas , Consciência no Peroperatório/epidemiologia , Pessoa de Meia-Idade , Propofol/efeitos adversos , Estudos Prospectivos , Sufentanil/efeitos adversos , Adulto JovemAssuntos
Neoplasias dos Ductos Biliares/diagnóstico , Ductos Biliares/lesões , Neuroma/diagnóstico , Complicações Pós-Operatórias/diagnóstico , Idoso , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/cirurgia , Ductos Biliares/patologia , Ductos Biliares/cirurgia , Biópsia , Colangiopancreatografia por Ressonância Magnética , Humanos , Jejunostomia , Masculino , Neuroma/patologia , Neuroma/cirurgia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/cirurgia , Valor Preditivo dos TestesRESUMO
We previously reported that overexpression of HSP25 delayed cell growth, increased the level of p21(waf), reduced the levels of cyclin D1, cyclin A and cdc2, and induced radioresistance in L929 cells. In this study, we demonstrated that HSP25 induced-radioresistance was abolished by transfection with plasmids containing antisense hsp25 cDNA. Extracellular regulated kinase (ERK) and MAP kinase/ERK kinase (MEK) expressions as well as their activation (phospho-forms) were inhibited by hsp25 overexpression. Furthermore, when control vector transfected cells were treated with PD98059, MEK inhibitor, they became resistant to radiation, suggesting that inhibition of ERK1/2 activities was essential for radioresistance in L929 cells. To confirm the relationship between ERK1/2 and hsp25-mediated radioresistance, ERK1 or ERK2 cDNA was transiently transfected into the hsp25 overexpressed cells and their radioresistance was examined. HSP25-mediated radioresistance was abolished by overexpression of ERK2, but not by overexpression of ERK1. Alteration of cell cycle distribution and cell cycle related protein expressions (cyclin D, cyclin A and cdc2) by hsp25 overexpression were also recovered by ERK2 cDNA transfection. Increase in Bcl-2 protein by hsp25 gene transfection was also reduced by subsequent ERK2 cDNA-transfection. Taken together, these results suggest that downregulation of ERK2 is essential for the inhibition of radiation-induced cell death in HSP25 overexpressed cells.
Assuntos
Apoptose , Regulação para Baixo , Proteínas de Choque Térmico , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Apoptose/efeitos da radiação , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Expressão Gênica , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Tolerância a Radiação , TransfecçãoRESUMO
Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL-resistant or -sensitive Jurkat clones and examined cross-responsiveness of the clones between TRAIL and radiation. Treatment with gamma-radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross-resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL-resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL-resistant cells was associated with cleavage of caspase-8 and the proapoptotic Bid protein, resulting in the activation of caspase-9 and caspase-3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl-2 or Bax were found after treatment. The caspase inhibitor z-VAD-fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and gamma-radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL-resistant cells through TRAIL receptor-independent synergistic activation of the cascades of the caspase-8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL-resistant cancer cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Glicoproteínas de Membrana/farmacologia , Radiação Ionizante , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/metabolismo , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Morte Celular/efeitos dos fármacos , Células Clonais , Humanos , Células Jurkat , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Ligante Indutor de Apoptose Relacionado a TNF , Proteína X Associada a bcl-2RESUMO
PURPOSE: The aim of this study was to determine whether gamma-rays can affect Ca2+-induced differentiation in normal and neoplastic mouse epidermal cells. METHODS AND MATERIALS: After gamma-ray irradiation, primary and v-rasHa transformed mouse keratinocytes were cultured for 48 h in 0.12 mM Ca2+-containing media, and cellular translocation from cytosolic to particulated fraction of each PKC isozyme and expressions of differentiation markers were examined. RESULTS: Morphological difference was seen at 48 h after irradiation in both Ca2+-shifted normal and v-rasHa transformed cells; v-rasHa cells were more resistant to the radiation than normal cells. Radiation potentiated granular cell-differentiation marker expressions (filaggrin, loricrin, and SPR-1) in both normal and v-rasHa transformed cells. In the case of spinous cell markers, the expression of keratins K1 and K10, which are usually blocked in v-rasHa cells was increased after irradiation. However, there was no change of K8 expression level, which can be seen only after v-rasHa transfection. Cellular fractionation and immunoblot analysis with antibodies against PKCalpha, delta, epsilon, eta, and xi revealed that PKCalpha was responsible for the differentiation marker expression. CONCLUSIONS: These findings suggest that PKCalpha is an important component of the signaling pathway regulating radiation-induced differentiation in both normal and neoplastic epidermal cells.
Assuntos
Cálcio/farmacologia , Diferenciação Celular/efeitos da radiação , Raios gama , Isoenzimas/metabolismo , Queratinócitos/efeitos da radiação , Proteína Quinase C/metabolismo , Animais , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Indução Enzimática/efeitos da radiação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Tolerância a RadiaçãoRESUMO
BACKGROUND AND PURPOSE: Ginkgo biloba leaf extract (GBE) is known to increase peripheral blood circulation. The hypothesis that GBE may be able to enhance radiosensitivity of tumor by improving tumor blood flow and thus decreasing hypoxic fraction was tested. MATERIALS AND METHODS: Fibrosarcoma (FSaII) growing in C3H mouse leg muscle was used as a tumor model. GBE was given i.p. 1 h before irradiation with or without priming dose given 1 day earlier. Effect on tumor and normal tissue radiation reaction was investigated. RESULTS: Tumor growth delay by radiation was more elongated after two doses (1-day interval) of GBE than after a single dose. Radiation dose for 3-day tumor growth delay was decreased from 12.45 (10.97-13.93) Gy to 6.06 (3.89-8.22) Gy by two doses of GBE [enhancement ratio = 2.06 (1.32-2.79)]. Hypoxic cell fraction was 10.6% (6.3-18.2%) for control, 7.2% (3.8-14.0%) after a single dose (P = 0.18) and 2.7% (1.5-5.0%) after two doses (P < 0.001). Radiation effect on normal tissue, estimated by acute skin reaction and jejunal crypt assay, was not affected by GBE. CONCLUSION: Ginkgo biloba extract enhances radiation effect on tumor without increasing acute normal tissue radiation damage in this model system probably by increasing tumor blood flow and further investigation for this possible radiosensitizer is needed.
Assuntos
Fibrossarcoma/radioterapia , Neoplasias Musculares/radioterapia , Extratos Vegetais/uso terapêutico , Radiossensibilizantes/uso terapêutico , Animais , Hipóxia Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Doses de RadiaçãoRESUMO
Previous studies in this laboratory have shown that gamma-ray ionizing radiation in combination with oltipraz, a radioprotective agent, enhances hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) expression. The present study was designed to investigate the effects of dexamethasone on the radiation-inducible expression of mEH and rGST genes and on the vitamin C and E-induced radioprotective effects in association with the expression of the genes. Treatment of rats with a single dose of dexamethasone (0.01-1 mg/kg, p.o.) caused a dose-dependent decrease in the constitutive mEH gene expression at 24 hr. The radiation-inducible mEH mRNA level (threefold increase after 3 Gy gamma-irradiation) was decreased by 21% and 88% by dexamethasone at the doses of 0.1 and 1 mg/kg, respectively. Although dexamethasone alone caused 2- to 5-fold increases in the hepatic rGSTA2 mRNA level, rats treated with dexamethasone prior to 3 Gy irradiation exhibited 80%-93% suppression in the radiation-inducible increases in the rGSTA2 mRNA level. The inducible rGSTA3 and rGSTA5 mRNA levels were also significantly decreased by dexamethasone, whereas the rGSTM1 mRNA level was reduced to a lesser extent. Vitamin C and/or E, however, failed to enhance the radiation-inducible increases in hepatic mEH and rGST mRNA levels. Whereas rats exposed to 3 Gy irradiation with or without vitamin C treatment (30 or 200 mg/kg/day, p.o., 2 days) exhibited approximately threefold increases in the mEH and rGSTA2/3/5 mRNA levels relative to untreated animals, dexamethasone treatment (1 mg/kg, p.o.) resulted in 64%-96% decreases in the mRNA levels at 24 hr. The inducible rGSTM1/2 mRNA levels in the vitamin C/E-treated rats were approximately 50% suppressed by dexamethasone. Although vitamin C and/or E treatment (200 mg/kg/day, p.o., 2 days) improved the 30-day survival rates of the 8 Gy gamma-irradiated mice from 39% up to 74%, the improved survival rate of gamma-irradiated animals was reduced to 30% by dexamethasone pretreatment (1 mg/kg/day, 2 days). The mean survival time of dexamethasone-treated animals was reduced to approximately 2 days from 14 days in the animals with total body irradiation alone. No significant hematologic changes were observed in mice at 10 days after dexamethasone plus gamma-irradiation, as compared with irradiation alone. These results demonstrate that: dexamethasone substantially suppresses radiation-inducible mEH, rGSTA and rGSTM expression in the liver; vitamins C/E exhibit radioprotective effects without enhancing radiation-inducible mEH and GST gene expression; and inhibition of radiation-inducible mEH and rGST gene expression in the vitamin C- and E-treated animals by dexamethasone was highly correlated with reduction in the survival rate and the mean survival time of gamma-irradiated animals.
Assuntos
Dexametasona/farmacologia , Epóxido Hidrolases/genética , Glutationa Transferase/genética , Microssomos Hepáticos/efeitos da radiação , Lesões Experimentais por Radiação/mortalidade , Protetores contra Radiação/farmacologia , Animais , Ácido Ascórbico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vitamina E/farmacologiaRESUMO
We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.
Assuntos
Apoptose/efeitos da radiação , Ciclo Celular/efeitos da radiação , Proteínas de Choque Térmico HSP70/metabolismo , Células 3T3 , Animais , Apoptose/fisiologia , Proteína Quinase CDC2/metabolismo , Ciclo Celular/fisiologia , Ciclina A/metabolismo , Ciclina B/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Citometria de Fluxo , Raios gama , Regulação da Expressão Gênica/fisiologia , Immunoblotting , Camundongos , Testes de Precipitina , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.
Assuntos
Anticarcinógenos/farmacologia , Epóxido Hidrolases/genética , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Pirazinas/farmacologia , Animais , Epóxido Hidrolases/metabolismo , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Masculino , Microssomos Hepáticos/enzimologia , RNA Mensageiro/genética , Ratos , Tionas , Tiofenos , Fatores de TempoRESUMO
Park, S-H., Lee, S-J., Chung, H-Y., Kim, T-H., Cho, C-K., Yoo, S-Y. and Lee, Y-S. Inducible Heat-Shock Protein 70 Is Involved in the Radioadaptive Response. The thermoresistant (TR) clone of radiation-induced fibrosarcoma (RIF) cells showed an adaptive response, i.e. a reduced effect, after exposure to a higher challenging dose (4 Gy) when the priming dose (1 cGy) was given 4 or 7 h earlier, but RIF cells did not. Since inducible Hsp70 expression was different in cells of these two cell lines, the role of inducible Hsp70 in the adaptive response was examined. When inducible Hsp70 was transfected into RIF cells, the adaptive response was acquired. Transfection of inducible Hsp70 to NIH 3T3 mouse embryo cells also conferred radioresistance to the cells as assayed by clonogenic survival, [(3)H]thymidine incorporation, and an ELISA cell death detection kit. An increased tendency for the induction of an adaptive response was also observed. Interestingly, basal levels of Ca(2+)-dependent and independent Pkc activities were increased by transfection with inducible Hsp70 compared to those of control vector cells. Irradiation with gamma rays induced activation of Pkc within minutes in control vector cells, while transfection with inducible Hsp70 did not. Cellular redistribution to particulate fractions of Pkca, d and z after exposure gamma rays also was not detected. Furthermore, radioresistance by transfection with inducible Hsp70, as tested by clonogenic survival, disappeared after pretreatment with Pkc inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and GF109203X. Taken together, these data suggest that radioresistance inducible by Hsp70 is associated with an elevated level of Pkc activity.
Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Tolerância a Radiação/fisiologia , Células 3T3 , Animais , Relação Dose-Resposta à Radiação , Ativação Enzimática , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP70/biossíntese , Camundongos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/biossíntese , Proteína Quinase C/metabolismoRESUMO
We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.
Assuntos
Apoptose , Ciclo Celular , Proteínas de Choque Térmico , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Tolerância a Radiação/fisiologia , Animais , Apoptose/efeitos da radiação , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Ensaio de Unidades Formadoras de Colônias , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Genes bcl-2 , Células L/citologia , Células L/metabolismo , Células L/efeitos da radiação , Camundongos , Mimosina/farmacologia , Chaperonas Moleculares , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Tolerância a Radiação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
We investigated the effect of vasoactive intestinal peptide (VIP) and its combination with acetylcholine (ACh) on the erectile response in 52 adults rats. Intracavernous injection of VIP (10(-8) to 10(-5)M, ACh (10(-9) to 10(-5), or a combination of VIP (10(-7) to 10(-5) with ACh (10(-6 M) additively enhanced that erection but did not lead to a full erection. VIP-antagonist (10(-9) to 10(-5 M), as well as atropine alone (10 (-7) to 10(-5 M), partially suppressed full erection induced by cavernous nerve stimulation (1 Hz, 3-6 V) in a dose-dependent manner, and the combination of VIP-antagonist (10(-9) to 10(-5 M) with atropine (10(-6 M) showed an additive effect. The results indicate that although VIP as well as ACh in involved in penile erection, they are not likely to be principal neurotransmitters. Their clinical application in the treatment of impotence may be confined to use in conjunction with another vasoactive agent.
Assuntos
Acetilcolina/farmacologia , Ereção Peniana/efeitos dos fármacos , Peptídeo Intestinal Vasoativo/farmacologia , Acetilcolina/administração & dosagem , Animais , Atropina/farmacologia , Interações Medicamentosas , Estimulação Elétrica , Injeções , Masculino , Antagonistas Muscarínicos/farmacologia , Pênis/efeitos dos fármacos , Pênis/inervação , Pênis/fisiologia , Pressão , Ratos , Ratos Sprague-Dawley , Peptídeo Intestinal Vasoativo/administração & dosagemRESUMO
Multiple biological effects are induced by ionizing radiation through dysfunction of cellular organelles, direct interaction with nucleic acids and production of free radical species. The expression of cytochrome P450s was assessed in the livers of 60Co gamma-irradiated rats. Three gray (G) of gamma-irradiation caused CYP2E1 induction with a 3.6-fold increase in the mRNA at 24 h, whereas the expression of CYP1A2 and CYP3A was not changed. Pharmacokinetics of chlorzoxazone, a specific substrate of CYP2E1, was studied in 3 G-irradiated rats. The area under the plasma concentration-time curve from time zero to infinity of 6-hydroxychlorzoxazone and the amount of 6-hydroxychlorzoxazone excreted in 8 h urine were both significantly greater than those in control rats. Hepatic CYP2E1 was not induced in rats exposed to 0.5-1 G of gamma-rays. Rats irradiated at 6-9 G accumulated doses of gamma-rays exhibited smaller increases in the mRNA due to liver injury than those irradiated at a single dose of 3 G gamma-rays. The plasma glucose and insulin levels were not altered in rats with 3 G of gamma-irradiation. As the exposure level of gamma-irradiation increased, the activity of hepatic aconitase, a key enzyme in energy metabolism in mitochondria, was 30-90% decreased. The amount of mitochondrial DNA per gram of wet liver was 50% decreased in rats exposed to 3 G of gamma-rays. These results demonstrated that gamma-ray irradiation at the exposure level inducing organelle dysfunction induced CYP2E1 in the liver, which might be associated with mitochondrial damage, but not with alterations in glucose or insulin levels.
Assuntos
Clorzoxazona/farmacocinética , Citocromo P-450 CYP2E1/biossíntese , Raios gama , Microssomos Hepáticos/efeitos da radiação , Mitocôndrias Hepáticas/efeitos da radiação , Relaxantes Musculares Centrais/farmacocinética , Aconitato Hidratase/metabolismo , Alanina Transaminase/sangue , Animais , Área Sob a Curva , Glicemia/efeitos da radiação , Clorzoxazona/metabolismo , Cromatografia Líquida de Alta Pressão , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/efeitos da radiação , Meia-Vida , Insulina/sangue , Masculino , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Relaxantes Musculares Centrais/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The dose response of the number of micronuclei in cytokinesis-blocked (CB) lymphocytes after in vitro irradiation with y-rays and neutrons in the 5 dose range was studied in a heterogeneous population of 4 donors. One thousand binucleated cells were systematically scored for micronuclei. Measurements performed after irradiation showed a dose-related increase in micronuclei (MN) frequency in each of the donors studied. The dose-response curves were analyzed by a linear-quadratic model; frequencies per 1000 CB cells were (0.31 +/- 0.049)D + (0.0022 +/- 0.0002)D2 + (13.19 +/- 1.854) (r2 = 1.000, x2 = 0.7074, p = 0.95) following y-irradiation, and (0.99 +/- 0.528)D + (0.0093 +/- 0.0047)D2 + (13.31 +/- 7.309) (r2 = 0.996, x2 = 7.6834, p = 0.11) following neutron irradiation (D is irradiation dose in cGy). The relative biological effectiveness (RBE) of neutrons compared with y-rays was estimated by best fitting linear-quadratic model. In the micronuclei frequency between 0.05 and 0.8 per cell, the RBE of neutrons was 2.37 +/- 0.17. Since the MN assay is simple and rapid, it may be a good tool for evaluating the y-ray and neutron response.
Assuntos
Linfócitos/efeitos da radiação , Testes para Micronúcleos/métodos , Adolescente , Adulto , Relação Dose-Resposta à Radiação , Humanos , Linfócitos/ultraestrutura , MasculinoRESUMO
The usefulness of apoptotic fragments assay for investigating radiation response of hair follicles was examined. Frequency was defined as the ratio of the total number of apoptotic fragments to the number of hair follicles per section examined. The curve of dose-effect relationship for the data of apoptotic fragments was obtained by fitting the linear-quadratic model y= a+bD+cD2. When plotting on a linear scale against radiation dose, the line of best fit was y= 0.549 +/- 1.775) + 3.578 +/- 1.236)D + (-0.124 +/- 0.139)D2. The dose-response curves were linear-quadratic and a significant relationship was found between the frequency of apoptotic fragments and dose. The morphological findings of the irradiated groups were typical apoptotic fragments in the matrix region of hair follicles, but the spontaneous occurrence of apoptotic fragments was not observed. Since the apoptotic fragments was not observed. Since the apoptotic fragment assay is simple and reproducible in the whole body irradiation range of 0.5 to 8 Gy, it may be a good tool for evaluating the dose response of low dose radiation in vivo and provide a potentially valuable biological dosimeter for dose distribution determinations following accidental exposure.
Assuntos
Apoptose/efeitos da radiação , Folículo Piloso/efeitos da radiação , Animais , Relação Dose-Resposta à Radiação , Raios gama , Folículo Piloso/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
PURPOSE: Protective effect of small heat-shock protein (sHSP) against gamma-radiation, which associated with HSP25-induced cell-cycle delay and Bcl-2 induction. We further extended our studies on the possible role of HSP25 on ionizing radiation-induced cell-cycle regulation. MATERIALS AND METHODS: Flow-cytometric analyses were performed for cell-cycle distribution and Western blotting. Kinase or immunocomplex kinase assay were performed for detection of cell-cycle protein expression or activation. RESULTS: Pronounced arrest of G1, S and G2/M phase was observed by 4Gy radiation and these arrests were augmented by hsp25 overexpression. Inhibition of cyclin-D1, and cyclin-E and induction of p21Waf by radiation, which was more pronounced in hsp25 overexpressed cells than control cells, which is associated with increased binding activity of CDK2. S-phase regulator, cyclin-A and its associated CDK2 and CDC2 kinase activities were also increased by irradiation and hsp25 overexpression attenuated these phenomena. In addition, cyclin-B1 expression and its associated kinase activity, which are responsible for the transition of G2 to M phase, were increased by radiation and hsp25 overexpression also decreased these phenomena. CONCLUSION: HSP25 augmented radiation-induced cell-cycle arrest (G1, S, and G2/M phase) may be caused by the HSP25-mediated cell-growth delay and is associated with radioresistance.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/efeitos da radiação , Proteínas de Choque Térmico , Proteínas de Neoplasias/biossíntese , Radiação Ionizante , Animais , Western Blotting , Proteína Quinase CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Ciclina D1/antagonistas & inibidores , Ciclina E/antagonistas & inibidores , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Camundongos , Chaperonas Moleculares , Fosfotransferases/metabolismo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/biossíntese , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Although protein kinase C (PKC) plays an important role in cellular response to radiation, little is known about the specific role of each isoform in the radiation induced cellular response. In this study, the induction of apoptosis and subcellular distribution of PKC isoforms after gamma-ray irradiation were examined in three kinds of mouse epidermal cells with different stages of carcinogenesis (normal mouse keratinocytes, PK: v-rasHa transfected mouse keratinocytes, ras-PK; and neoplastic cells from mouse skin papilloma, 308 cells). The induction of apoptosis was different in normal and neoplastic cells; in normal cells after 16 Gy of radiation, apoptosis was 2-10 times higher than that in ras-PK or 308 cells, and was rapidly induced; other cells died more slowly, depending on the stage of carcinogenesis. The responses of each PKC, especially rapid translocation of PKCdelta and no response of PKCepsilon by radiation in normal cells may influence the induction of apoptosis by radiation.
Assuntos
Apoptose/efeitos da radiação , Transformação Celular Neoplásica , Epiderme/efeitos da radiação , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Animais , Transporte Biológico , Células Epidérmicas , Epiderme/enzimologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Frações Subcelulares/enzimologiaRESUMO
We have examined the effects of ginseng on the induction and repair of gamma-ray-induced DNA double strand breaks (dsb) using neutral filter elution technique at pH 9.6 in cultured murine spleen lymphocytes. Ginseng water extract 500 micrograms/ml was added to the culture medium either for 48 hours prior to irradiation. Ginseng extract showed protective effect against the formation of dsb when it was treated for 48 hours before 100 Gy gamma-ray-irradiation. While repair was almost completed until 220.2 minutes after irradiation, DNA repair of irradiated cells in the presence of ginseng extract was did not return to the corresponding control levels even after 621.8 minutes. From these data, it could be calculated that ginseng reduced the relative strand scission factor (RSSF) by about 2. Therefore, it could be concluded that ginseng has radioprotective effect against gamma-ray induced DNA dsb and repair in cultured mouse lymphocytes.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/efeitos da radiação , Linfócitos/efeitos da radiação , Panax , Plantas Medicinais , Protetores contra Radiação/farmacologia , Animais , Feminino , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Adaptive responses induced by low dose gamma-ray irradiation in human melanoma cells were examined using a clonogenic assay. Survival fractions were significantly increased in cells irradiated with low dose gamma-rays then 4 hrs later with high dose gamma-ray as compared to cells irradiated only with high dose gamma-rays. When low dose irradiation was given 20 hrs prior to high dose irradiation, however, no adaptive response was induced. Changes in protein biosynthesis in human melanoma cells were observed under the same conditions. Significant changes in protein biosynthesis occurred in the nuclear and membrane proteins of cells first irradiated with a low dose then a high dose of gamma-rays after 4 hrs. No such changes were found in cells irradiated with low dose gamma-rays 20 hrs prior to high dose irradiation, consistent with the results of the clonogenic assay. Our findings suggest that prior treatment with low dose gamma-rays induces an adaptive response that has a significant effect on the induction of the nuclear and membrane protein biosynthesis caused by high dose gamma-ray irradiation of human melanoma cells.