RESUMEN
Manganese superoxide dismutase (SOD2)-mediated adaptive processes that protect against radiation-induced micronucleus formation can be induced in cells after a 2-Gy exposure by previously exposing them to either low-dose ionizing radiation (10cGy) or WR1065 (40µM), the active thiol form of amifostine. Although both adaptive processes culminate in elevated levels of SOD2 enzymatic activity, the underlying pathways differ in complexity, with the tumor necrosis factor α (TNFα) signaling pathway implicated in the low-dose radiation-induced response, but not in the thiol-induced pathway. The goal of this study was the characterization of the effects of TNFα receptors 1 and 2 (TNFR1, TNFR2) on the adaptive responses induced by low-dose irradiation or thiol exposure using micronucleus formation as an endpoint. BFS-1 wild-type cells with functional TNFR1 and 2 were exposed 24h before a 2-Gy dose of ionizing radiation to either 10cGy or a 40µM dose of WR1065. BFS2C-SH02 cells, defective in TNFR1, and BFS2C-SH22 cells, defective in both TNFR1 and TNFR2 and generated from BFS2C-SH02 cells by transfection with a murine TNFR2-targeting vector and confirmed to be TNFR2 defective by quantitative PCR, were also exposed under similar conditions for comparison. A 10-cGy dose of radiation induced a significant elevation in SOD2 activity in BFS-1 (P<0.001) and BFS2C-SH02 (P=0.005) but not BFS2C-SH22 cells (P=0.433), compared to their respective untreated controls. In contrast, WR1065 significantly induced elevations in SOD2 activity in all three cell lines (P=0.001, P=0.007, P=0.020, respectively). A significant reduction in the frequency of radiation-induced micronuclei was observed in each cell line when exposure to a 2-Gy challenge dose of radiation occurred during the period of maximal elevation in SOD2 activity. However, this adaptive effect was completely inhibited if the cells were transfected 24h before low-dose radiation or thiol exposure with SOD2 siRNA. Under the conditions tested, TNFR1 and 2 inhibition negatively affected the low-dose radiation-induced but not the thiol-induced adaptive responses observed to be mediated by elevations in SOD2 activity.
Asunto(s)
Mercaptoetilaminas/farmacología , Superóxido Dismutasa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Amifostina/análogos & derivados , Amifostina/química , Animales , Línea Celular Tumoral , Activación Enzimática/genética , Activación Enzimática/efectos de la radiación , Mercaptoetilaminas/química , Ratones , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Pruebas de Micronúcleos , ARN Interferente Pequeño/genética , Radiación Ionizante , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Superóxido Dismutasa/genéticaRESUMEN
Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria wall-lipopolysaccharide (LPS). Endothelial permeability was monitored by changes in transendothelial electrical resistance. Cytoskeletal remodelling and reactive oxygen species (ROS) production was examined by immunofluorescence. Cell signalling was assessed by Western blot. Measurements of Evans blue extravasation, cell count and protein content in bronchoalveolar lavage fluid were used as in vivo parameters of lung vascular permeability. Hydrogen peroxide, LPS and interleukin-6 caused cytoskeletal reorganisation and increased permeability in the pulmonary endothelial cells, reflecting endothelial barrier dysfunction. These disruptive effects were inhibited by pre-treatment with amifostine and linked to the amifostine-mediated abrogation of ROS production and redox-sensitive signalling cascades, including p38, extracellular signal regulated kinase 1/2, mitogen-activated protein kinases and the nuclear factor-kappaB pathway. In vivo, concurrent amifostine administration inhibited LPS-induced oxidative stress and p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and neutrophil recruitment to the lungs. The present study demonstrates, for the first time, protective effects of amifostine against lipopolysaccharide-induced lung vascular leak in vitro and in animal models of lipopolysaccharide-induced acute lung injury.
Asunto(s)
Amifostina/farmacología , Permeabilidad Capilar/efectos de los fármacos , Pulmón/efectos de los fármacos , Protectores contra Radiación/farmacología , Animales , Antioxidantes/metabolismo , Líquido del Lavado Bronquioalveolar , Citoesqueleto/metabolismo , Inflamación , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno , Transducción de SeñalAsunto(s)
Traumatismos por Radiación/etiología , Traumatismos por Radiación/prevención & control , Protección Radiológica/métodos , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/fisiología , Protectores contra Radiación/administración & dosificación , Radioterapia/efectos adversos , HumanosRESUMEN
The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFkappaB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFalpha. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFkappaB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFkappaB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50-p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFkappaB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFkappaB activation by SH. Thus, while oxidative damage is known to activate NFkappaB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFkappaB. There does not appear to be a significant role, if any, for the production of H(2)O(2) as an intermediate step in the activation of NFkappaB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFkappaB. In contrast to TNFalpha, exposure of cells to either 40 microM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.
Asunto(s)
Acetilcisteína/análogos & derivados , Endotelio Vascular/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , FN-kappa B/biosíntesis , Superóxido Dismutasa/biosíntesis , Acetilcisteína/farmacología , Northern Blotting , Catalasa/farmacología , Línea Celular Transformada/efectos de los fármacos , Dimerización , Endotelio Vascular/metabolismo , Inducción Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Intercelular/genética , Mercaptoetilaminas/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/química , FN-kappa B/genética , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Profármacos/metabolismo , Piruvatos/farmacología , ARN Mensajero/biosíntesis , Protectores contra Radiación/farmacología , Piel/irrigación sanguínea , Superóxido Dismutasa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.
Asunto(s)
Glutatión Peroxidasa/fisiología , Animales , Células CHO/enzimología , Células CHO/efectos de la radiación , Bovinos , Línea Celular/enzimología , Línea Celular/efectos de la radiación , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Cricetulus , Citosol/enzimología , ADN Complementario/genética , Inducción Enzimática , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Humanos , Linfocitos/enzimología , Linfocitos/efectos de la radiación , Estrés Oxidativo , Tolerancia a Radiación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/efectos de la radiación , TransfecciónRESUMEN
PURPOSE: This study tests the hypothesis that p53 status, i.e. wild type versus mutant form, is a determinant in radiation protection of human glioma cells by WR-1065, the active thiol form of amifostine (WR-2721). MATERIALS AND METHODS: The cytoprotective effectiveness of WR-1065 when present during irradiation was investigated using four well-characterized human glioma cell lines. The p53 positive lines were U87 and D54, and the mutant p53 lines were U251 (mutant at codon 273; CGT/CAT; Arg/His) and A172 (mutant at codon 242; TGC/TTC; Cys/Phe). Treatment conditions included exposure of cells to a range of doses (0-10Gy) alone or in combination with 4mM of WR-1065 added 30min prior to irradiation. Resultant survival curves were obtained using a clonogenic assay and protection factors, the ratio of terminal slopes +/- WR-1065, were determined for each glioma cell line. RESULTS: The Do values of wild-type U87 and D54 were 1.62 and 1.89Gy while those of p53 mutants U251 and A172 were 1.64 and 1.68 Gy, respectively. Protection factors were determined to be 2.4 and 1.9 for U87 and D54, and 2.6 and 2.8 for U251 and A172, respectively. CONCLUSIONS: The p53 status of the four human glioma cell lines tested was not a predictor for either their relative sensitivity to ionizing radiation or ability to be protected by WR-1065. It is concluded that cytoprotection exhibited by cells exposed to WR-1065 during irradiation is independent of their p53 status.
Asunto(s)
Glioma/tratamiento farmacológico , Glioma/metabolismo , Glioma/radioterapia , Mercaptoetilaminas/farmacología , Protectores contra Radiación/farmacología , Proteína p53 Supresora de Tumor/biosíntesis , Amifostina/farmacología , Supervivencia Celular/efectos de la radiación , Citoprotección , Relación Dosis-Respuesta en la Radiación , Genes p53/genética , Glioma/genética , Humanos , Mutación , Células Tumorales CultivadasRESUMEN
It has recently been proposed that the thiol form of the cytoprotective drug amifostine that is designated WR-1065 [2-((aminopropyl)amino)ethanethiol] exerts its cytoprotective effects in part via a catalytic inhibition of DNA topoisomerase II (topo II)alpha. This in turn leads to the subsequent accumulation of cells in G2 phase and a prolongation of the cell cycle. We have used a Chinese hamster V79 cell-based micronucleus assay to further evaluate this hypothesis. It is demonstrated that WR-1065 strongly inhibits the clastogenesis of the topo II poisons etoposide and clinafloxacin at clinically attained exposure levels while having no effect on clastogenesis induced by topo II-noninteractive chemicals. These findings are consistent with the hypothesis that WR-1065 is a catalytic inhibitor of topo II in mammalian cells. These studies also suggest that WR-1065 might be expected to reduce the toxicity and clastogenicity in clinical applications of etoposide or quinolone antibiotics in dose-limiting normal tissues.
Asunto(s)
Mercaptoetilaminas/farmacología , Protectores contra Radiación/farmacología , Inhibidores de Topoisomerasa II , Animales , Catálisis , Ciclo Celular/efectos de los fármacos , Línea Celular , Cricetinae , Pruebas de MicronúcleosRESUMEN
Amifostine is an important drug in the new field of cytoprotection. It was developed by the Antiradiation Drug Development Program of the US Army Medical Research and Development Command as a radioprotective compound and was the first drug from that Program to be approved for clinical use in the protection of dose limiting normal tissues in patients against the damaging effects of radiation and chemotherapy. Its unique polyamine-like structure and attached sulfhydryl group give it the potential to participate in a range of cellular processes that make it an exciting candidate for use in both cytoprotection and chemoprevention. Amifostine protects against the DNA damaging effects of ionizing radiation and chemotherapy drug associated reactive species. It possesses anti-mutagenic and anti-carcinogenic properties. At the molecular level, it has been demonstrated to affect redox sensitive transcription factors, gene expression, chromatin stability, and enzymatic activity. At the cellular level it has important effects on growth and cell cycle progression. This review focuses on relating its unique chemical design to mechanisms of action that underlie its broad usefulness as both a cytoprotective and chemopreventive agent for use in cancer therapy.
Asunto(s)
Amifostina/farmacología , Anticarcinógenos/farmacología , Protectores contra Radiación/farmacología , Adolescente , Animales , Antineoplásicos/efectos adversos , Cricetinae , Cricetulus , Reparación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Femenino , Humanos , Masculino , Ratones , Medicina Militar , Neoplasias Primarias Secundarias/tratamiento farmacológico , Ratas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Using very low frequency in vivo electron paramagnetic resonance (EPR), we have compared, for the first time, the average microviscosity of the total aqueous compartment of murine fibrosarcomas and that of normal leg tissue in a living animal. EPR spectra from dissolved nitroxide spin probes report the solvent microviscosity. The tumor aqueous microviscosity, 1.8 +/- 0.1 centipoise, was significantly lower than that of the corresponding normal tissue, 2.9 +/- 0.3 centipoise, a difference of 38 +/- 7%. These results confirm the commonly observed increase in the water proton transverse relaxation times (T2) in magnetic resonance imaging of hyperproliferative states, for example, malignancy. The specificity of the localization of the EPR signal indicates a substantial portion of the T2 increase seen in magnetic resonance imaging derives from decreased bulk-water viscosity. The effect of this microviscosity differences may be the basis of several physiological differences between tumors and normal tissues which could confer a growth rate advantage to tumor tissue.
Asunto(s)
Agua Corporal/química , Neoplasias Experimentales/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Ratones , Ratones Endogámicos C3H , Neoplasias Experimentales/fisiopatología , ViscosidadRESUMEN
Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O6-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO(AGT)) and pcDNA3 (CHOpcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO(AGT) is 7- or 20-fold less sensitive to the toxic effects of 30 microM 4-HC or 300 microM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHO(AGT) cells treated with 20 microM 4-HC or 200 microM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 microM acrolein, the cell survival for CHO(AGT) was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10(6) cells in CHOpcDNA3 compared with no mutants in CHO(AGT). In contrast, CHO(AGT) and CHOpcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Supervivencia Celular/efectos de los fármacos , Ciclofosfamida/toxicidad , Transferasas Alquil y Aril/genética , Animales , Células CHO , Supervivencia Celular/efectos de la radiación , Cricetinae , Ciclofosfamida/análogos & derivados , Reparación del ADN , Humanos , Mutágenos/toxicidad , Mostazas de Fosforamida/toxicidad , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
In addition to the cytoprotective benefits of amifostine (Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) to normal cells, it also prevents the induction of somatic mutations that can lead to therapy-induced second cancers. The mutagenic effects of cyclophosphamide, an agent that is known to be mutagenic to normal cells, were determined in mouse splenocytes using a mutational assay system. Cyclophosphamide 100 mg/kg increased mutant frequencies 10-fold. In contrast, amifostine 100 mg/kg, whether administered 30 minutes before or 2 hours after cyclophosphamide administration, resulted in eightfold lowered mutant frequencies. To address potential cytoprotective effects on tumors exposed to this dose, amifostine was administered to tumor-bearing mice either 30 minutes before or 2 hours after the administration of cyclophosphamide. Cyclophosphamide (range, 10 to 100 mg/kg) was administered intraperitoneally into mice 4 days following the injection of 3.5 x 10(5) viable fibrosarcoma (FSa) cells. At this time, microcolonies of FSa tumors containing 50 to 200 cells were present in the lung. The number of FSa lung nodules formed at the end of 14 days in control animals was compared with that of animals treated with cyclophosphamide +/- amifostine. No cytoprotection of murine FSa tumors by amifostine was observed across the entire cyclophosphamide dose range tested, regardless of time of administration, demonstrating the utility of amifostine as a chemopreventive drug under conditions that do not allow cytoprotection for tumor cells.
Asunto(s)
Amifostina/farmacología , Anticarcinógenos/farmacología , Antimutagênicos/farmacología , Antineoplásicos Alquilantes/efectos adversos , Ciclofosfamida/efectos adversos , Citoprotección , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Primarias Secundarias/prevención & control , Sustancias Protectoras/farmacología , Animales , Antineoplásicos Alquilantes/uso terapéutico , Carcinógenos , Ciclofosfamida/uso terapéutico , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Hipoxantina Fosforribosiltransferasa , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C3H , Mutagénesis , Mutágenos , Trasplante de Neoplasias , Neoplasias Primarias Secundarias/inducido químicamente , Bazo/citologíaRESUMEN
Thiol containing compounds exhibiting antioxidant properties are currently being evaluated for use in cytoprotection and chemoprevention. Many of these have also been found to be effective in inhibiting cell cycle progression and cellular proliferation. N-Acetyl-L-cysteine (L-NAC), along with its nonmetabolically active stereoisomer N-acetyl-D-cysteine (D-NAC), together with captopril and dithiothreitol (DTT) were investigated to assess their effects on cell cycle progression as determined by flow cytometry. Topoisomerase-IIa (topo-II alpha) activity, an enzyme involved in DNA synthesis, was also monitored as a function of drug dose using a kinetoplast DNA (kDNA) decatenation assay. Chinese hamster ovary (CHO) AA8 cells were exposed to each thiol at concentrations ranging from 4 microM to 4 mM for a period of 3 h. Following the removal of the thiols, cell cultures were followed for an additional 5 h to assess changes in cell cycle progression. L-NAC, which also serves as a precursor for glutathione (GSH) synthesis, effectively inhibited topo-IIa activity by at least 50% at all concentrations tested. Associated with this reduction in enzyme activity was a sixfold increase in the relative number of cells accumulating in G2phase. D-NAC, which is unable to participate in GSH synthesis, was only half as effective as L-NAC at each concentration tested in inhibiting topo-IIa activity as well as perturbing cell progression through G2. In comparison, captopril, an inhibitor of angiotensin converting enzyme (ACE), had little effect on the progression of cells into G2 phase. In contrast to the repressive effects of L-NAC and D-NAC, it enhanced topo-IIa activity over control values by approximately 20%. DTT, a well characterized thiol known to be capable of reducing disulphides in proteins, was observed to be relatively ineffective in either perturbing cell cycle progression or affecting topo-IIa activity. This suggests an involvement of a mechanism(s) in addition to thiol mediated affects on reduction/oxidation processes. The inhibitory effects of L-NAC and D-NAC on topo-IIa activity, in contrast to the other two thiols, may be due in part to the presence of amine groups which could allow for their participation in polyamine related processes. The difference in the magnitude of the effect exhibited by L-NAC, as compared to D-NAC, on the repression topo-IIa activity also suggests a role for GSH in this process. Inhibition of cellular progression and proliferation by thiols can therefore be mediated by diverse mechanisms which include both cycle-phase specific (i.e. L-NAC and D-NAC) and non cell cycle specific (i.e. captopril) processes.
Asunto(s)
Ciclo Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Compuestos de Sulfhidrilo/farmacología , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antígenos de Neoplasias , Células CHO , Captopril/farmacología , Extractos Celulares , Cricetinae , ADN de Cinetoplasto/metabolismo , Proteínas de Unión al ADN , Ditiotreitol/farmacología , Citometría de Flujo , Isoenzimas/antagonistas & inhibidores , Estereoisomerismo , Factores de Tiempo , Inhibidores de Topoisomerasa IIRESUMEN
The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) II alpha activity, and topo II alpha phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 microM of WR-1065 for 30 min did not effect cell cycle progression nor topo II alpha activity and phosphorylation status. However, concentrations ranging from 4 microM to 4 mM were equally effective in significantly altering these three end points. Cell cycle progression was analysed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G2. Topo II alpha activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 microM to 4 mM dose range tested. Likewise, topo II alpha phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo II alpha activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G2 phase of the cell cycle.
Asunto(s)
Amifostina/metabolismo , Ciclo Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Mercaptoetilaminas/farmacología , Protectores contra Radiación/farmacología , Animales , Antígenos de Neoplasias , Células CHO , Separación Celular , Cricetinae , Proteínas de Unión al ADN , Citometría de Flujo , Fosforilación/efectos de los fármacosRESUMEN
The clinically approved cytoprotector amifostine, designated WR-2721, [S-2-(3-aminopropylamino)ethylphosphorothioic acid], protects against both radiation and drug-induced mutagenesis in animal systems. These effects extend over a wide concentration range making amifostine a strong candidate for evaluation as a possible cancer chemopreventive agent. To better identify and develop potential intermediate biomarkers for chemoprevention at the molecular level we applied the technique of differential display RT-PCR to assess the effects of both the thiol (SH), i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of amifostine on gene expression in CHO-AA8 cells. Cells were exposed to either 40 microM or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in corresponding untreated control cells. One band that showed a differential response was sequenced and was found to have 78% homology with a segment of the human pHL-1 cDNA clone contained in GenBank. This clone contains a COX III mitochondrial DNA insert and two exons of human c-myc. Northern blot analyses were performed by using the cloned human c-myc exon 1 probe to confirm whether c-myc gene expression was affected. Repression of c-myc expression was observed under all of the conditions evaluated. An exposure of cells to 40 microM of the disulfide form of amifostine was the most effective in repressing c-myc, i.e. 27% of control level. A concentration of 4 mM of the disulfide form reduced gene expression to 45% of the control level, while the thiol form was less effective, with 4 mM and 40 microM concentrations reducing c-myc gene expression to 65% and 46% of control levels, respectively.
Asunto(s)
Amifostina/farmacología , Genes myc , Amifostina/química , Animales , Células CHO , Cricetinae , Disulfuros , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Oxidación-Reducción , ARN Mensajero/genética , Compuestos de SulfhidriloRESUMEN
Mice bearing the autosomal recessive mutation 'wasted' (wst/wst) express a disease syndrome characterized by neurologic dysfunction, immunodeficiency, and increased sensitivity to the killing effects of ionizing radiation relative to normal littermates (wst/-) and to parental control mice (BCF1, BALB/c, and C57BL/6). Many of these abnormalities, evident as early as 21 days of age, have been localized to thymic tissues and T-lymphocyte populations. Comparison of two-dimensional gel electrophoresis patterns of proteins from wst/wst and control mouse thymus revealed that an acidic protein with a molecular mass of approximately 30 kDa was consistently expressed at lower levels in wasted mice than in controls. Microsequencing of this protein revealed a sequence of 19 N-terminal amino acids identical to the sequence of murine proliferating cell nuclear antigen (PCNA). Northern blot analyses of PCNA expression in thymus and spleen demonstrated lower accumulation of PCNA-specific transcripts in wasted mice compared with that in controls. Because PCNA expression is associated with cell cycle progression, the percentages of thymic and splenic cells in each stage of the cell cycle were examined; there were no differences in the cell stage distribution of lymphocytes freshly isolated from wasted mice compared with littermate or parental controls. After activation with concanavalin A, however, splenocytes from wst/wst mice showed a lower percentage of cells in S phase compared with that in controls. Southern blots with PCNA probes showed that the PCNA loci from the wasted mice and their normal littermates have the same restriction maps. While differences in polymerase chain reaction (PCR) priming were obtained, these could be attributed to strain-specific differences in mouse PCNA pseudogenes. These results suggest the presence of an alteration in the pathway leading to PCNA expression in radiation-sensitive tissues of wasted mice.
Asunto(s)
Antígeno Nuclear de Célula en Proliferación/biosíntesis , Tolerancia a Radiación/fisiología , Timo/fisiología , Síndrome Debilitante/metabolismo , Animales , Secuencia de Bases , Electroforesis en Gel Bidimensional , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fenotipo , Homología de Secuencia de Ácido Nucleico , Timo/metabolismo , Síndrome Debilitante/genéticaRESUMEN
In order to evaluate the anti-mutagenic effects of the potential chemoprotective compounds selenium and (S)-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-1065), CHO AA8 cells were exposed to both compounds either individually or in combination prior to irradiation. Mutation frequency following exposure to 8 Gy was evaluated by quantitation of the mutations detected at the hprt locus of these cells. Protection against radiation-induced mutation was observed for both 30 nM sodium selenite or 4 mM WR-1065. In addition, the protection against mutation induction provided by the combination of these agents appeared additive. In contrast, sodium selenite did not provide protection against radiation toxicity when provided either alone or in conjunction with WR-1065. In order to evaluate the possible mechanisms of the anti-mutagenic effects observed in these cells, glutathione peroxidase (GPx) activity was evaluated following exposure to the chemopreventative compounds. The addition of sodium selenite to the culture media resulted in a 5-fold increase in GPx activity, which was unaltered by the presence of the WR-1065. Northern analysis of RNA derived from these cells indicated that selenium supplementation resulted in a marginal increase in the mRNA for the cytosolic GPx (GSHPx-1) which was insufficient to account for the stimulation of GPx activity observed in cellular extracts. These results suggest that selenium and WR-1065 offer protection via independent mechanisms and that GPx stimulation remains a possible mechanism of the anti-mutagenic effect of selenium.
Asunto(s)
Mercaptoetilaminas/farmacología , Mutagénesis/efectos de los fármacos , Mutagénesis/efectos de la radiación , Protectores contra Radiación/farmacología , Selenio/farmacología , Animales , Northern Blotting , Células CHO , Células Cultivadas , Cricetinae , Glutatión Peroxidasa/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Pruebas de Mutagenicidad , ARN Mensajero/análisisRESUMEN
To test the hypothesis that attenuation in G2-phase checkpoint control leads to elevated frequencies of unrejoined chromosome breaks in mitosis, the relationship between G2-phase cell cycle checkpoint control and unrejoined chromosome break frequencies after radiation exposure was examined in cells of 10 human tumor cell lines: 8 squamous cell carcinoma cell lines and 2 lymphoblastoid cell lines. Most of the delay in progression through the cell cycle seen in the first cell cycle after radiation exposure in these cell lines was due to blocks in G2 phase, and there were large cell line-dependent variations in the length of the G2-phase block. There was a highly significant inverse correlation between the length of G2-phase delay after radiation exposure and the frequency of induced unrejoined chromosome breaks seen as chromosome terminal deletions in mitosis. This observation supports the hypothesis that the signal for G2-phase delay in mammalian cells is an unrejoined chromosome break and that attenuation of G2-phase checkpoint control allows cells with unrejoined breaks to progress into mitosis. Attenuation in G2-phase checkpoint control was not associated with alterations in the frequency of induced chromosome rearrangements, suggesting that most chromosome rearrangements develop prior to G2 phase, and there was no significant relationship between the length of G2-phase delay and inherent radiation sensitivity, suggesting that unrejoined chromosome breaks are not the primary toxic lesion induced by radiation in mammalian cells.
Asunto(s)
Aberraciones Cromosómicas , Fase G2 , Neoplasias/genética , Tolerancia a Radiación , Genes p53/fisiología , Humanos , Células Tumorales CultivadasRESUMEN
The radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (amifostine; WR-2721) was evaluated for its ability to protect against cyclophosphamide-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in mouse splenocytes under conditions that do not interfere with cyclophosphamide's therapeutic effectiveness against fibrosarcoma lung tumors. Mutations at the HPRT locus increase in frequency as a function of the dose of cyclophosphamide used. With a spontaneous mutation frequency in C3H mice of 1.5 x 10(-6), mutation frequencies increased from 6.2 x 10(-6) to 2.0 x 10(-5) as the cyclophosphamide dose increased from 50 to 200 mg/kg. C3H male mice had 3.5 x 10(5) viable fibrosarcoma cells injected into their tail veins. This resulted in an average of 68 tumor colonies per mouse. Four days following injection, animals received cyclophosphamide 100 mg/kg, which provided significant tumor cell killing and a reduction in tumor colony number to an average of less than one per animal. Amifostine at a concentration of 100 mg/kg did not affect cyclophosphamide's therapeutic efficacy. However, amifostine 100 mg/kg was effective in reducing cyclophosphamide-induced HPRT mutation frequency in mice from 160 to 35 per 10(5) viable cells regardless of whether it was administered 30 minutes before or 2 hours after the cyclophosphamide.
Asunto(s)
Amifostina/uso terapéutico , Ciclofosfamida/efectos adversos , Mutagénesis/efectos de los fármacos , Protectores contra Radiación/uso terapéutico , Animales , Quimioterapia Combinada , Humanos , Masculino , Ratones , Ratones Endogámicos C3HRESUMEN
We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response.