RESUMO
Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Técnicas Citológicas , Luz , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/efeitos da radiação , Animais , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Engenharia Celular/métodos , Dano ao DNA , DNA Ligase Dependente de ATP , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
OBJECTIVE: The objective of this study was to investigate the correlation between in vivo δ-tocotrienol (DT3) pharmacokinetics, pharmacodynamics and radiation protection, and to evaluate the effect of DT3 pre-treatment on radiation-induced alterations in apoptotic and autophagic pathways. METHODS: We evaluated pharmacokinetics (plasma, 0.5 to 12 h) and pharmacodynamics (peripheral blood indices; day 3, 7, 10 and 14) after a single subcutaneous injection of 300 mg kg(-1) DT3 in unirradiated CD2F1 mice. Next, we monitored 30-day post-irradiation survival (9.25 Gy) and haematopoietic recovery of DT3-treated mice (7 Gy) exposed to cobalt-60 γ-irradiation. The effects of DT3 on irradiated bone marrow apoptosis and autophagy were determined by analyses of key caspases (3, 7, 9 and 8), beclin-1 and light chain 3 conversion. RESULTS: Plasma concentration of DT3 reached â¼195 µM (Cmax) 1 h after injection (Tmax), and DT3 was eliminated from plasma 12 h later. In unirradiated mice, DT3 significantly increased white blood cells (WBCs), neutrophils, lymphocytes (day 3 post DT3 injection) and platelets (day 7) by 1.5- to 2-fold, over vehicle-treated control. DT3 pre-treatment improved 30-day survival to 100% (â¼15% in control) and accelerated recovery of reticulocytes, platelets, WBCs, neutrophils, lymphocytes and monocytes in peripheral blood. DT3 reduced activation of caspase-8, caspase-3 and caspase-7, inherent to apoptosis, while increasing autophagy-related beclin-1 expression in irradiated bone marrow. CONCLUSION: These data indicate that DT3 stimulates multilineage haematopoiesis, protects against radiation-induced apoptosis downstream of the mitochondria and stimulates cytoprotective autophagy. Apart from a potent antioxidant activity, DT3 may elicit survival advantage following irradiation by enhancing haematopoiesis and modulating signalling pathways.
Assuntos
Protetores contra Radiação/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vitamina E/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Caspases/metabolismo , Caspases/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Citocromos c/metabolismo , Citocromos c/efeitos da radiação , Contagem de Eritrócitos , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Contagem de Leucócitos , Masculino , Camundongos , Pancitopenia/tratamento farmacológico , Contagem de Plaquetas , Protetores contra Radiação/análise , Protetores contra Radiação/farmacocinética , Vitamina E/sangue , Vitamina E/farmacocinética , Vitamina E/farmacologiaRESUMO
The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Enteropatias/genética , Intestinos/efeitos da radiação , Lesões por Radiação/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Caspases/metabolismo , Caspases/efeitos da radiação , Ciclo Celular/efeitos da radiação , Morte Celular/efeitos da radiação , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Histonas/metabolismo , Histonas/efeitos da radiação , Enteropatias/metabolismo , Intestinos/patologia , Camundongos , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Irradiação Corporal TotalRESUMO
Abstract Recent reports suggest that mobile phone radiation may diminish male fertility. However, the effects of this radiation on human spermatozoa are largely unknown. The present study examined effects of the radiation on induction of apoptosis-related properties in human spermatozoa. Ejaculated, density-purified, highly motile human spermatozoa were exposed to mobile phone radiation at specific absorption rates (SARs) of 2.0 and 5.7 W/kg. At various times after exposure, flow cytometry was used to examine caspase 3 activity, externalization of phosphatidylserine (PS), induction of DNA strand breaks, and generation of reactive oxygen species. Mobile phone radiation had no statistically significant effect on any of the parameters studied. This suggests that the impairment of fertility reported in some studies was not caused by the induction of apoptosis in spermatozoa.
Assuntos
Telefone Celular/estatística & dados numéricos , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Caspase 3/efeitos da radiação , Caspases/metabolismo , Caspases/efeitos da radiação , Fertilidade , Humanos , Infertilidade Masculina/diagnóstico por imagem , Masculino , Estresse Oxidativo , Exposição Paterna , Cintilografia , Espermatozoides/enzimologia , Espermatozoides/fisiologia , Espermatozoides/efeitos da radiaçãoRESUMO
These experiments were designed to investigate transcriptional effects in Atlantic salmon (Salmo salar) after exposure in vivo to ionizing gamma radiation combined with subtoxic levels of aluminum (Al) and cadmium (Cd). Juvenile fish (35 g) in freshwater with or without Al and Cd (255 microg Al/L + 6 microg Cd/L) were exposed to a 75 mGy dose of gamma-irradiation, and induced responses were compared to those of controls. The transcriptional levels of eight genes encoding proteins known to respond to stress in fish were quantified in liver of fish exposed for 5 h to gamma radiation, to Al and Cd or to the combination of Al, Cd and gamma radiation. The studied genes were caspase 3B, caspase 6A, caspase 7, p53 (apoptosis), glutathione reductase (GR), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), (oxidative stress), metallothionein (MT-A) (metal stress) and ubiquitin (Ubi) (protein degradation). The results showed that gamma-irradiation alone induced significant upregulation of caspase 6A, GR, GSH-Px, MT-A and Ubi compared to the control group, while 5 h exposure to Al+Cd alone did not induce any of the studied genes compared to the control. No significant upregulation of the series of investigated genes could be observed in fish exposed to gamma-irradiation in combination with Al+Cl. In conclusion, the results suggest that the presence of Al+Cd in the water counteracted the gamma-irradiation effect by modifying the transcription of genes encoding proteins involved in the defense mechanisms against free radicals in the cells.
Assuntos
Raios gama , Metais/farmacologia , Salmo salar/fisiologia , Alumínio/toxicidade , Animais , Cádmio/toxicidade , Caspases/efeitos dos fármacos , Caspases/genética , Caspases/efeitos da radiação , Radioisótopos de Cobalto/farmacologia , Dano ao DNA , Exposição Ambiental , Água Doce , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Metalotioneína/efeitos dos fármacos , Metalotioneína/genética , Metalotioneína/efeitos da radiação , Reação em Cadeia da Polimerase/métodos , Proteínas/efeitos dos fármacos , Proteínas/genética , Proteínas/efeitos da radiação , RNA/sangue , RNA/efeitos dos fármacos , RNA/genética , Salmo salar/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Ubiquitina/efeitos dos fármacos , Ubiquitina/genética , Ubiquitina/efeitos da radiaçãoRESUMO
Glioblastoma is the most common astrocytic brain tumor in humans. Current therapies for this malignancy are mostly ineffective. Photodynamic therapy (PDT), an exciting treatment strategy based on activation of a photosensitizer, has not yet been extensively explored for treating glioblastoma. We used 5-aminolevulinic acid (5-ALA) as a photosensitizer for PDT to induce apoptosis in human malignant glioblastoma U87MG cells and to understand the underlying molecular mechanisms. Trypan blue dye exclusion test showed a decrease in cell viability after exposure to increasing doses of 5-ALA for 4h followed by PDT with a broad spectrum blue light (400-550 nm) at a dose of 18J/cm(2) for 1h and then incubation at 37 degrees C for 4h. Following 0.5 and 1mM 5-ALA-based PDT (5-ALA-PDT), Wright staining and ApopTag assay showed occurrence of apoptosis morphologically and biochemically, respectively. After 5-ALA-PDT, down regulation of nuclear factor kappa B (NFkappaB) and baculovirus inhibitor-of-apoptosis repeat containing-3 (BIRC-3) protein indicated inhibition of survival signals. Besides, 5-ALA-PDT caused increase in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF). Activation of calpain, caspase-9, and caspase-3 occurred in course of apoptosis. Calpain and caspase-3 activities cleaved alpha-spectrin at specific sites generating 145kD spectrin breakdown product (SBDP) and 120kD SBDP, respectively. The results suggested that 5-ALA-PDT induced apoptosis in U87MG cells by suppression of survival signals and activation of proteolytic pathways. Thus, 5-ALA-PDT can be an effective strategy for inducing apoptosis in glioblastoma.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Peptídeo Hidrolases/efeitos dos fármacos , Fotoquimioterapia/métodos , Ácido Aminolevulínico/química , Ácido Aminolevulínico/uso terapêutico , Apoptose/fisiologia , Apoptose/efeitos da radiação , Fator de Indução de Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Fator de Indução de Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/efeitos da radiação , Proteína 3 com Repetições IAP de Baculovírus , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatologia , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Calpaína/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Glioblastoma/metabolismo , Glioblastoma/fisiopatologia , Humanos , Proteínas Inibidoras de Apoptose/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose/efeitos da radiação , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , NF-kappa B/efeitos da radiação , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/efeitos da radiação , Ubiquitina-Proteína LigasesRESUMO
Stress-induced heat shock protein 70 (Hsp70) effectively protects cells against apoptosis, although the anti-apoptotic mechanism is still undefined. Exposure of human melanocytes to heat and subsequent UVB irradiation increased the level of Hsp70 and pre-heating reduced UVB induced apoptosis. Immunofluorescence staining of Hsp70 in combination with staining of lysosomes (Lamp2) or mitochondria (Mitotracker) in pre-heated UVB exposed cells showed co-localization of Hsp70 with both lysosomes and mitochondria in the surviving cell population. Furthermore, UVB induced apoptosis was accompanied by lysosomal and mitochondrial membrane permeabilization, detected as release of cathepsin D and cytochrome c, respectively, which were prevented by heat pre-treatment. In purified fractions of lysosomes and mitochondria, recombinant Hsp70 attached to both lysosomal and mitochondrial membranes. Moreover, in apoptotic cells Bax was translocated from a diffuse cytosolic location into punctate mitochondrial-like structures, which was inhibited by Hsp70 induction. Such inhibition of Bax translocation was abolished by transfection with Hsp70 siRNA. Furthermore, Hsp70 siRNA eliminated the apoptosis preventive effect observed after pre-heating. These findings show Hsp70 to rescue melanocytes from UVB induced apoptosis by preventing release of cathepsins from lysosomes, Bax translocation and cytochrome c release from mitochondria.
Assuntos
Apoptose/efeitos da radiação , Catepsinas/metabolismo , Citocromos c/metabolismo , Proteínas de Choque Térmico HSP70/fisiologia , Melanócitos/fisiologia , Animais , Caspases/metabolismo , Caspases/efeitos da radiação , Catepsinas/efeitos da radiação , Ativação Enzimática , Temperatura Alta , Humanos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/efeitos da radiação , Fígado/fisiologia , Lisossomos/fisiologia , Lisossomos/efeitos da radiação , Melanócitos/efeitos da radiação , Mitocôndrias/efeitos da radiação , Mitocôndrias Hepáticas/fisiologia , Ratos , Proteínas Recombinantes/metabolismo , Pele/efeitos da radiação , Raios UltravioletaRESUMO
OBJECTIVE: To investigate the effect of X-rays on expression of caspase-3 and p53 protein in EL-4 cells and its implications in induction of apoptosis and polyploid cells. METHODS: Mouse lymphoma cell line (EL-4 cells) was used. Fluorescent staining and flow cytometry analysis were employed for measurement of protein expression, apoptosis, cell cycle, and polyploid cells. RESULTS: The expression of caspase-3 protein increased significantly at 8 h and 12 h, compared with that of sham-irradiated control (P<0.05, respectively) and the expression of p53 protein increased significantly at 2, 4, 8, 12, and 24 h, compared with that of sham-irradiated control (P<0.05-P<0.01) in EL-4 cells after 4.0 Gy X-irradiation. Apoptosis of EL-4 cells was increased significantly at 2, 4, 8, 12, 24, 48, and 72 h after 4.0 Gy exposure, compared with that of sham-irradiated control (P<0.05-P<0.001). G2 phase cells were increased significantly at 4, 8, 12, 24, 48, and 72 h (P<0.05-P<0.001). However, no marked change in the number of 8 C polyploid cells was found from 2 to 48 h after 4.0 Gy exposure. CONCLUSION: The expressions of caspase-3 and p53 protein in EL-4 cells are induced by X-rays, which might play an important role in the induction of apoptosis, and the molecular pathway for polyploid formation might be p53-independent.
Assuntos
Caspase 3/efeitos da radiação , Caspases/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos da radiação , Animais , Caspase 3/metabolismo , Caspases/metabolismo , Caspases Iniciadoras , Linhagem Celular Tumoral , Camundongos , Proteína Supressora de Tumor p53/metabolismo , Raios XRESUMO
Malignant gliomas are almost uniformly fatal and display exquisite radiation resistance. Glioma cells lacking wild-type (WT) p53 function are more susceptible to radiation-induced apoptosis than their isogenic counterparts expressing WT p53. We explored the mechanisms of such apoptosis and found that, in the absence of WT p53, radiation increases caspase-8 expression and activity. Inhibition of caspase-8 expression using caspase-8 antisense or small interfering RNA (siRNA) oligonucleotides partially blocks radiation-induced apoptosis. In contrast, inhibition of the mitochondrial death pathway by expression of Bcl-2 has no effect on radiation-induced caspase-8 activity or apoptosis. Our data indicate that, in contrast to commonly accepted models of p53-dependent radiation-induced apoptosis, in our cell system, radiation relies on caspase-8 activity to help mediate p53-independent cell death. In a system of inducible E2F1 activity, E2F1 activated caspase-8 and, accordingly, decreased cellular viability, effects that were abolished by caspase-8 siRNA. In this model, in the absence of WT p53, p21Cip1 is not induced, and E2F1 activity is sustained and allows transcription and activation of caspase-8. This model may explain why p53 mutations in adult gliomas paradoxically correlate with improved survival and enhanced response to radiation.
Assuntos
Apoptose/efeitos da radiação , Caspases/fisiologia , Glioma/enzimologia , Apoptose/fisiologia , Caspase 8 , Caspases/biossíntese , Caspases/genética , Caspases/efeitos da radiação , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator de Transcrição E2F1/metabolismo , Indução Enzimática/efeitos da radiação , Glioma/patologia , Glioma/radioterapia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transcrição Gênica/efeitos da radiação , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Two tetrasubstituted (RLP024 and RLP040) and one monosubstituted (MRLP101) Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse embryo fibroblasts and HaCaT human keratinocytes) upon 1 h-incubation with 0.5-5 microM phthalocyanine concentrations. The affinity was markedly larger for the tetra- as compared with the mono-substituted phthalocyanine, even though smaller phthalocyanine amounts were generally recovered from keratinocytes. As a consequence, the two tetra-substituted phthalocyanines exhibited a higher phototoxicity against all the three cell lines. Typically, the cell survival decreased by at least 80% after 1 min irradiation with 600-700 nm light at a fluence-rate of 50 mW/cm2 in the presence of 5 microM phthalocyanine. Fluorescence microscopy and caspase-3 activation studies indicate that cell death of fibroblasts largely occurred by a random-necrotic process while the keratinocytes underwent cell death predominantly via apoptosis in spite of a very similar pattern of subcellular distribution of the phthalocyanines.
Assuntos
Fibroblastos/efeitos dos fármacos , Indóis/uso terapêutico , Queratinócitos/efeitos dos fármacos , Compostos Organometálicos/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Dermatopatias/radioterapia , Células 3T3 , Animais , Benzimidazóis/farmacologia , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Linhagem Celular , Fibroblastos/efeitos da radiação , Humanos , Isoindóis , Queratinócitos/efeitos da radiação , Camundongos , Microscopia de Fluorescência , Radiossensibilizantes/farmacologia , Relação Estrutura-Atividade , Compostos de ZincoRESUMO
The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m(2) UVB and 100 J/m(2) UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.
Assuntos
Apoptose/efeitos da radiação , Queratinócitos/citologia , Queratinócitos/efeitos da radiação , Dímeros de Pirimidina/análise , Raios Ultravioleta , Proteínas Reguladoras de Apoptose , Caspases/efeitos da radiação , Linhagem Celular , Citocromos c/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
The organic thiophosphate, amifostine, is a promising pharmacological compound showing selective protection in many tissues against the toxic side-effects of radiation and cytotoxic drugs. The aim of the present study was to assess the radioprotective effects of amifostine on ovarian follicles. Three-week-old female mice, with or without pretreatment with amifostine, were irradiated with 6.42 Gy of gamma-ray. Reduced proliferation of granulosa cells was verified with BrdU staining and the incidences of follicular degeneration increased in ovarian follicles in the gamma-ray-irradiated mice compared to that of the control or amifostine-treated group. Biochemical changes caused by gamma-irradiation provoked a rise of p53 and Bax protein and a decline of the inactive form in caspase-3 and PARP protein. Caspase-3 and PARP cleaved into active peptides during apoptosis. This process was confirmed by the result of this study, which was that the amount of the stable form decreased immediately after irradiation. In the amifostine treatment group before irradiation, the increased rate of p53 and Bax was suppressed, particularly in the LDs-treated group. The relationship between PARP and caspase-3 levels showed the effect of amifostine exposure before irradiation. In conclusion, amifostine had an inhibitory effect on ovarian programmed cell death induced by gamma-ray, affecting the expression of apoptotic signaling molecules and the level of proliferation of the granulosa cells.
Assuntos
Amifostina/farmacologia , Ovário/efeitos da radiação , Protetores contra Radiação/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Bromodesoxiuridina , Caspase 3 , Caspases/metabolismo , Caspases/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Feminino , Raios gama , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/efeitos da radiação , Camundongos , Camundongos Endogâmicos ICR , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/efeitos da radiação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Maturidade Sexual , Proteína Supressora de Tumor p53/efeitos da radiação , Proteína X Associada a bcl-2RESUMO
As components of the apoptosome, a caspase-activating complex, cytochrome c (Cyt c) and Apaf-1 are thought to play critical roles during apoptosis. Due to the obligate function of Cyt c in electron transport, its requirement for apoptosis in animals has been difficult to establish. We generated "knockin" mice expressing a mutant Cyt c (KA allele), which retains normal electron transfer function but fails to activate Apaf-1. Most KA/KA mice displayed embryonic or perinatal lethality caused by defects in the central nervous system, and surviving mice exhibited impaired lymphocyte homeostasis. Although fibroblasts from the KA/KA mice were resistant to apoptosis, their thymocytes were markedly more sensitive to death stimuli than Apaf-1(-/-) thymocytes. Upon treatment with gamma irradiation, procaspases were efficiently activated in apoptotic KA/KA thymocytes, but Apaf-1 oligomerization was not observed. These studies indicate the existence of a Cyt c- and apoptosome-independent but Apaf-1-dependent mechanism(s) for caspase activation.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Citocromos c/genética , Ativação Enzimática/fisiologia , Proteínas/metabolismo , Animais , Apoptose/efeitos da radiação , Fator Apoptótico 1 Ativador de Proteases , Caspase 1 , Caspases/efeitos da radiação , Transporte de Elétrons/fisiologia , Transporte de Elétrons/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/efeitos da radiação , Raios gama , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Linfócitos T/metabolismoRESUMO
Inorganic arsenic is an environmental toxin and a human carcinogen. Being a co-mutagen, arsenic enhances carcinogenesis of ultraviolet irradiation on the mouse skin. Apoptosis, a well-regulated cell death process, is essential for cell development and tissue homeostasis. Dysregulation of apoptosis will lead to various kinds of pathological conditions, such as cancers. The purpose of this study is to investigate the apoptotic effect induced by the interactions of arsenic and UVB on cultured human keratinocytes. Cultured keratinocytes were treated with sodium arsenite (1 microM) and/or UVB 50 mJ/cm2 irradiation in different combinations, including arsenic alone (As group), UVB alone (UVB group), arsenic followed by UVB (As/UVB group), and UVB followed by As (UVB/As group) treatments. Our results revealed that a low concentration of sodium arsenite did not induce keratinocytes apoptosis. The UVB group showed obvious elevation of caspase-8, -9, and -3 activities in addition to strong induction of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling (TUNEL) assay. Similar pro-apoptotic effects were observed in the UVB/As group. In contrast, only subtle changes of cell morphology and survival rate were noticed in the As/UVB group. In addition, the results of Western blot and activity assay of caspase-8, -9, and -3 revealed that neither the receptor nor the mitochondrial apoptotic signaling pathway was activated in the As/UVB group. Therefore, we conclude that the pretreatment of keratinocytes with sodium arsenite decreased the pro-apoptotic effects induced by UVB. This finding corroborated with the animal model studying the effects of arsenic and UVB on carcinogenesis. The molecular mechanisms by which arsenic decreased UVB-induced apoptosis remain to be elucidated.
Assuntos
Apoptose , Arsenitos/toxicidade , Queratinócitos , Compostos de Sódio/toxicidade , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Western Blotting , Carcinógenos/toxicidade , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Células Cultivadas , Cocarcinogênese , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Humanos , Marcação In Situ das Extremidades Cortadas , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiaçãoRESUMO
Despite significant evidence of a role of hypoxia in cellular resistance to ionizing radiation-induced toxicity, the underlying molecular mechanisms remain unclear. This study focused on the influence of hypoxia on radiation-induced signals in TK6 human lymphoblastoid cells. Hypoxic (<10 ppm oxygen) and aerobic cells were exposed to equilethal doses of ionizing radiation, radiation dose ratio, 3:1 (hypoxia:air). Hypoxia alone or radiation treatment under aerobic or hypoxic conditions led to increased levels of phospho-p44/42 mitogen-activated protein kinase. Levels of phospho-p38 mitogen-activated protein kinase did not change as a result of either hypoxia or irradiation. Hypoxia alone had no effect on expression of phospho-stress-activated protein kinase (SAPK), wild-type p53, or cleaved caspase 3. Irradiation under aerobic conditions resulted in an increase in the phospho-SAPK signal, whereas hypoxia suppressed the irradiation-induced increase in the level of phospho-SAPK. Both hypoxic and aerobic cells showed increases in p53 levels in response to radiation. Hypoxia blocked radiation-induced cleavage of caspase 3 and poly-ADP-ribose polymerase. Irradiation of aerobic and hypoxic TK6 cells using 6 and 18 Gy, respectively, resulted in a similar and significant increase in fraction of apoptotic cells within 24 hours postirradiation. In contrast, basal levels of apoptosis were observed at 24 hours postirradiation in aerobic and hypoxic NH32 cells, a p53 null derivative of TK6 cells. These results suggest that radiation-induced apoptosis under hypoxia occurs independent of phospho-SAPK and caspase 3, and the p53 response is an obligatory apoptotic signal in TK6 cells.
Assuntos
Caspases/metabolismo , Linfócitos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Aerobiose , Apoptose/fisiologia , Apoptose/efeitos da radiação , Caspase 3 , Caspases/efeitos da radiação , Hipóxia Celular/fisiologia , Linhagem Celular , Citometria de Fluxo , Humanos , Linfócitos/enzimologia , Linfócitos/efeitos da radiação , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/efeitos da radiação , Proteína Quinase 3 Ativada por Mitógeno/efeitos da radiação , Fosforilação/efeitos da radiação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing wound etc. Yet, the mechanism of LPLI remains unclear. In order to determine the effects of high fluence LPLI on cell growth and caspase-3 activity, we have measured the dynamics of caspase-3 activity during cell apoptosis induced by high fluence LPLI treatment. STUDY DESIGN/MATERIALS AND METHODS: He-Ne laser was used to irradiate human lung adenocarcinoma cells (ASTC-a-1). Cell Counting Kit-8 was used for cytotoxicity assay. A fluorescent microscope was used to perform fluorescence resonance energy transfer (FRET) imaging. A luminescence spectrometer was used to acquire the fluorescent emission spectrum. Statistical analysis was performed with Student's paired t-test. RESULTS: Cytotoxicity assay showed that when light irradiation fluence exceeded 60 J/cm2, LPLI treatment induced ASTC-a-1 cell apoptosis in a fluence-dependent manner. FRET imaging and spectrofluorometric analysis demonstrated that caspase-3 was activated during high fluence LPLI-induced cell apoptosis. CONCLUSIONS: Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.
Assuntos
Apoptose/fisiologia , Apoptose/efeitos da radiação , Caspases/metabolismo , Caspases/efeitos da radiação , Terapia com Luz de Baixa Intensidade , Adenocarcinoma , Caspase 3 , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias PulmonaresRESUMO
Exposure to ultraviolet radiation (UVR) and reactive oxygen species (ROS) can damage the human lens and contribute to cataract formation. Recent evidence suggests that apoptosis in lens epithelial cells (LEC) is an initiating event in noncongenital cataract formation in humans and animals. The present study examines the cellular and molecular mechanisms by which environmental (ultraviolet B [UVB]) and chemical (hydrogen peroxide [H(2)O(2)], t-butyl hydroperoxide [TBHP]) stress induces cell death in an SV-40 immortalized human lens epithelial (HLE) cell line. Treatment of HLE cells with UVB, H(2)O(2), and TBHP significantly decreased cell density with LD50 values of 350 J/m(2), 500 muM, and 200 muM, respectively. Cellular morphology, DNA fragmentation, and annexin/propidium iodide staining consistent with apoptosis was observed only in UVB-treated cells, whereas lactate dehydrogenase (LDH) release was significantly higher in H(2)0(2)- and TBHP-treated cells. In addition, activation of apoptotic stress-signaling proteins, including c-Jun NH2-terminal kinase (JNK), caspase-3, and DNA fragmentation factor 45 (DFF45) was observed only in UVB-treated cells. Inhibition of JNK activity increased UVB-induced cell death, suggesting that this pathway may serve a prosurvival role in HLE cells. These findings suggest UVB predominantly induces apoptosis in HLE cells, whereas H(2)O(2) and TBHP induce necrosis.
Assuntos
Apoptose/efeitos da radiação , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Cristalino/citologia , Cristalino/efeitos da radiação , Estresse Oxidativo , Anexinas/metabolismo , Apoptose/efeitos dos fármacos , Caspases/efeitos da radiação , Contagem de Células , Linhagem Celular Transformada , Epitélio/efeitos dos fármacos , Epitélio/efeitos da radiação , Epitélio/ultraestrutura , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/efeitos da radiação , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Cristalino/efeitos dos fármacos , Dose Letal Mediana , Necrose , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Raios Ultravioleta , terc-Butil Hidroperóxido/farmacologiaRESUMO
OBJECTIVE: To discuss the expression and significance of caspase-3 gene in the apoptotic muscle cells in gamma-radiation-induced muscle cell lines. METHODS: The caspase-3 mRNA in the control and gamma-radiation induced apoptotic muscle cells was analysed by RT-PCR. RESULTS: The expression of caspase-3 gene transcript was higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct, and apoptotic muscle cells were higher in 103Pd radioactive stent dog bile duct than in general stent dog bile duct. CONCLUSIONS: The high level expression of caspase-3 gene may help to understand the muscle cells sensitivity to gamma-radiation apoptosis. 103Pd radioactive stent may increase the expression of caspase-3 gene in dog bile duct and prevent the billiary narrow when dog bile duct was injured by balloon.
Assuntos
Apoptose/efeitos da radiação , Ductos Biliares/efeitos da radiação , Caspases/efeitos da radiação , Miócitos de Músculo Liso/efeitos da radiação , Paládio/administração & dosagem , Radioisótopos/administração & dosagem , Stents , Animais , Proteínas Reguladoras de Apoptose , Ductos Biliares/enzimologia , Caspase 3 , Caspases/genética , Cães , Miócitos de Músculo Liso/citologia , RNA Mensageiro/genética , RNA Mensageiro/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although arsenic and ultraviolet light B (UVB) are both causes for skin cancers, lesions of arsenic-induced Bowen's disease are often confined to sun-protected skin. UVB may play a modulatory role in skin carcinogenesis by arsenic. The purpose of this study was to evaluate the effects and interactions of arsenic and UVB on cell cycle progression and apoptosis. Cultured human keratinocytes were treated with sodium arsenite (1 microM) and/or UVB (50 mJ/cm(2)) irradiation in different combinations: (i) arsenic alone, (ii) UVB alone, (iii) arsenic followed by UVB (As-UVB), and (iv) UVB followed by arsenic (UVB-As) treatments. Cell cycle analysis and BrdU pulsing revealed S phase arrest in all treatment groups and growth arrest in As-UVB and UVB-As groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling assay showed a higher apoptosis rate in the UVB-As group as compared to that of the As-UVB and UVB groups. UVB irradiation significantly decreased Bcl-2 expression. In either the As-UVB or the UVB-As group, the expression of Bcl-2 was further suppressed as compared to the UVB group. The caspase-3, -8, and -9 relative activities were all increased in the UVB group; however, arsenic significantly enhanced caspase-8 and -3 relative activities in UVB-irradiated keratinocytes (the UVB-As group). Pretreatment with the caspase inhibitor(s) rescued the keratinocytes viability to different degrees with the least in the UVB-As group. Our findings revealed that arsenic enhances UVB-induced keratinocyte apoptosis via suppression of Bcl-2 expression and stimulation of caspase-8 activity. Combined UVB and arsenic treatment resulted in the antiproliferative and proapoptotic effects in keratinocytes. Our results provide the explanation for the rare occurrences of arsenical cancers in the sun-exposed skin and the potential therapeutic role of UVB in arsenic-induced Bowen's disease.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Arsênio/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Raios Ultravioleta , Caspases/efeitos dos fármacos , Caspases/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos da radiação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Humanos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos da radiação , Proteína X Associada a bcl-2RESUMO
Using primary cultures of neural precursor cells of cortex from developing rat brain, we demonstrated the involvement of caspase-3 in the apoptotic process induced by gamma irradiation. The precursor nature of cells was confirmed by nestin and GFAP immunoreactivity and by the capacity of differentiation in neuronal and glial cells after 5 days in culture. Neural precursors were irradiated with single doses ranging from 0.1 to 4Gy. Cellular death, determined 24 h post-irradiation (pi) was dose-dependent and the induction of apoptosis was confirmed by nuclear condensation, DNA fragmentation and hypodiploid DNA peak represented by the "sub G1" region. For the higher doses, apoptosis was evident after 4-6 h pi and increased during 24 h. Caspase-3 activity increased with doses and was maximal at 4-6 h pi with 3Gy and remained similar with 4Gy. The protection from radiation-induced apoptosis by caspase-3 inhibitor, zDEVD-fmk, confirmed that this enzyme is involved in the apoptotic mechanism in this system. The possibility of using this tissue culture system for studying the effects of ionizing radiation on morphological and molecular differentiation was considered.