RESUMO
Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.
Assuntos
Apoptose , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteína bcl-X/fisiologia , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/farmacologia , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína bcl-X/genéticaRESUMO
Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak-/-Bax-/- haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak-/-Bax-/- cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.
Assuntos
Retículo Endoplasmático/metabolismo , Metaboloma , Estresse Fisiológico , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Imageamento por Ressonância Magnética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Tunicamicina/toxicidade , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Numerous reports have raised the level of national concern that chemicals found in the environment may have adverse effects on the health of humans and wildlife. Environmental exposure to pollutants, such as dioxin, has been implicated in gonadal tumor formation in Maine softshell clams (Mya arenaria). Prevalence of these tumors is as high as 40% in some populations. Although their etiology is still unknown, investigations into the mechanisms of tumor formation have revolved around a hypothesis of dioxin-induced toxicity. The aryl hydrocarbon receptor (AHR) was initially investigated, but was later determined to not bind the prototypical ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suggesting that dioxin toxicity is mediated through an AHR-independent pathway. An alternative mechanism of tumor formation has been investigated, involving a protein with significant sequence similarity to mammalian E6AP, a HECT (homologous to E6AP carboxy terminus) E3 ubiquitin-protein ligase. E6AP, in association with the high-risk human papillomavirus (HPV) E6 protein, is involved in the abnormal degradation of the p53 tumor suppressor protein in human cervical cancer. Tumorigenic clam reproductive tissue revealed higher M. arenaria E3 (MaE3) protein levels concomitant with lower M. arenaria p53 (Map53) levels. While the function of MaE3 as a HECT E3 was verified, results from three methods agree that MaE3 does not associate with Map53. However, alteration in Map53 levels may still play a role in clam gonadal tumorigenesis. Due to upregulation of MaE3 in neoplastic reproductive tissue, further investigations will focus on determining the proteolytic targets of MaE3. In conjunction with our previous findings that dioxin toxicity in the softshell clam is not mediated by AHR, the results from our current investigation suggest a complex etiology for the clam germinomas.
Assuntos
Bivalves/genética , Genes p53/efeitos dos fármacos , Proteínas Repressoras , Ubiquitina-Proteína Ligases/farmacologia , Animais , Feminino , Gônadas , Masculino , Mutagênese Sítio-Dirigida , Neoplasias/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Bcl-2 proteins are major regulators of cellular responses to intrinsic and extrinsic apoptotic stimuli. Among them, overexpression of the antiapoptotic protein Bcl-x(L) modulates intracellular Ca(2+) homeostasis and organelle-specific apoptotic signaling pathways. However, the specific activities of Bcl-x(L) at mitochondria and the endoplasmic reticulum (ER) have not been fully defined. To further explore this, we generated mouse embryonic fibroblast (MEF) cell lines deficient in Bcl-x(L) expression (Bcl-x-KO). Deficiency in Bcl-x(L) expression did not induce compensatory changes in the expression of other Bcl-2 proteins, and Bcl-x-KO MEF cells showed increased sensitivity to various apoptotic stimuli compared with wild-type MEF cells. Targeting Bcl-x(L) at mitochondria but not at the ER restored apoptosis protection in Bcl-x-KO MEF cells to the degree observed in wild-type MEF cells. However, expression of ER-targeted Bcl-x(L) but not mitochondrially targeted Bcl-x(L) was required to restore Ca(2+) homeostasis in Bcl-x-KO MEF cells. Of importance, ER-targeted Bcl-x(L) was able to protect cells against death stimuli in the presence of endogenous Bcl-x(L). These data indicate that mitochondrial Bcl-x(L) can regulate apoptosis independently of ER Bcl-x(L) and that when localized exclusively at the ER, Bcl-x(L) impinges on Ca(2+) homeostasis but does not affect apoptosis unless Bcl-x(L) is present in additional cellular compartments.
Assuntos
Apoptose , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteína bcl-X/metabolismo , Animais , Caspases/metabolismo , Sobrevivência Celular , Células Cultivadas , Expressão Gênica , Perfilação da Expressão Gênica , Homeostase , Humanos , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteína bcl-X/genéticaRESUMO
Many types of cancer cells possess the ability to evade apoptosis, leading to their rapid and uncontrolled proliferation. As major regulators of apoptosis, Bcl-2 proteins serve as emerging targets for novel chemotherapeutic strategies. In this study, we examined the involvement of Bcl-2 proteins in apoptosis induced by the chemotherapeutic agent actinomycin D. A dramatic decrease in anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) mRNA and protein expression was detected upon actinomycin D treatment. Further, Mcl-l over-expression caused resistance to cell death upon treatment with actinomycin D, implicating a role for the down-regulation of Mcl-1 in actinomycin D-induced apoptosis. We also explored the therapeutic potential of actinomycin D in combination with ABT-737, an experimental agent that inhibits anti-apoptotic Bcl-2 proteins. Actinomycin D sensitized cells to ABT-737 treatment in a Bak- or Bax-dependent manner. Importantly, low concentrations of actinomycin D and ABT-737 were more effective in inducing cell death in transformed cells than their untransformed counterparts. A synergistic effect of actinomycin D and ABT-737 on cell death was observed in several human tumor cell lines. Like actinomycin D treatment, knocking down Mcl-1 expression greatly sensitized tumor cells to ABT-737, and Mcl-1 over-expression abrogated the cytotoxic effect induced by ABT-737 and actinomycin D. These results suggest that the down-regulation of Mcl-1 by actinomycin D is likely responsible for the observed synergistic effect between the two drugs. Overall, our studies provide compelling evidence that the combination of actinomycin D and ABT-737 may lead to an effective cancer treatment strategy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose , Compostos de Bifenilo/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Dactinomicina/farmacologia , Nitrofenóis/farmacologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Expressão Gênica , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fragmentos de Peptídeos , Piperazinas/farmacologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismoRESUMO
A high prevalence of germinomas has been observed in certain populations of Mya arenaria from eastern Maine. The etiology of these tumors is unknown. We are investigating the hypothesis that exposure to environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contributes to gonadal carcinogenesis. Clams were exposed to TCDD with or without the initiating compound diethylnitrosamine (DEN) in an attempt to induce germinomas. A TCDD-dependent alteration in gametogenesis was observed in which 32.5+/-6.5% of individuals exhibited undifferentiated gonads. Analyses of AhR and p53 expression were carried out to identify similarities between naturally occurring neoplastic and TCDD (+/-DEN)-altered reproductive tissues. Neoplastic tissues had significantly less p53 protein than matched controls, whereas TCDD-induced undifferentiated samples exhibited no difference in p53 protein levels compared to controls. No gender-specific differences were observed in AhR mRNA, but there were significant differences in protein levels. AhR was undetectable in male gonadal tissue whereas females exhibited a significant positive relationship between AhR protein levels and stage of ovogenesis. Despite exhibiting some morphological similarity, we conclude the TCDD-induced pathology is not a germinoma. We further suggest the change in reproductive tissue is due to inhibition of cell differentiation and/or development by an AhR-independent mechanism.