RESUMO
The envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) can induce apoptosis by a cornucopia of distinct mechanisms. A soluble Env derivative, gp120, can kill cells through signals that are transmitted by chemokine receptors such as CXCR4. Cell surface-bound Env (gp120/gp41), as present on the plasma membrane of HIV-1-infected cells, can kill uninfected bystander cells expressing CD4 and CXCR4 (or similar chemokine receptors, depending on the Env variant) by at least three different mechanisms. First, a transient interaction involving the exchange of lipids between the two interacting cells ('the kiss of death') may lead to the selective death of single CD4-expressing target cells. Second, fusion of the interacting cells may lead to the formation of syncytia which then succumb to apoptosis in a complex pathway involving the activation of several kinases (cyclin-dependent kinase-1, Cdk1; checkpoint kinase-2, Chk2; mammalian target of rapamycin, mTOR; p38 mitogen-activated protein kinase, p38 MAPK; inhibitor of NF-kappaB kinase, IKK), as well as the activation of several transcription factors (NF-kappaB, p53), finally resulting in the activation of the mitochondrial pathway of apoptosis. Third, if the Env-expressing cell is at an early stage of imminent apoptosis, its fusion with a CD4-expressing target cell can precipitate the death of both cells, through a process that may be considered as contagious apoptosis and which does not involve Cdk1, mTOR, p38 nor p53, yet does involve mitochondria. Activation of some of the above- mentioned lethal signal transducers have been detected in patients' tissues, suggesting that HIV-1 may indeed trigger apoptosis through molecules whose implication in Env-induced killing has initially been discovered in vitro.
Assuntos
Apoptose , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1 , Receptores de Quimiocinas/efeitos dos fármacos , Animais , Antígenos CD4/efeitos dos fármacos , Células Cultivadas , Produtos do Gene vpr/farmacologia , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/patogenicidade , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores CCR5/efeitos dos fármacos , Receptores CXCR4/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Transdução de Sinais , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The cyclin-dependent kinase 1 (Cdk1), formerly called Cdc2 (or p34(Cdc2)), interacts with cyclin B1 to form an active heterodimer. The activity of Cdk1 is subjected to a complex spatiotemporary regulation, required to guarantee its scheduled contribution to the mitotic prophase and metaphase. Moreover, the activation of Cdk1 may be required for apoptosis induction in some particular pathways of cell killing. This applies to several clinically important settings, for instance to paclitaxel-induced killing of breast cancer cells, in which the ErbB2 receptor kinase can mediate apoptosis inhibition through inactivation of Cdk1. The activation of Cdk1 participates also in HIV-1-induced apoptosis, upstream of the p53-dependent mitochondrial permeabilization step. An unscheduled Cdk1 activation may contribute to neuronal apoptosis occurring in neurodegenerative diseases. Finally, the premature activation of Cdk1 can lead to mitotic catastrophe, for instance after irradiation-induced DNA damage. Thus, a cell type-specific modulation of Cdk1 might be taken advantage of for the therapeutic correction of pathogenic imbalances in apoptosis control.
Assuntos
Apoptose/genética , Proteína Quinase CDC2/metabolismo , Células Eucarióticas/metabolismo , Mitose/genética , Animais , Proteína Quinase CDC2/genética , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Células Eucarióticas/citologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismoRESUMO
Infection by human immunodeficiency virus-1 (HIV-1) is associated with a progressive decrease in CD4 T-cell numbers and the consequent collapse of host immune defenses. The major pathogenic mechanism of AIDS is the massive apoptotic destruction of the immunocompetent cells, including uninfected cells. The latter process, also known as by-stander killing, operates by various mechanisms one of which involves the formation of syncytia which undergo cell death by following a complex pathway. We present here a detailed and curated map of the syncytial apoptosis signaling network, aimed at simplifying the whole mechanism that we have characterized at the molecular level in the last 15 years. The map was created using Systems Biology Graphical Notation language with the help of CellDesigner software and encompasses 36 components (proteins/genes) and 54 interactions. The simplification of this complex network paves the way for the development of novel therapeutic strategies to eradicate HIV-1 infection. Agents that induce the selective death of HIV-1-elicited syncytia might lead to the elimination of viral reservoirs and hence constitute an important complement to current antiretroviral therapies.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Células Gigantes/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Transdução de Sinais/genética , Apoptose/genética , Efeito Espectador/genética , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/patologia , Fusão Celular , Regulação da Expressão Gênica , Células Gigantes/patologia , Proteína gp160 do Envelope de HIV/genética , Infecções por HIV/genética , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Mapeamento de Interação de Proteínas , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Purinérgicos/genética , Receptores Purinérgicos/metabolismoRESUMO
The protozoan parasite Cryptosporidium parvum causes persistent diarrhea and malnutrition in children and the diarrhea-wasting syndrome in AIDS. No therapy exists for eliminating the parasite in the absence of a healthy immune response. Although it had been reported that infection of intestinal cell lines with C. parvum leads to host cell death, the mechanisms of cytolysis have not been characterized. We show here that infection with C. parvum leads to typical apoptotic nuclear condensation and DNA fragmentation in host cells. Both nuclear condensation and DNA fragmentation are inhibited by a caspase inhibitor, showing that caspases are involved in this type of apoptosis. Finally, blocking apoptosis with the caspase inhibitor increases the percentage of infected cells, suggesting that parasites may use apoptosis to exit from the infected cell or that the infected cells may eliminate the parasite through apoptosis. These results suggest that apoptosis could be involved in the pathogenesis of C. parvum infections in vivo, and raise the possibility that therapeutic interference with host cell death could alter the course of the pathology in vivo.
Assuntos
Apoptose , Caspases/fisiologia , Criptosporidiose/patologia , Cryptosporidium parvum , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Inibidores de Caspase , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA , Células HeLa , Humanos , Microscopia de FluorescênciaRESUMO
Noninvasive nuclear magnetic resonance was used to measure the relaxation decay curves of naturally occurring 23Na ions in several biological systems. Experimental results showed an increase of membrane bound population for pathologic samples as compared with control. The bound sodium population was put in evidence using singular value decomposition method. Thus, the singular values that are obtained without any a priori from the fitting the relaxation decay curves are a new parameter in characterizing the cellular state. In the presence of artificial biological membranes, 23Na bound strongly to membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS), but not to membranes consisting of only PC. A large bound population also appeared in the presence of apoptotic epithelial cells, which are known to translocate PS to the cell surface. A role for PS was confirmed by showing that sodium binds to the surface of epithelial cells infected with Chlamydia psittaci, and the amplitude of the bound population increases with a time-course similar to the appearance of PS on the surface of dying cells. Finally, this approach could distinguish between normal perfused liver and liver undergoing ischemia, due most likely to the exposure of surface PS on apoptotic and necrotic cells in the damaged tissue. Taken together, these studies demonstrate that the analysis of 23Na relaxation decay curves could reveal the presence of cells undergoing apoptosis and/or necrosis in living tissues. Noninvasive 23Na NMR measurements could thus be envisioned for controlling the quality of organs before transplantation, for the detection of asymptomatic infections that result in death of the host cell or inflammation of the tissue, and for characterizing the efficiency of novel apoptosis-inducing drugs to treat cancer.
Assuntos
Compartimento Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Isquemia/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sódio/metabolismo , Animais , Apoptose , Membrana Celular/metabolismo , Chlamydophila psittaci , Simulação por Computador , Células HeLa , Humanos , Isquemia/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Membranas Artificiais , Camundongos , Necrose , Perfusão , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Fatores de TempoRESUMO
Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos da radiação , Raios gama , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/radioterapia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transplante HeterólogoRESUMO
Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-XL inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence.
Assuntos
Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Neoplasias Pulmonares/radioterapia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Radiossensibilizantes/administração & dosagem , Carcinoma de Pequenas Células do Pulmão/radioterapia , Sulfonamidas/administração & dosagem , Proteína bcl-X/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Cisplatino/administração & dosagem , Terapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tolerância a Radiação , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase- and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.
Assuntos
Trifosfato de Adenosina/metabolismo , Antineoplásicos/toxicidade , Morte Celular/efeitos dos fármacos , Animais , Proteína 5 Relacionada à Autofagia , Morte Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Conexinas/antagonistas & inibidores , Conexinas/genética , Conexinas/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína HMGB1/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/antagonistas & inibidores , Proteína 1 de Membrana Associada ao Lisossomo/genética , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
A new efficient type of gadolinium-based theranostic agent (AGuIX®) has recently been developed for MRI-guided radiotherapy (RT). These new particles consist of a polysiloxane network surrounded by a number of gadolinium chelates, usually 10. Owing to their small size (<5 nm), AGuIX typically exhibit biodistributions that are almost ideal for diagnostic and therapeutic purposes. For example, although a significant proportion of these particles accumulate in tumours, the remainder is rapidly eliminated by the renal route. In addition, in the absence of irradiation, the nanoparticles are well tolerated even at very high dose (10 times more than the dose used for mouse treatment). AGuIX particles have been proven to act as efficient radiosensitizers in a large variety of experimental in vitro scenarios, including different radioresistant cell lines, irradiation energies and radiation sources (sensitizing enhancement ratio ranging from 1.1 to 2.5). Pre-clinical studies have also demonstrated the impact of these particles on different heterotopic and orthotopic tumours, with both intratumoural or intravenous injection routes. A significant therapeutical effect has been observed in all contexts. Furthermore, MRI monitoring was proven to efficiently aid in determining a RT protocol and assessing tumour evolution following treatment. The usual theoretical models, based on energy attenuation and macroscopic dose enhancement, cannot account for all the results that have been obtained. Only theoretical models, which take into account the Auger electron cascades that occur between the different atoms constituting the particle and the related high radical concentrations in the vicinity of the particle, provide an explanation for the complex cell damage and death observed.
Assuntos
Gadolínio , Nanopartículas , Neoplasias/tratamento farmacológico , Radiossensibilizantes , Animais , Meios de Contraste , Humanos , Imageamento por Ressonância Magnética , Camundongos , Modelos Teóricos , Neoplasias/radioterapia , Radiossensibilizantes/química , SiloxanasAssuntos
Apoptose/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Proteínas Imediatamente Precoces/farmacologia , Proteínas Inibidoras de Apoptose/farmacologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas Virais/farmacologia , Apoptose/fisiologia , Cálcio/metabolismo , Ceramidas/farmacologia , Citomegalovirus , Células Gigantes/efeitos dos fármacos , Células Gigantes/patologia , Células Gigantes/ultraestrutura , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/fisiologia , Nelfinavir/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologia , Proteína Supressora de Tumor p53/fisiologiaAssuntos
Aneuploidia , Apoptose/genética , Antígenos CD4/genética , Ciclo Celular/efeitos dos fármacos , Fusão Celular , Sobrevivência Celular/genética , Coloração Cromossômica , DNA/análise , Citometria de Fluxo , Produtos do Gene env/genética , Células Gigantes/citologia , Células Gigantes/metabolismo , HIV-1/genética , Células HeLa , Humanos , Nocodazol/farmacologia , TransfecçãoRESUMO
p53 binding protein-1 (53BP1) participates in checkpoint signaling during the DNA damage response (DDR) and during mitosis. In this study we report that 53BP1 aggregates in nuclear foci within syncytia elicited by the human immunodeficiency virus (HIV)-1 envelope. 53BP1 aggregation occurs as a consequence of nuclear fusion (karyogamy (KG)). It colocalizes partially with the promyelomonocytic leukemia protein (PML), and the ataxia telangiectasia mutated kinase (ATM), the two components of the DDR that mediate apoptosis induced by the HIV-1 envelope. ATM-dependent phosphorylation of 53BP1 on serines 25 and 1778 (53BP1S25P and 53BP1S1778P) occurs at these DNA damage foci. 53BP1S25P was also detected in syncytia present in the lymph nodes or frontal brain sections from HIV-1-infected carriers, as well as in peripheral blood mononucleated cells from HIV-1-infected individuals, correlating with viral load. Knockdown of 53BP1 caused HIV-1 envelope-induced syncytia to enter abnormal mitoses, leading to their selective destruction through mitochondrion-dependent and caspase-dependent pathways. In conclusion, depletion of 53BP1 triggers the demise of HIV-1-elicited syncytia through mitotic catastrophe.
Assuntos
HIV-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adulto , Apoptose/genética , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Gigantes/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Mitose/genética , Mitose/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Apoptosis-inducing factor (AIF) has important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. In this study, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria in which they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 on exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been 'designed' to be retained in mitochondria and to minimize its potential neurotoxic activity.
Assuntos
Fator de Indução de Apoptose/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
The anti-apoptotic transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in CD34(+) myeloblasts from high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Inhibition of NF-kappaB by suppressing the canonical NF-kappaB activation pathway, for instance by knockdown of the three subunits of the inhibitor of NF-kappaB (I kappaB) kinase (IKK) complex (IKK1, IKK2 and NEMO) triggers apoptosis in such cells. Here, we show that an MDS/AML model cell line exhibits a constitutive interaction, within the nucleus, of activated, S1981-phosphorylated ataxia telangiectasia mutated (ATM) with NEMO. Inhibition of ATM with two distinct pharmacological inhibitors suppressed the activating autophosphorylation of ATM, blocked the interaction of ATM and NEMO, delocalized NEMO as well as another putative NF-kappaB activator, PIDD, from the nucleus, abolished the activating phosphorylation of the catalytic proteins of the IKK complex (IKK1/2 on serines 176/180), enhanced the expression of I kappaB alpha and caused the relocalization of NF-kappaB from the nucleus to the cytoplasm, followed by apoptosis. Knockdown of ATM with small-interfering RNAs had a similar effect that could not be enhanced by knockdown of NEMO, PIDD and the p65 NF-kappaB subunit, suggesting that an ATM inhibition/depletion truly induced apoptosis through inhibition of the NF-kappaB system. Pharmacological inhibition of ATM also induced the nucleocytoplasmic relocalization of p65 in malignant myeloblasts purified from patients with high-risk MDS or AML, correlating with the induction of apoptosis. Altogether, these results support the contention that constitutively active ATM accounts for the activation of NF-kappaB in high-risk MDS and AML.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Mutadas de Ataxia Telangiectasia , Células da Medula Óssea , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dano ao DNA , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Ensaio de Desvio de Mobilidade Eletroforética , Imunofluorescência , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Leucemia Mieloide Aguda/patologia , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , NF-kappa B/genética , Fosforilação , Transporte Proteico , Fatores de Risco , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Células Tumorais CultivadasRESUMO
Promyelomonocytic leukemia (PML) is a prominent oncosuppressor whose inactivation is involved in the pathogenesis of hematological and epithelial cancers. Here, we report that PML aggregated in nuclear bodies in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. PML aggregation occurred after the fusion of nuclei (karyogamy) within syncytia but before the apoptotic program was activated. The aggregation of PML was detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Using a range of specific inhibitors of PML (the oncogenic PML/RARalpha fusion product or specific small interfering RNAs), we demonstrated that, in Env-elicited syncytia, PML was required for activating phosphorylation of ataxia telangiectasia mutated (ATM), which colocalized with PML in nuclear bodies, in a molecular complex that also involved topoisomerase IIbeta-binding protein 1. PML knockdown thus inhibited the ATM-dependent DNA damage response that culminates in the activation of p53, p53-dependent transcription of pro-apoptotic genes and cell death. Infection of CD4-expressing cells with HIV-1 also induced syncytial apoptosis, which could be suppressed by inhibiting PML. Altogether, these data indicate that PML activation is a critical early event that participates in the apoptotic demise of HIV-1-elicited syncytia.
Assuntos
Apoptose , HIV-1 , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Gigantes/virologia , Células HeLa , Humanos , Proteína da Leucemia Promielocítica , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.
Assuntos
Apoptose , Infecções por Chlamydia/patologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Macrófagos/microbiologia , Macrófagos/patologia , Humanos , Células Tumorais CultivadasRESUMO
The pathology observed during Chlamydia infection is due initially to localized tissue damage caused by the infection itself, followed by deleterious host inflammatory responses that lead to permanent scarring. We have recently reported that the infection by Chlamydia in vitro results in apoptosis of epithelial cells and macrophages and that infected monocytes secrete the proinflammatory cytokine interleukin-1beta. At the same time, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can also trigger apoptosis of susceptible cells. To study the possible relationship between Chlamydia trachomatis infection and apoptosis in vivo, we used the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling technique to determine whether infection may cause apoptosis in the genital tract of mice and, conversely, whether cytokines produced during the inflammatory response may modulate the level of apoptosis. Our results demonstrate that infected cells in the endocervix at day 2 or 7 after infection are sometimes apoptotic, although there was not a statistically significant change in the number of apoptotic cells in the endocervix. However, large clumps of apoptotic infected cells were observed in the lumen, suggesting that apoptotic cells may be shed from the endocervix. Moreover, there was a large increase in the number of apoptotic cells in the uterine horns and oviducts after 2 or 7 days of infection, which was accompanied by obvious signs of upper tract pathology. Interestingly, depletion of TNF-alpha led to a decrease in the level of apoptosis in the uterine horns and oviducts of animals infected for 7 days, suggesting that the inflammatory cytokines may exert part of their pathological effect via apoptosis in infected tissues.
Assuntos
Apoptose , Colo do Útero/metabolismo , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Tubas Uterinas/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Útero/metabolismo , Animais , Linhagem Celular , Colo do Útero/microbiologia , Colo do Útero/patologia , Fragmentação do DNA , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Tubas Uterinas/microbiologia , Tubas Uterinas/patologia , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Interleucina-1/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Útero/microbiologia , Útero/patologiaRESUMO
Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Infecções por Chlamydia/metabolismo , Chlamydophila psittaci/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Marcadores de Afinidade/farmacologia , Animais , Apoptose/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Infecções por Chlamydia/patologia , Infecções por Chlamydia/fisiopatologia , Chlamydophila psittaci/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/fisiologia , Espaço Extracelular/metabolismo , Células HeLa , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Magnésio/farmacologia , Camundongos , Nucleotídeos/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2X7RESUMO
The third reading frame of the envelope gene from HIV-1 codes for a protein homologous to the human selenoprotein glutathione peroxidase (GPX). Cells stably or transiently transfected with a HIV-1 GPX construct are protected against the loss of the mitochondrial transmembrane potential and subsequent cell death induced by exogenous reactive oxygen species (ROS) as well as mitochondrion-generated ROS. However, HIV-1 GPX does not confer a general apoptosis resistance, because HIV-1 GPX-transfected cells were not protected against cell death induced by staurosporine or oligomycin. The inhibition of cell death induced by the ROS donor tert-butylhydroperoxide was also observed in cells depleted from endogenous glutathione (GSH), suggesting that GSH is not the sole electron acceptor for HIV-1 GPX. Clinical HIV-1 isolates from long-term non-progressors (untreated patients with diagnosed HIV-1 infection for > 10 years, with CD4 T cell count of > 500 cells/mm3) mostly possess an intact GPX gene (with only 18% of loss-of-function mutations), while HIV-1 isolates from patients developing AIDS contain non-functional GPX mutants in 9 out of 17 cases (53%). Altogether, these data suggest that HIV-1 GPX possesses a cytoprotective, pathophysiologically relevant function.
Assuntos
Apoptose/fisiologia , Glutationa Peroxidase/metabolismo , HIV-1/enzimologia , Sequência de Aminoácidos , Sequência Conservada , Glutationa/metabolismo , Glutationa Peroxidase/genética , HIV-1/genética , HIV-1/metabolismo , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Espécies Reativas de Oxigênio/metabolismo , TransfecçãoRESUMO
Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.