RESUMO
Glioblastoma multiform (GBM), the most common and aggressive primary brain tumor, is characterized by a high degree of hypoxia and resistance to therapy because of its adaptation capacities, including autophagy and growth factors signaling. In this study, we show an efficient hypoxia-induced survival autophagy in four different GBM cell lines (U87MG, M059K, M059J and LN-18) and an activation of a particular neurotrophin signaling pathway. Indeed, the enhancement of both TrkC and NT-3 was followed by downstream p38MAPK phosphorylation, suggesting the occurrence of a survival autocrine loop. Autophagy inhibition increased the hypoxia-induced expression of TrkC and its phosphorylated form as well as the phosphorylation of p38, suggesting a complementary effect of the two processes, leading to cell survival. Alone, autophagy inhibition reduced cellular growth without inducing cell death. However, the double inhibition of autophagy and TrkC signaling was necessary to bring cells to death as shown by PARP cleavage, particularly important in hypoxia. Moreover, a very high expression of TrkC and NT-3 was found in tumor sections from GBM patients, highlighting the importance of neurotrophic signaling in GBM tumor cell survival. These data suggest that a combined treatment targeting these two pathways could be considered in order to induce the death of GBM cells.
Assuntos
Autofagia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Fatores de Crescimento Neural/metabolismo , Receptor trkC/metabolismo , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Glioblastoma/metabolismo , Humanos , Hipóxia , Neurotrofina 3 , Fosforilação , Poli(ADP-Ribose) Polimerase-1/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
This Letter reports the synthesis and the characterization of two new water-stable and soluble photosensitizer-conjugated magnetic nanoparticles (PS-MNPs) composed of an iron oxide magnetic core coated with a biocompatible dextran shell bearing polyaminated chlorin p6. Designed to improve cancer cell targeting, these photosensitizers were assayed for their antitumour activity against two variants of B16 mouse melanoma cell line (B16F10 and B16G4F, with or without melanin, respectively). Cell viability measurements demonstrated that PS-MNPs were more phototoxic than PEI-chlorin p6 making these photosensitizers promising for further in vitro and in vivo investigations.
Assuntos
Antineoplásicos/química , Nanopartículas de Magnetita/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Luz , Nanopartículas de Magnetita/ultraestrutura , Melanoma Experimental/tratamento farmacológico , Camundongos , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , TemperaturaRESUMO
The development of hypoxic areas often takes place in solid tumors and leads cells to undergo adaptive signalization like autophagy. This process is responsible for misfolded or aggregated proteins and nonfunctional organelle recycling, allowing cells to maintain their energetic status. However, it could constitute a double-edged pathway leading to both survival and cell death. So, in response to stress such as hypoxia, autophagic and apoptotic cells are often mixed. To specifically study and characterize autophagic cells and the process, we needed to develop a method able to (1) isolate autophagic subpopulation and (2) respect apoptotic and autophagic status. Sedimentation field-flow fractionation (SdFFF) was first used to monitor physical parameter changes due to the hypoxia mimetic CoCl(2) in the p53 mutated SKNBE2(c) human neuroblastoma cell line. Second, we showed that "hyperlayer" elution is able to prepare autophagic enriched populations, fraction (F3), overexpressing autophagic markers (i.e., LC3-II accumulation and punctiform organization of autophagosomes as well as cathepsin B overactivity). Conversely, the first eluted fraction exhibited apoptotic markers (caspase-3 activity and Bax increased expression). For the first time, SdFFF was employed as an analytical tool in order to discriminate apoptotic and autophagic cells, thus providing an enriched autophagic fraction consecutively to a hypoxic stress.
Assuntos
Autofagia , Separação Celular/métodos , Fracionamento por Campo e Fluxo/métodos , Caspase 3/metabolismo , Catepsina B/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Cobalto/metabolismo , Humanos , Mutação , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.
Assuntos
Autofagia/fisiologia , Diferenciação Celular/fisiologia , Queratinócitos/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenilato Quinase/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteína Beclina-1 , Cálcio/farmacologia , Catepsina B/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Meios de Cultura Livres de Soro/farmacologia , Humanos , Integrina beta1/metabolismo , Queratina-10/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Sirtuína 1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/metabolismoRESUMO
Pro- and anti-apoptotic members of the Bcl-2 family are fundamental in the control of apoptosis. Among them, Bax plays a key role in apoptosis induction by mediating the release of apoptogenic factors from mitochondria to the cytosol. In this report, we investigated, by immunohistofluorescence, the in vivo distribution of Bax in normal human epidermis before and 24 h after exposure to solar-simulated radiation. Bax expression was evaluated with three different, Western blot pretested, anti-Bax antibodies (Ab) and correlated with markers of keratinocyte differentiation and apoptosis using anti-beta(1) integrin and anti-active caspase-3 Abs respectively. Using anti-Bax N20 and A-3533 polyclonal Ab, we found that, whereas undifferentiated keratinocytes of the basal proliferative compartment contained Bax in the cytosol, the differentiated suprabasal cells had Bax mainly in the nucleus. This immunoreactivity pattern was not modified by skin irradiation. Interestingly, the well known apoptosis-related Bax redistribution to mitochondria in response to a cell death signal, could be detected only with yet another, the 2D2 monoclonal Ab. This relocalization occurred specifically in apoptotic, active caspase-3 positive cells of irradiated epidermis. Our data highlight the differentiation- and apoptosis-associated changes in the pattern of Bax subcellular and cellular distribution as uncovered by different anti-Bax Abs and suggest that Bax undergoes successive activation that progresses in parallel with keratinocyte differentiation and apoptosis.
Assuntos
Apoptose/efeitos da radiação , Epiderme/metabolismo , Epiderme/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Raios Ultravioleta , Proteína X Associada a bcl-2/metabolismo , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Biópsia , Caspase 3/metabolismo , Diferenciação Celular/efeitos da radiação , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Epiderme/efeitos da radiação , Humanos , Queratinócitos/efeitos da radiação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos da radiação , Proteína X Associada a bcl-2/imunologiaRESUMO
It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Mitocôndrias/metabolismo , Anticorpos Monoclonais , Western Blotting , Núcleo Celular/metabolismo , Citometria de Fluxo , Humanos , Células K562 , Microscopia ConfocalRESUMO
Glioblastoma multiforme is the most common and the most aggressive primary brain tumor. It is characterized by a high degree of hypoxia and also by a remarkable resistance to therapy because of its adaptation capabilities that include autophagy. This degradation process allows the recycling of cellular components, leading to the formation of metabolic precursors and production of adenosine triphosphate. Hypoxia can induce autophagy through the activation of several autophagy-related proteins such as BNIP3, AMPK, REDD1, PML, and the unfolded protein response-related transcription factors ATF4 and CHOP. This review summarizes the most recent data about induction of autophagy under hypoxic condition and the role of autophagy in glioblastoma.
Assuntos
Autofagia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Animais , Autofagia/genética , Neoplasias Encefálicas/genética , Hipóxia Celular/genética , Sobrevivência Celular/genética , Glioblastoma/genética , HumanosRESUMO
More than other tissues, skin is exposed to numerous external stresses generating ROS that, in addition to endogenous oxygen radicals, cause keratinocyte alterations and contribute in part to photocarcinogenesis and aging. Recent evidence suggests a differentiation-dependent susceptibility of keratinocytes to apoptosis. We explored hydrogen peroxide-induced cell death in normal human keratinocytes according to their differentiation. On H(2)O(2)-exposed skin explants, caspase-3 was strongly activated in basal keratinocytes double stained with beta(1) integrin, whereas DNA fragmentation occurred in suprabasal cells only without caspase-3 activation. In addition, isolated basal keratinocytes, selected by adhesion to type IV collagen, were more sensitive than nonadherent cells to H(2)O(2)-induced apoptosis with regard to mitochondrial transmembrane potential (Deltapsi(mt)) collapse and membrane integrity. Similarly, necrotic/late apoptotic cells were present at low levels only in the adherent epidermal population. Furthermore, in primary cultures of undifferentiated keratinocytes H(2)O(2)-induced cell death appeared via a mitochondrial failure. Deltapsi(mt) collapse was associated with a strong early activation of the initiatory caspase-8, then the executive caspase-3, and, to a lesser extent, the inflammatory caspase-1. Finally, undifferentiated basal cells possess a higher sensitivity than differentiated suprabasal cells to H(2)O(2)-induced cell death, and apoptosis in human keratinocytes occurs via different pathways depending on the cell's differentiation state.
Assuntos
Diferenciação Celular/fisiologia , Peróxido de Hidrogênio/farmacologia , Queratinócitos/efeitos dos fármacos , Adulto , Apoptose/efeitos dos fármacos , Caspase 1/efeitos dos fármacos , Caspase 1/metabolismo , Caspase 3 , Caspase 8 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo IV/fisiologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Queratinócitos/citologia , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Oxidantes/farmacologia , Pele/citologia , Pele/efeitos dos fármacos , Fatores de TempoRESUMO
Doxorubicin (DOX), a widely used antitumoral drug, induces numerous modifications in sensitive cells, interacting with nuclear and mitochondrial DNA. In previous studies achieved in two K562 DOX-resistant sublines (K562/0.2R and K562/0.5R), we have shown stable mitochondrial damage comparatively with sensitive parental cells, such as decrease of cytochrome c oxidase activity (COX; EC 1.9.3.1) and cytochrome aa3 content. In order to explain these data, we have studied several COX genes and their expression, in relationship with altered COX activity and multidrug resistance (MDR) phenotype. We have observed a lower expression of the catalytic subunits COX I and II in MDR sublines, which was neither related to mutations in the corresponding mitochondrial genes, nor to a reduced transcription rate. In contrast, we have noticed an increase in both MDR K562 variants, in the mRNA expression of the catalytic subunit COX III, related to an increase in the half-life of these transcripts. Moreover, the doxorubicin resistance phenotype in K562 cells was accompanied by modifications of the expression and steady-state mRNA levels of several nuclear-encoded regulatory COX subunits. Thus, doxorubicin-resistant K562 cells represent an interesting model to study stable modifications concomitant to MDR phenotype. Our results seem to indicate compensatory mechanisms which highlight the complexity of regulatory systems of COX enzyme, involving coordinate regulation of both nuclear and mitochondrial subunit expression.
Assuntos
Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Leucemia/metabolismo , Catálise , DNA Mitocondrial/análise , Complexo IV da Cadeia de Transporte de Elétrons/genética , Expressão Gênica , Humanos , Células K562 , RNA Mensageiro/biossínteseRESUMO
Solid tumors like neuroblastoma exhibit hypoxic areas, which can lead both to cell death or aggressiveness increase. Hypoxia is a known stress able to induce stabilization of p53, implicated in cell fate regulation. Recently, p53 appeared to be involved in autophagy in an opposite manner, depending on its location: when nuclear, it enhanced transcription of pro-autophagic genes whereas when cytoplasmic, it inhibited the autophagic process. Today, we used cobalt chloride, a hypoxia mimetic that inhibits proteasomal HIF-1 degradation and generates reactive oxygen species (ROS). We focused on CoCl2-induced cell death in a DNA-binding mutated p53 neuroblastoma cell line (SKNBE(2c)). An autophagic signaling was evidenced by an increase of Beclin-1, ATG 5-12, and LC3-II expression whereas the p53(mut) presence decreased with CoCl2 time exposure. Activation of the pathway seemed to protect cells from ROS production and, at least in part, from death. The autophagic inhibitors activated the apoptotic signaling and the death was enhanced. To delineate the eventual implication of the p53(mut) in the autophagic process in response to hypoxia, we monitored signaling in p53(WT)SHSY5Y cells, after either shRNA-p53 down-regulation or transcriptional activity inhibition by pifithrin alpha. We did not detect autophagy neither with p53(wt) nor when p53 was lacking whereas such a response was effective with a mutated or inactivated p53. To conclude, mutated p53 in neuroblastoma cells could be linked with the switch between apoptotic response and cell death by autophagy in response to hypoxic mimetic stress.
Assuntos
Autofagia , Cobalto/farmacologia , Mutação , Neuroblastoma/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Photodynamic therapy (PDT) is now a well-established treatment modality for cutaneous carcinomas and is based on the administration of a light-activated drug followed by illumination of the pathological area. The treatment of metastatic melanoma remains a therapeutic challenge. To define the possible role of melanin in relative phototoxicity of 5-aminolevulinic acid (5-ALA), a photosensitizer used in PDT in vivo, we studied cell death in two variants (with or without melanin, B16F10 and B16G4F cells, respectively) of a melanoma cell line. Concentrations of 5-Ala up to 10 mM induced similar cytostatic effects in the B16G4F and B16F10 cells. PDT and high 5-ALA concentrations induced photocytotoxicity in both melanoma cell lines (at 10 mM for B16F10 cells and at 5 mM for B16G4F cells). Cell death corresponded to p53-dependent apoptotic signaling in pigmented B16F10 cells, whereas an autophagic response leading to a caspase-independent death was detected in non-pigmented B16G4F cells. Therefore, the PDT-induced cell death pathway appeared to correlate with melanin synthesis capacity in melanoma cells. To reduce the cytotoxicity of 5-ALA without irradiation, a low drug concentration could be used. Consequently, in combination with current therapeutics, a moderate concentration of 5-ALA and PDT may constitute a supplementary promising approach to eliminate metastatic melanoma.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Melaninas/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Caspases/metabolismo , Catepsina B/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Melanoma Experimental , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fotoquimioterapia , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cobalt chloride (CoCl2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 ( SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (∆Ψmt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.
Assuntos
Apoptose/efeitos dos fármacos , Cobalto/farmacologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Genes p53 , Humanos , Mutação , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
UNLABELLED: The large number of studies devoted to the effect of ultraviolet light on biological systems, contrasts with the lack of experimental data concerning the direct effects of visible light. It has been shown that blue light inhibited the growth of B16F10 melanoma cell lines and reduced the percentage of S phase cells. Yet these effects are poorly understood. MATERIALS AND METHODS: Two cell lines and irradiation with blue light were used. Cell mortality and a possible mechanism of action were investigated. RESULTS: Exposure of B16F10 melanoma and bovine endothelial cells to blue light (wavelength 450 nm, 10 J/cm(2) from a Waldman lamp) induced a rapid and large reduction in viability followed by the death of virtually all the irradiated cells within 24 h. These results led us to expose a patient with haemorrhagic cutaneous melanoma metastasis to blue light. Irradiation led to an immediate arrest of haemorrhage, an inhibition of tumour growth and extensive tumour necrosis 24h after irradiation. CONCLUSION: Exposure to blue light may offer new approaches to the treatment of superficial skin carcinomas in humans.
Assuntos
Células Endoteliais/efeitos da radiação , Melanoma Experimental/terapia , Fototerapia/métodos , Animais , Bovinos , Sobrevivência Celular/efeitos da radiação , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Luz , Peroxidação de Lipídeos/efeitos da radiação , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , CamundongosRESUMO
BACKGROUND: Age-related changes in the antibody response have been classically associated with alterations in T-cell help, but increasing evidence shows that intrinsic B-cell defects exist. This article analyzes the apoptotic susceptibility of peripheral B-cells in aged and young control mice. MATERIALS AND METHODS: Freshly isolated lymphocytes from spleen and Peyer's patches (PPs) were labeled for B-cell lineage (B220(+) cells) and germinal center B subset (GCs, B220(+)/PNA(+) cells). Alternatively, splenic B-cells purified by MACS were used. Apoptosis was monitored by the Annexin V binding, incorporation of 3,3(')-dihexyloxacarbocyanine iodide (DiOC(6)(3)), propidium iodide (PI) staining, and morphological changes. Moreover, intracellular Bcl-2 expression and Bad phosphorylation status were also analyzed in B-cells. RESULTS: We showed in aging mice an enhanced Annexin V(+)/PI(-) cell percentage in splenic B-lymphocytes, which was correlated with a lower DeltaPsi(m). By contrast, no change in apoptosis was observed in compartments known to be enriched in activated B-cells (GCs and PPs). Analysis of Bcl-2 levels revealed no modification. When using B-cells purified by MACS, we strongly confirm data obtained on staining cells. Moreover, enhanced spontaneous apoptosis of splenic B-cells in aged mice was found to be correlated with a reduced phosphorylated Bad expression. CONCLUSION: Increased apoptosis of resting B-cells in old mice may be determined by an altered Bad phosphorylation, which in turn contributes to cell death by lowering the mitochondrial threshold for apoptosis.
Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Linfócitos B/fisiologia , Animais , Anexina A5/metabolismo , Linfócitos B/citologia , Biomarcadores/metabolismo , Células Cultivadas , Feminino , Humanos , Antígenos Comuns de Leucócito/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/citologia , Fenótipo , Fosforilação , Baço/citologia , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
BACKGROUND: Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Deltapsimt) and membrane integrity fluorochromes. Rhodamine 123 and DiOC6(3) remain controversial to identify cells displaying a low Deltapsimt. JC-1 constitutes a good Deltapsimt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Deltapsimt. Nevertheless, it is not feasible to analyze it simultaneously with propidium iodide. Among available fluorescent probes, TOTO-3 seems to be a good candidate for double staining with JC-1. METHODS: Cell death of HaCaT cells was induced by H2O2 and FasL. Samples were stained with DiOC6(3)/IP or JC-1/TOTO-3 then analyzed by flow cytometry. Results were supported by confocal microscopy analyses of mitochondrial membrane potential. Moreover, cell morphology was determined on the sorted subpopulations defined on the basis of staining (JC-1 versus TOTO-3). RESULTS: We found that JC-1 is a more efficient mitochondrial probe than DiOC6(3). After stress induction, the fluorescence level of JC-1 and TOTO-3 clearly defined three fluorescent subpopulations, respectively: (1) JC-1high and TOTO-3low, (2) JC-1low and TOTO-3medium, and (3) JC-1low and TOTO-3high. Their morphologic aspects after cell sorting indicated that they corresponded to three functional states (intact, apoptotic, and necrotic cells), and data were supported by caspase activity measurements. CONCLUSIONS: We propose a reliable and efficient staining, with JC-1 and TOTO-3 to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry.