RESUMO
Shear stress is determined by three physical components described in a famous triad: blood flow, blood viscosity and vessel geometry. Through the direct action on endothelium, shear stress is able to radically interfere with endothelial properties and the physiology of the vascular wall. Endothelial cells (ECs) have also to sustain biochemical stresses represented by chemokines, growth factors, cytokines, complement, hormones, nitric oxide (NO), oxygen and reactive oxygen species (ROS). Many growth factors, cytokines, chemokines, hormones, and chemical substances, like NO, act and regulate endothelium functions and homeostasis. Among these cytokines Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) has been assigned a regulatory role in ECs physiology and physiopathology. Thus, the aim of this review is to provide a general overview of the endothelial response pathways after different types of biomechanical and biochemical stress in in vitro models and to analyze the crucial role of TRAIL under pathological conditions of the cardiocirculatory system like atherosclerosis, coronary artery disease, and diabetes.
Assuntos
Artérias/metabolismo , Células Endoteliais/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Artérias/citologia , Artérias/patologia , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Fenômenos Biomecânicos , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/fisiopatologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Células Endoteliais/patologia , Humanos , Técnicas In Vitro , Estresse FisiológicoAssuntos
Alopurinol/farmacologia , Resistencia a Medicamentos Antineoplásicos , Furosemida/farmacologia , Extratos Vegetais/farmacologia , Sarcoma/patologia , Neoplasias Uterinas/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Linhagem Celular Tumoral , Doxorrubicina , Feminino , Humanos , Sarcoma/tratamento farmacológico , Neoplasias Uterinas/tratamento farmacológicoRESUMO
Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to protect normal cells from chemotherapy-induced cytotoxixity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Colagogos e Coleréticos/farmacologia , Cinamatos/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , S-Adenosilmetionina/metabolismo , Sarcoma/metabolismo , Neoplasias Uterinas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antibióticos Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/patologiaRESUMO
Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 µM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 µM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 µM doxo in the presence of the lowest concentration of DEHP (3 µM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 µM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 µM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Dietilexilftalato/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Plastificantes/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Dietilexilftalato/uso terapêutico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Plastificantes/uso terapêutico , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/patologiaRESUMO
Morphological features of granulosa cells can reflect their functional status. The present study was aimed at comparing possible differences in the fine structure of human granulosa cells exposed to gonadotrophin-releasing hormone (GnRH) agonist or antagonist treatment during ovarian stimulation. Cells were obtained from follicular aspirates of 21 women treated with recombinant follicle-stimulating hormone (rFSH) plus either a GnRH agonist or a GnRH antagonist. Conventional light microscopy procedures and computerized image analysis systems were used to identify different cell type morphological patterns and to quantify different cells distribution. Two morphologically distinct granulosa cell populations, defined as large/pale and small/dark cells, were identified and a different distribution in the two groups of women under investigation was found: a significantly higher percentage in large/pale cells was detected in the agonist-treated women (P<0.05), whereas the percentage of small/dark cells was significantly higher in the antagonist-treated group (P<0.05). Ultrastructural observations showed the presence in both cell populations of typical hallmarks of steroidogenic cells, highlighting signs of functional activity in the large/pale cell population. Further investigations are needed to define the possible clinical significance of these morphological findings.
Assuntos
Hormônio Liberador de Gonadotropina/agonistas , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Células da Granulosa/efeitos dos fármacos , Adulto , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/citologia , Células da Granulosa/ultraestrutura , Humanos , Microscopia Eletrônica , Proteínas Recombinantes/farmacologiaRESUMO
BACKGROUND: Hypoxia and aging determine on mammalian cells a stress response which implies modified production of oxidants, reactive oxygen species or reactive nitrogen species at the mitochondrial level, interfering with cell-signaling proteins and inducing mitochondrial damage, apoptosis occurrence and functional consequences. OBJECTIVE: Here we report the effects of hypoxia on the in vivo morphological and biochemical response of young and aged Wistar rat hearts. METHODS: Left ventricles were excised from each experimental point and processed. Investigations of vascular endothelial growth factor (VEGF) expression and apoptotic events, mitochondrial damage, were performed by light and electron microscopy, respectively; endothelial, inducible and neuronal NOS, PKCα, pPKCα, caspase-3 expression and Apaf-1/cytochrome c complex formation were assessed by Western blotting and co-immunoprecipitation analyses, respectively. RESULTS: Besides morphological modifications, which confirm mitochondrial suffering upon hypoxia exposure in both young and aged hearts, the role played by PKCα in controlling nitric oxide synthase (NOS) protein level was investigated. Downstream PKCα activation, a dramatic iNOS expression increase, concomitant to enhanced apoptotic cell percentage and Apaf-1/cytochrome c co-immunoprecipitation, is evident in the hypoxic young, suggesting iNOS-mediated activation of the mitochondrial apoptotic pathway. CONCLUSIONS: Moreover, overexpression of iNOS and VEGF in the hypoxic young rat hearts suggests that an increased VEGF level may allow coordinated development of the lymphatic and blood vasculature, necessary for fluid homeostasis and to counteract oxidative stress. Thus the inhibition of such growth factor proposes new therapeutic possibilities for diseases associated to vascular function and for solid tumors which show pathological angiogenesis and lymphoangiogenesis.
Assuntos
Apoptose , Ventrículos do Coração/metabolismo , Mitocôndrias Cardíacas/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Envelhecimento/metabolismo , Animais , Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 3/metabolismo , Hipóxia Celular , Senescência Celular , Citocromos c/metabolismo , Ventrículos do Coração/patologia , Redes e Vias Metabólicas , Neovascularização Patológica/metabolismo , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Wistar , Estresse Fisiológico , Fator A de Crescimento do Endotélio VascularRESUMO
During heart development, cell hyperplasia and hypertrophy are the main mechanisms by which cardiac mass grows. Both these processes along with programmed cell death lead to complete growth and function. In addition, since the establishment of cardiac function depends on the relationship between oxygen supply and demand, we investigated some of the molecular mechanisms at the basis of rat myocardial cell response to hypoxic stress at different times of neonatal life. In particular, the role played by hypertrophic and survival factors like NF-kB and IAP-1 (Inhibiting Apoptosis Protein) and by death factors ASK-1 (Apoptosis Signal Regulating Kinase), JNK/SAPK (Jun-N-Terminal-Kinase/Stress-Activated Protein Kinase) pathways in regulating caspase-3 expression and activity has been evaluated by immunohistochemical and Western blotting analyses, respectively. Level of phosphorylation of IkBalpha and IAP-1 expression were substantial in 8-day-old hypoxic hearts, suggesting the persistence of NF-kB driven hypertrophic signal along with a rescue attempt against hypoxic stress. In contrast, ASK-1 mediated JNK/SAPK activation, regulating Bcl(2) levels, allows Bax homodimerization and caspase-3 activation in the same experimental conditions. Thus, a regulation carried out by NF-kB and JNK/SAPK pathways on caspase-3 activation at day 8 of neonatal life can be suggested as the main factor for the heart 'adaptive' response to hypoxia.
Assuntos
Cardiomegalia/patologia , Caspases/metabolismo , Ativação Enzimática/fisiologia , Coração/crescimento & desenvolvimento , Hipóxia/patologia , Animais , Animais Recém-Nascidos/fisiologia , Apoptose/fisiologia , Western Blotting , Caspase 3 , Tamanho Celular , Feminino , Proteínas I-kappa B/fisiologia , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Inibidoras de Apoptose , MAP Quinase Quinase Quinase 5/fisiologia , Miocárdio/metabolismo , NF-kappa B/fisiologia , Consumo de Oxigênio/fisiologia , Gravidez , Proteínas/fisiologia , Ratos , Ratos Wistar , Estresse Fisiológico/fisiopatologia , Análise de SobrevidaRESUMO
Here we provide evidence for a role of phosphatidylinositol-3-kinase (PI-3-kinase) and for its product phosphatidylinositol-3,4, 5-triphosphate (PI3,4,5P3) in the occurrence of the metabolic differentiation state induced by DMSO in murine Friend erythroleukemia cells. Of note, the activation of PI-3-kinase correlated with the modulation of the activation of another enzyme, the atypical protein kinase C zeta (aPKC zeta). In particular, the expression of PI-3-kinase was substantially unaffected by DMSO treatment while its phosphorylation and the production of PI3,4,5P3 was strongly increased within 24 h of DMSO. Such a result was paralleled by an evident phosphorylation of a PKC zeta. Treatment of the cells with the two unrelated PI-3-kinase inhibitors wortmannin and LY 294002 impaired the recovery of the number of differentiated cells, therefore indicating that PI-3-kinase might be involved in the induction of erythroid differentiation, possibly engaging a protein kinase C zeta as downstream effector.
Assuntos
Hematopoese/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Animais , Cromonas/farmacologia , Dimetil Sulfóxido/metabolismo , Dimetil Sulfóxido/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hematopoese/efeitos dos fármacos , Leucemia Eritroblástica Aguda , Camundongos , Modelos Biológicos , Morfolinas/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Células Tumorais Cultivadas , WortmaninaRESUMO
The present investigation was aimed at studying the effects of dimethylsulfoxide (DMSO) in combination with high dose (15 and 60 Gy) ionising radiation on the growth and differentiation of murine erythroleukemia cells (MEL). The incubation with DMSO was performed for 96 h starting immediately after exposure to radiation and resulted only in a slight inhibition of cell growth and in a high increase in cell death with the induction of both necrosis and apoptosis. The enhancement of radiation cytotoxicity was directly related to dose, time in culture and degree of differentiation as demonstrated by the severe and multiple aberrations observed in light and electron microscopy. Of interest was the observation in induced cells of a marked rearrangement of the plasma membrane architecture as well as that of the nuclear envelope, with a massive translocation and/or decrease in the nuclear pore complexes.
Assuntos
Dimetil Sulfóxido/farmacologia , Leucemia Eritroblástica Aguda/patologia , Radiação Ionizante , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Núcleo Celular/ultraestrutura , Relação Dose-Resposta à Radiação , Camundongos , Membrana Nuclear/ultraestrutura , Células Tumorais CultivadasRESUMO
The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.
Assuntos
Núcleo Celular/fisiologia , Reparo do DNA/fisiologia , Fosfatidilinositóis/fisiologia , Transdução de Sinais/fisiologia , Animais , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Camundongos , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Células Tumorais CultivadasRESUMO
The development and growth of the rat heart implies hyperplasia, which stops at birth, and hypertrophy, allowing cardiac mass to grow in response to programmed genetic events along with to haemodynamic overload. Moreover, hypertrophy is accomplished to apoptosis which controls the final number of myocardial cells, deletes vestigial structures, and takes part in remodelling the organ. Since at the basis of all these processes, which lead to the complete development of the heart, the activation of specific signalling pathways underlies, attention has been addressed to the role played in vivo by Protein Kinase C zeta (PKC zeta) in regulating NF-kB signalling system and intrinsic mitochondrial apoptotic route at days 1, 4, 10 and 22 of rat life. In fact, a role has been assigned to PKC zeta in indirectly phosphorylating IKBa, which peaks between 10 and 22 days, through a IKK determining, in turn, NF-kB activation, concomitantly to cytochrome c/Apaf 1 co-localization in the cytoplasm and caspase-9/caspase-3 activation, which leads to the occurrence of apoptosis. Thus a key role for PKC zeta in regulating the hypertrophic and apoptotic events leading to establishment of complete function in rat neonatal heart is here suggested.
Assuntos
Apoptose/fisiologia , Cardiomegalia/enzimologia , Coração/crescimento & desenvolvimento , Proteína Quinase C/fisiologia , Animais , Animais Recém-Nascidos/fisiologia , Western Blotting , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Microscopia Imunoeletrônica , Miocárdio/citologia , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Fatores de TranscriçãoRESUMO
The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process.
Assuntos
Envelhecimento/fisiologia , Subpopulações de Linfócitos T/citologia , Adulto , Idoso , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Citometria de Fluxo , Humanos , Microscopia Eletrônica , Fito-Hemaglutininas/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/ultraestruturaRESUMO
To examine the effects of age and training on the active T subpopulation we considered elderly amateur cyclists over 65 in comparison with young amateur cyclists and young and aged sedentary healthy controls. Significant differences were observed between trained and sedentary elderly subjects consisting of an increase in the percentage of active E rosettes after 4 and 24 h of in vitro PHA stimulation, and of a decrease in the in vitro phosphorylation of phosphatydylinositol 4,5-bisphosphate (PtdInsP2) and a corresponding increase in phosphatydylinositol 4-phosphate (PtdInsP) in the early steps of the mitotic response. Our findings support the hypothesis of the involvement of inositol lipids in controlling the expression of lymphocyte surface receptors.
Assuntos
Envelhecimento/imunologia , Fosfatos de Fosfatidilinositol/sangue , Educação Física e Treinamento , Fito-Hemaglutininas/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Adulto , Idoso , Estudos de Casos e Controles , Humanos , Masculino , Formação de Roseta , Estimulação Química , Subpopulações de Linfócitos T/metabolismoRESUMO
From birth to aging the heart undergoes functional changes reflecting biochemical and ultrastructural modifications which imply apoptosis. This is a physiological process resulting from genetic programs closely associated with development and aging. During development apoptosis eliminates redundant cells leading to heart remodeling, while during aging it eliminates damaged or exhausted cells. In the present paper we analyze some molecular mechanisms involved with heart morphological modifications, especially in the neonatal heart which displays different features in the subendocardial and myocardial area. The high number of subendocardial apoptotic cells and the inverted ratio of Bcl-2/Bax molecule expression in the two heart compartments led us to hypothesize a different metabolism in the myocardium as compared with subendocardium. Moreover, we propose that PKC zeta may mediate this different response by activating Nf-kB pathway and by maintaining the balance between hypertrophic growth and apoptosis involved with remodeling of neonatal heart. Further, we underline that in the aged heart, where this pathway is not activated, such balance is not maintained.
Assuntos
Envelhecimento/metabolismo , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Apoptose , Endocárdio/metabolismo , Endocárdio/fisiologia , Masculino , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Proteína X Associada a bcl-2RESUMO
Ionizing radiation induces a series of multiple intracellular events which can lead to activation of caspases, cytoplasmic proteases involved in the occurrence of apoptosis. The response of leukemic cells to ionizing radiation is amplified when they have been pre-treated with the anticancer drug etoposide, therefore the aim of this work has been to establish the lowest etoposide concentration combined with the lowest ionizing radiation dose to obtain the best antineoplastic response. Two leukemic cell lines, HL-60 and Jurkat, employed in this study demonstrated different sensitivities to ionizing radiation and to etoposide treatment, with Jurkat T cells requiring a higher dose (1 microM) to display cell cycle perturbation and apoptotic DNA damage similar to those seen in HL-60. We hypothesize that this kind of response could be mediated by mitochondrial release of apoptogenic factors and by SAPK/JNK metabolic pathway activation, both leading to caspase-3 cleavage. All in all these results provide insight into the sensitivity or resistance of leukemic cells to antineoplastic agents and identify molecular targets for rational therapeutic intervention strategies.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/fisiologia , Caspases/biossíntese , Caspases/genética , Etoposídeo/uso terapêutico , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Leucemia/metabolismo , Mitocôndrias/enzimologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3 , Fracionamento Celular , Terapia Combinada , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Células HL-60 , Humanos , Imunoprecipitação , Células Jurkat , Leucemia/tratamento farmacológico , Leucemia/radioterapia , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Inibidores da Topoisomerase IIRESUMO
Tumor irradiation induces modifications in the interaction of surviving target cells with immune cells. This interaction is mediated by adhesion molecules, whose expression can be strongly altered by radiation treatment. Here the probably of K562 tumor cells for lymphocyte binding was studied after exposure of target cells to different doses of gamma-radiation. Results were correlated to the expression of ICAM-1 and LFA-3 adhesion molecules on target cells. Radiation treatment enhanced the expression of both ICAM-1 and LFA-3 on the surface of target cells in a dose and time of culture-dependent fashion, reaching a maximum 24 hrs postirradiation, when also lymphocyte binding was increased. 10-30 Gy irradiation of K562 cells in vitro induces after 24 hrs, an up-regulation of ICAM-1 and LFA-3 expression that, in turn, increase lymphocyte binding, making tumor cells more exposed to cytotoxic attack. The progressive morphological damage induced by radiation, documented by the scattering singlas in flow cytometry and by electron microscopy analysis of irradiated K562 cells, induced, particularly at delayed times of culture in high doses irradiated cells, alterations of the target cell surface that might prevent the correct interaction with immune cells.
Assuntos
Antígenos CD58/biossíntese , Molécula 1 de Adesão Intercelular/biossíntese , Linfócitos/imunologia , Adulto , Anticorpos Monoclonais , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Citometria de Fluxo , Humanos , Técnicas In Vitro , Linfócitos/efeitos da radiação , Linfócitos/ultraestrutura , Masculino , Microscopia Eletrônica , Células Tumorais Cultivadas , Regulação para Cima/efeitos da radiaçãoRESUMO
In aged rats a decrease in axosomatic synapses of granule cells as well as a decrease in the number of synaptic vesicles of giant synapses was found. These phenomena were supposed to be correlated on the basis of a feed-back circuit existing at the level of the dentate gyrus. In fact the axosomatic synapses of the granules are inhibitory gamma-aminobutyric acid-ergic terminals of interneurons. Interneurons receive excitatory afferences from granules via the giant synapses of the mossy fibre collaterals. This results in a feed-back regulation of granule cell activity. The long-term administration of acetyl-L-carnitine to aged rats restores a synaptic pattern comparable to that of young rats. This effect on synaptic plasticity is transient.
Assuntos
Acetilcarnitina/farmacologia , Carnitina/análogos & derivados , Hipocampo/ultraestrutura , Animais , Hipocampo/efeitos dos fármacos , Microscopia Eletrônica , RatosRESUMO
Radiation causes damage to cell surface membranes, cytoplasmic organelles, and the nuclear process of DNA synthesis and repair, and this eventually results in different modes of cell death. In this study we examined murine erythroleukemia (MEL) cells, exposed to 15 and 60 Gy of 10 MeV photonic energy, and left in culture for up to 96 hours. Electron microscopical analysis was performed on conventionally embedded samples and freeze-fracture replicas, in order to detect ultrastructural patterns of cell damage and death. Of interest was the observation of chromatin condensates, nuclear membrane associations and nuclear pore redistribution during early apoptosis. Pronounced rearrangements of transmembrane particles during late stages of cellular necrosis were also found. The morphological damage induced by both doses of radiation as a function of time after exposure was only quantitatively but not qualitatively different.
Assuntos
Cromatina/ultraestrutura , Dano ao DNA/efeitos da radiação , Leucemia Eritroblástica Aguda/patologia , Animais , Apoptose/efeitos da radiação , Morte Celular/efeitos da radiação , Cromatina/efeitos da radiação , Técnica de Fratura por Congelamento , Raios gama , Leucemia Eritroblástica Aguda/radioterapia , Camundongos , Células Tumorais CultivadasRESUMO
Tumour necrosis factor alpha (TNF-alpha) is a cytokine that induces physiological and pathophysiological effects in the immune system. In this study we analyzed its action on a human lymphoma cell line (Daudi cells) after 1 h, 6 h and 24 h of incubation. Using vital DNA stains, DNA gel electrophoresis, in situ nick translation, transmission electron microscopy and flow cytometry we showed that as early as after 6 h of treatment, target cells were able to undergo death by apoptosis. This was associated with cell shrinkage, chromatin condensation, apoptotic bodies in the cytoplasm without the typical DNA fragmentation into low molecular weight nucleosomes. Of interest was the observation of a significant number (60%) of cells positive to the nick translation in specimens treated for 6 h, decreasing to 40% in samples treated for 24 h, when most of the cells were in late apoptosis. In addition, no subdiploid peak was evident in flow cytometry regardless of the time of incubation with TNF. Our study on Daudi cells clearly supports the existence of alternative forms of apoptosis in which DNA degradation does not result only in oligonucleosomal fragmentation.
Assuntos
Apoptose , Fator de Necrose Tumoral alfa/farmacologia , DNA , Eletroforese em Gel de Ágar , Citometria de Fluxo , Humanos , Linfoma , Microscopia Eletrônica , Propídio/química , Coloração e Rotulagem , Células Tumorais CultivadasRESUMO
A morphological study of DNA repair and apoptotic patterns in relationship with cell cycle events was performed on murine erythroleukemia cells. The presence and distribution of DNA replicon sites were evaluated through the BrdU-anti BrdU immunofluorescence and immunogold techniques in light and electron microscopy. Different patterns of labelling and percentages of BrdU positive cells were observed depending on irradiation dose (up to 60 Gy) and time in post-irradiation culture (up to 24 hours). An enlargement of the S phase of the cell cycle was evidenced 18 hours post-irradiation as determined by flow cytometry analysis. The high resolution approach showed that, in spite of several morphological alterations, BrdU labelling was present even in cells displaying early and late apoptotic features.