RESUMO
Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neuritos , Fosfolipases A2/metabolismo , Frações Subcelulares/efeitos dos fármacos , Animais , Microscopia Confocal , Microscopia de Fluorescência , Neurogênese , Células PC12 , Ratos , Frações Subcelulares/enzimologiaRESUMO
Episodic ataxia type 1 (EA1) is a rare human neurological syndrome characterized by continuous myokymia and attacks of generalized ataxia that can be triggered by abrupt movements, emotional stress and fatigue. An Italian family has been identified where related members displayed continuous myokymia, episodes of ataxia, attacks characterized by myokymia only, and neuromyotonia. A novel missense mutation (F414C), in the C-terminal region of the K(+) channel Kv1.1, was identified in the affected individuals. The mutant homotetrameric channels were non-functional in Xenopus laevis oocytes. In addition, heteromeric channels resulting from the co-expression of wild-type Kv1.1 and Kv1.1(F414C), or wild-type Kv1.2 and Kv1.1(F414C) subunits displayed reduced current amplitudes and altered gating properties. This indicates that the pathogenic effect of this KCNA1 mutation is likely to be related to the defective functional properties we have identified.
Assuntos
Ataxia/genética , Saúde da Família , Canal de Potássio Kv1.1/genética , Mutação de Sentido Incorreto/genética , Mioquimia/genética , Adulto , Animais , Ataxia/complicações , Fenômenos Biofísicos , Cromossomos Humanos Par 12/genética , Cisteína/genética , Análise Mutacional de DNA , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Humanos , Itália , Canal de Potássio Kv1.2/genética , Masculino , Potenciais da Membrana/genética , Microinjeções/métodos , Modelos Moleculares , Mioquimia/complicações , Oócitos , Técnicas de Patch-Clamp/métodos , Fenilalanina/genética , Proteínas de Xenopus/genética , Xenopus laevis , Adulto JovemAssuntos
Pesos e Medidas Corporais/normas , Gráficos de Crescimento , Adolescente , Pesos e Medidas Corporais/métodos , Criança , Desenvolvimento Infantil/fisiologia , Pré-Escolar , Sistemas de Apoio a Decisões Clínicas/normas , Deficiências do Desenvolvimento/diagnóstico , Endocrinologia/métodos , Endocrinologia/normas , Feminino , Transtornos do Crescimento/diagnóstico , Humanos , Internacionalidade , Masculino , Seleção de Pacientes , Pediatria/métodos , Pediatria/normas , Padrões de Referência , Valores de Referência , Organização Mundial da Saúde , Adulto JovemRESUMO
We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.
Assuntos
Éxons/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Promielocítica Aguda/genética , Mutação/genética , Proteínas Nucleares/genética , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Citoplasma/metabolismo , Análise Mutacional de DNA , Humanos , Cariotipagem , Leucemia Promielocítica Aguda/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , NucleofosminaRESUMO
We have reported previously that among human prostate cancer cell lines LNCaP but not PC-3 cells undergo apoptosis after treatment with the protein kinase inhibitor staurosporine (STS). We have now further investigated this model to uncover the molecular mechanism causing resistance to STS-induced apoptosis in PC-3 cells. S-100 lysates of both cell lines showed biochemical changes typical of apoptosis after the addition of cytochrome c and dATP, suggesting that the postmitochondrial phase of apoptosis was intact. Upon addition of STS, the proapoptotic molecules Bax and Bad became predominantly mitochondrial in both cell lines. This, in turn, was followed by loss of mitochondrial transmembrane potential, translocation of cytochrome c to the cytosol, activation of caspase-9, -3, and -7, and cleavage of the apoptotic targets, DNA fragmentation factor and poly(ADP-ribose) polymerase, in LNCaP but not in PC-3 cells. Components of the mitochondrial permeability transition pore, adenine nucleotide transporter and voltage-dependent anion channel, were normally expressed in the correct subcellular fraction of both cell lines. Overexpression of the proapoptotic proteins Bax and Bad, fused to a green fluorescent protein but not of green fluorescent protein alone, induced apoptosis in >80% of PC-3 cells. These experiments suggested that a factor protecting the mitochondria of PC-3 cells mediates resistance to STS-induced apoptosis. A wide search among the antiapoptotic Bcl-2 family members was performed, and Bcl-X(L) was found to be overexpressed in PC-3 cells. Experiments down-regulating Bcl-X(L) expression by using the tyrosine kinase inhibitor genistein, sodium butyrate, or an antisense Bcl-X(L) oligonucleotide restored sensitivity to apoptosis in PC-3 cells. Thus, Bcl-X(L) overexpression is one of the mediators of resistance to STS-induced apoptosis in the prostate cancer cell line PC-3.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Inibidores Enzimáticos/farmacologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Estaurosporina/farmacologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Genisteína/farmacologia , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/fisiologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
We studied the molecular mechanisms of apoptosis in the prostate cancer cell line LNCaP and whether overexpression of caspase activity could force this cell line to undergo apoptosis. The inhibitor of phosphomevalonate decarboxylase, sodium phenylacetate, and the protein kinase inhibitor staurosporine induced (a) release of cytochrome c from the mitochondria to the cytosol; (b) reduction in mitochondrial transmembrane potential; (c) proteolytic processing of caspase-3 and -7 but not -2; (d) cleavage of the DEVD substrate and the death substrates poly(ADP-ribose) polymerase and DNA fragmentation factor; and (e) apoptosis. The panspecific inhibitor of caspase activation N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-FMK) prevented all of these events except release of mitochondrial cytochrome c into the cytosol. None of these apoptotic signaling events were elicited by staurosporine or sodium phenylacetate treatment of LNCaP-Bcl-2 cells that overexpress the oncoprotein Bcl-2. Because caspase-7 is activated in every model of apoptosis that we have characterized thus far, we wished to learn whether overexpression of this protease could directly cause apoptosis of LNCaP cells. By using a replication-defective adenovirus, overexpression of caspase-7 protein in both LNCaP and LNCaP-Bcl-2 cells was accompanied by induction of cleavage of the DEVD substrate and TUNEL. These studies have demonstrated that caspase-7 and -3 are critical mediators of apoptosis in LNCaP cells. Caspase-7 was proteolytically activated in every model of apoptosis that we have developed, and the overexpression of it induced apoptosis of LNCaP and LNCaP-Bcl-2 cells. Thus, adenoviral-mediated transfer of caspase-7 may offer a new effective approach for the treatment of prostate cancer.
Assuntos
Apoptose , Caspases/genética , Terapia Genética , Neoplasias da Próstata/terapia , Butiratos/farmacologia , Caspase 2 , Caspase 3 , Caspase 7 , Inibidores de Caspase , Caspases/metabolismo , Caspases/fisiologia , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Estaurosporina/farmacologia , Células Tumorais CultivadasRESUMO
The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which is implicated in the regulation of Ras, suggesting that Shc is involved in the intracellular transmission of growth signals from activated tyrosine kinases to Ras. Overexpression of Shc proteins in cultured fibroblasts induces a transformed phenotype. We now report that, in vitro, the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole Shc high affinity binding site for Grb2. Mutant Shc proteins with substitution of the Tyr317 by Phe lose the capacity to be highly phosphorylated on tyrosine upon growth factor receptor activation, to bind Grb2 and to induce neoplastic transformation. In contrast, Shc proteins that have an extensive aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc and support the notion that Shc proteins can deliver activation signals to RAS.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Transformação Celular Neoplásica , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Proteína Adaptadora GRB2 , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas/genética , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Tirosina/metabolismoRESUMO
Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor alpha (RAR alpha) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RAR alpha or PLZF/RAR alpha fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RAR alpha) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RAR alpha and compare it with the localization of PML and PML/RAR alpha. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RAR alpha is different to that of PLZF and RAR alpha. The nuclear distribution pattern of PLZF/RAR alpha is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RAR alpha did not provoke disruption of the PML-NBs. Co-expression of PML/RAR alpha and PLZF/RAR alpha in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.
Assuntos
Núcleo Celular/química , Proteínas de Ligação a DNA/análise , Proteínas de Neoplasias/análise , Proteínas Nucleares/análise , Fatores de Transcrição/análise , Imunofluorescência , Humanos , Fatores de Transcrição Kruppel-Like , Proteína da Leucemia Promielocítica , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores do Ácido Retinoico/análise , Proteínas Recombinantes de Fusão/análise , Receptor alfa de Ácido Retinoico , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.
Assuntos
Apoptose , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares , Fatores de Transcrição/fisiologia , Dedos de Zinco/fisiologia , Células 3T3 , Animais , Sítios de Ligação , Divisão Celular , Linhagem Celular Transformada , Sobrevivência Celular , Cisteína/genética , Cisteína/fisiologia , Citoplasma/metabolismo , Células HeLa , Histidina/genética , Histidina/fisiologia , Humanos , Isomerismo , Camundongos , Mutagênese , Proteínas de Neoplasias/genética , Proteína da Leucemia Promielocítica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Dedos de Zinco/genéticaRESUMO
PML/RAR alpha is the putative transforming sequence of acute promyelocytic leukemias. We investigated the effects of PML/RAR alpha on cell survival by expressing the fusion protein in the growth factor-dependent TF-1 cell line and analyzing the kinetics of cell death after GM-CSF deprivation. Results showed that PML/RAR alpha expression markedly delayed apoptotic cell death (3 weeks vs 1 week) without inducing growth factor independence. Growth factor deprivation caused rapid and massive apoptosis of control TF-1 cells (>95% apoptotic cells after 4-5 days). Factor-deprived control cells were synchronously and irreversibly committed to apoptosis as shown by their inability to re-enter the cell cycle after GM-CSF re-addition. The percentage of apoptotic cells in the PML/RAR alpha expressing cells was low (approximately 30-35%) and constant over the 4 weeks of factor deprivation. GM-CSF re-addition produced rapid increase in cell number at all time points during the 4 weeks of factor deprivation, suggesting that commitment to apoptosis was asynchronous and delayed in PML/RAR alpha-expressing TF-1 cells. We conclude that PML/RAR alpha interferes with the genetic pathways which regulate survival by reducing the frequency of commitment to apoptosis. This biological effect of PML/RAR alpha may contribute to its leukemogenetic potential.
Assuntos
Apoptose , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Apoptose/genética , Ciclo Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Acute promyelocytic leukaemia is characterized by an expansion of haematopoietic precursors arrested at the promyelocytic stage (1). The differentiation block can be reversed by retinoic acid, which induces blast differentiation both in vitro (2) and in vivo (3-4). Acute promyelocytic leukaemia is also characterized by a 15;17 chromosome translocation (5) with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and within the PML gene, that encodes a putative transcription factor of unknown function (6-7), on 15 (8-10). As a consequence of the translocation a PML/RAR alpha gene is formed. It is transcriptionally active and encodes a PML/RAR alpha fusion protein detectable in all APL cases (11-14). We expressed the PML/RAR alpha protein in U937 myeloid precursor cell line and show that they: 1) lose the capacity to differentiate under the action of different stimuli (vitamin D3, transforming growth factor beta 1); ii) acquire enhanced sensitivity to retinoic acid; iii) exhibit a higher growth rate that is due to a reduction in apoptotic cell death. These results provide the first evidence of biological activity of PML/RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.
Assuntos
Células-Tronco Hematopoéticas/citologia , Proteínas de Neoplasias , Proteínas Nucleares , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , DNA/análise , Humanos , Leucemia Promielocítica Aguda/patologia , Proteína da Leucemia Promielocítica , Receptores do Ácido Retinoico/análise , Sulfatos/farmacologia , Fatores de Transcrição/análise , Tretinoína/farmacologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Compostos de Zinco/farmacologia , Sulfato de ZincoRESUMO
The action that hyaluronic acid (HA) exerts on cell proliferation was investigated in embryonic chick skin fibroblasts at different ages (7-14 days) and in different cell-cycle stages evaluated by flow cytometry (cells maintained with and without serum). Proliferation was estimated by 3H-thymidine incorporation and cell counting. The results demonstrated hyaluronic acid inhibits cell multiplication in all different environmental conditions examined. The inhibitory effect of HA is more evident in 14-day than 7-day old fibroblasts. The ability of HA to modulate 3H-thymidine incorporation did not involve a change in the time required for cells entering the S phase of the replicating cycle, but is due to a smaller number of cells entering in this phase. As the relationships between components of the extracellular matrix (ECM) and the cytoskeleton are known, parallel studies were carried out on some cytoskeleton proteins. Furthermore, by modifying the capacity of cells to adhere to the substrate, HA induced alterations in cell shape and in cytoskeleton components involved in these processes. We may hypothesize that HA, binding specific membrane receptors, affects cell adhesion and morphology inducing less receptivity of fibroblasts to mitogenic stimuli by transmembrane interactions with cytoskeleton.
Assuntos
Citoesqueleto/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Actinina/análise , Actinina/metabolismo , Actinas/análise , Actinas/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Citoesqueleto/ultraestrutura , DNA/biossíntese , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Imunofluorescência , Pele/citologia , Timidina/metabolismo , Trítio , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismoRESUMO
Somatostatin (SS) and SS analogs have been shown to exert an antiproliferative effect on several transplantable tumors in animals and to reduce the growth of pancreatic, pituitary, and mammary tumor cells in vitro. We evaluated the effects that the SS analog SMS 201-995 exerts on growth, cell-cycle parameters, and suicidal cell death (apoptosis) of human breast cancer cells (MCF-7) in vitro. SMS 201-995 significantly reduced the MCF-7 cell growth induced by serum, estradiol, insulin, and insulin-like growth Factor-I in both short term and long term experiments. The effect was maximal when 10 nM estradiol was used as mitogen in long term cultures. SMS 201-995 treatment produced a slight but transient accumulation of cells in the G2/M phase but did not cause any noteworthy reduction in the percentage of proliferating cells. There was, instead, a time-related increase in the number of cells with the flow-cytometric characteristics of apoptosis in the cultures treated with the SS analog, which correlated well with its growth-inhibiting activity. It would, therefore, seem that SMS 201-995 exerts its inhibitory effect on MCF-7 cell growth in vitro mainly by enhancing the rate of programmed (or suicidal) cell death in the culture.
Assuntos
Neoplasias da Mama/patologia , Octreotida/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Células Tumorais CultivadasRESUMO
The effect of chronic administration of tolbutamide (150 mg/kg X day, orally, over 60 days) and glibenclamide (1 mg/kg X day, orally, over 60 days) on pancreatic A, B, and D cell function was investigated in male nondiabetic rats. In a first set of experiments pancreatic hormonal response to metabolic stimuli was evaluated during glucose (11.1 mM) or arginine (10 and 20 mM) infusion in the isolated perfused rat pancreas. The basal levels of insulin (IRI) and glucagon (IRG) were similar in control and in sulfonylurea-treated rats. Tolbutamide treatment markedly depressed the IRI response to glucose (P less than 0.005) or arginine (P less than 0.0005) infusion and the IRG response to arginine (P less than 0.01). After glibenclamide treatment, IRI decreased significantly (P less than 0.0005 and P less than 0.005, respectively) only in response to arginine infusion. This effect was still evident 10 days after the end of treatment. Furthermore, long term glibenclamide administration suppressed somatostatin (SRIF) response to glucose (P less than 0.0005) or arginine (P less than 0.0005). In a different group of rats treated with glibenclamide (1 mg/kg X day, orally, over 60 days) IRI, IRG, and SRIF plasma concentration and blood glucose levels were examined at the end of treatment. The results did not differ significantly from those of a control group. In the same animals, pancreatic IRI, IRG, and SRIF content, measured on acid-ethanol extracts, was reduced (P less than 0.1 vs. controls). These data clearly indicate that long term treatment with sulfonylurea drugs has a suppressive effect on pancreatic endocrine function in rats. The concomitant involvement of A, B, and D cell suggests that this effect is not specific.
Assuntos
Glibureto/administração & dosagem , Ilhotas Pancreáticas/metabolismo , Tolbutamida/administração & dosagem , Animais , Arginina/farmacologia , Glucagon/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos , Somatostatina/metabolismo , Fatores de TempoRESUMO
This study was performed in order to investigate the role of insulin in the modulation of pancreatic A cell response to glucose. The isolated perfused rat pancreas model was used: intraislet insulinopenia was induced in vitro by 0.56 mM alloxan infusion over 15 min. Alloxan caused a transitory insulin release but did not affect glucagon secretion. Exposure to alloxan completely abolished insulin response to 20 mM arginine, 1.6 mM glucose, and 11.1 mM glucose. Glucagon response to 20 mM arginine and 1.6 mM glucose was unchanged by alloxan pretreatment compared to control pancreata not treated with alloxan. However, the suppression of glucagon release by 11.1 mM glucose was abolished in the alloxan experiments. Twenty milliunits per ml of insulin infused during 11.1 mM glucose infusion restored glycemic suppression of glucagon release, but it produced only a slight inhibitory effect on A cell function in the presence of 3.9 mM glucose. Our study indicates that glucose is the physiological suppressor of the pancreatic A cell and that, in this regard, insulin exerts only a permissive effect.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Glucagon/metabolismo , Glucose/farmacologia , Insulina/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Aloxano/toxicidade , Animais , Arginina/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Perfusão , Ratos , Ratos EndogâmicosRESUMO
To investigate the site and mode of action of progesterone in inducing gonadotropin release, the effects of catecholamine-depleting (methyldopa) or dopamine agonist (bromocriptine) drugs on progesterone positive feedback and the gonadotropin response to a centrally acting noradrenergic drug (clonidine) were evaluated in estrogen-primed postmenopausal women. Progesterone administration induced a significant rise in LH, FSH, and PRL serum levels in the control group. Bromocriptine administration was followed by a marked suppression of PRL release but did not modify the gonadotropin response to progesterone. Methyldopa pretreatment significantly reduced the progesterone-induced LH surge, while PRL release was unaffected. After estrogen priming, clonidine administration did not result in an increase in serum LH or FSH concentrations. The dissociated responses of LH and PRL in bromocriptine-pretreated subjects and the significant reduction of the LH rise after progesterone in methyldopa-pretreated women seem to invalidate the hypothesis that a fall in endogenous dopamine is responsible for progesterone positive feedback and suggest that neural noradrenergic mechanisms are involved in progesterone-induced gonadotropin release. The ineffectiveness of a centrally acting noradrenergic agonist in inducing gonadotropin rise provides indirect evidence that an increased pituitary responsiveness may also be involved in progesterone positive feedback.
Assuntos
Bromocriptina , Gonadotropinas Hipofisárias/sangue , Menopausa , Metildopa , Progesterona , Idoso , Clonidina , Etinilestradiol , Retroalimentação , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Hormônio Luteinizante/sangue , Pessoa de Meia-Idade , Prolactina/sangueRESUMO
Genistein, a natural isoflavonoid phyto-oestrogen, inhibits the tyrosine kinase activity of growth factor receptors and oncogene products, as well as the in vitro growth of some tumour cell lines. The low incidence of breast cancer in countries with a flavonoid-rich soy-based diet and the protection afforded by soy-derived products against experimental mammary tumours in rats suggest that genistein and other isoflavonoid compounds may exert an anti-tumour activity. We analysed the effects of genistein on cell number and cell cycle progression (flow cytometric analysis of propidium iodide-stained nuclei) of human breast cancer cells (MCF-7) in vitro. Genistein produced a significant, dose-dependent inhibition of MCF-7 cell growth with an ID50 of approximately 40 microM after 72 h of incubation. Cell cycle analysis showed a reversible G2/M arrest in cell cycle progression at 10 microM genistein concentrations, whilst a marked fall in S-phase cell percentage associated with a persistent arrest in G2/M phase was observed in cultures treated with genistein doses equal to or greater than 50 microM. These effects were significant at 24 h of incubation; flow cytometric analysis at later times (48 and 72 h) revealed a population of cells with decreased DNA content and nuclear fragmentation characteristic of apoptosis. Thus, the growth inhibitory activity of genistein in MCF-7 cells results from the sum of cytostatic and apoptotic effects. Since the mitogenic action of insulin and insulin-like growth factor (IGF)-I in MCF-7 cells is a tyrosine kinase-dependent phenomenon, we analysed the genistein impact on S-phase entry produced by insulin in cultures partially synchronised in G0/G1 phase by serum deprivation. Insulin addition after a 36-h culture period in serum-free medium produced a strong increase in the percentage of S-phase cells (from 18.4 +/- 2.3 to 46.2 +/- 4.1 after 24 h) which was almost completely blocked by 100 microM genistein (20.1 +/- 3.1). Immunofluorescence analysis with a fluoresceine isothiocyanate (FITC)-conjugated anti-phosphotyrosine antibody revealed a strong increase in MCF-7 cell staining after insulin stimulation, but not when genistein was added with insulin. In conclusion, the dietary phyto-oestrogen genistein inhibits in vitro growth of MCF-7 human breast cancer cells through blocks in the "critical checkpoints" of cell cycle control and induction of apoptosis. These effects are likely to depend on impairment in the signal transduction pathway from tyrosine kinase receptor(s).
Assuntos
Neoplasias da Mama/patologia , Inibidores do Crescimento/farmacologia , Isoflavonas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Citometria de Fluxo , Imunofluorescência , Genisteína , Humanos , Antagonistas da Insulina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.
Assuntos
Inibidores do Crescimento/farmacologia , Isoflavonas/farmacologia , Mitocôndrias/metabolismo , Proteínas Tirosina Quinases/farmacologia , Rodaminas/metabolismo , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Neoplasias da Mama/metabolismo , Contagem de Células , Formazans/metabolismo , Genisteína , Humanos , Mitose/efeitos dos fármacos , Estimulação QuímicaRESUMO
Yondelis (trabectidin, ET-743) is a marine natural product that has shown activity both in preclinical systems and in human malignancies such as soft tissue sarcoma and ovarian cancers that are resistant to previous chemotherapies. Molecular pharmacological studies indicated that Yondelis interacts with DNA and DNA repair systems in a way that is different from Cisplatin (DDP). The current study was designed to investigate the effects of the combination of Yondelis and DDP in human cancer cell lines and in xenografts derived from different tumours. The in vitro studies performed in human TE-671 rhabdomyosarcoma, Igrov-1 and 1A9 human ovarian carcinoma cell lines showed additive effects or slight synergism. Several human tumour xenografts, such as TE-671 rhabdomyosarcoma, SK-N-DX neuroblastoma, FADU head and neck, LX-1 non-small cell lung cancer (NSCLC), H-187 melanoma and SKOV HOC 8 ovarian carcinoma, showed an antitumour effect for the combination that was greater than that of each drug when given as a single agent. No consistent changes in the activity were observed if Yondelis and DDP were given 1 h apart in sequence or simultaneously. An orthotopically transplanted human ovarian cancer HOC 8 growing in the peritoneal cavity of nude mice was used that is insensitive to Yondelis alone and only moderately sensitive to DDP alone. The combination of the two drugs produced a dramatic increase of survival lasting several months. In conclusion, the combination of Yondelis and DDP is synergistic in vivo (i.e. the antitumour effect is greater than that of each drug used as a single agent at the maximum tolerated dose (MTD)) in different human tumour xenografts. The two drugs can be combined at the MTD of each drug, thus indicating there are no overlapping toxicities. These results provide a rationale for testing the combination of Yondelis and DDP in the clinic.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Dioxóis/administração & dosagem , Dioxóis/efeitos adversos , Sinergismo Farmacológico , Feminino , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/efeitos adversos , Camundongos , Transplante de Neoplasias , Tetra-Hidroisoquinolinas , Trabectedina , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into oligonucleosomal subunits. This type of cell death (apoptosis), which physiologically occurs in the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colorimetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4 degrees C) incubation or cycloheximide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodium azide, a substance with cell-killing activity through non-apoptotic mechanisms, did not result in any variation in the normal DNA peak. The flow cytometric data showed an excellent correlation with the results obtained with both electrophoretic and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes.