RESUMO
Meiotic maturation of oocytes requires a variety of ATP-dependent reactions, such as germinal vesicle breakdown, spindle formation, and rearrangement of plasma membrane structure, which is required for fertilization. Mitochondria are accordingly expected be localized to subcellular sites of energy utilization. Although microtubule-dependent cellular traffic for mitochondria has been studied extensively in cultured neuronal (and some other somatic) cells, the molecular mechanism of their dynamics in mammalian oocytes at different stages of maturation remains obscure. The present work describes dynamic aspects of mitochondria in porcine oocytes at the germinal vesicle stage. After incubation of oocytes with MitoTracker Orange followed by centrifugation, mitochondria-enriched ooplasm was obtained using a glass needle and transferred into a recipient oocyte. The intracellular distribution of the fluorescent mitochondria was then observed over time using a laser scanning confocal microscopy equipped with an incubator. Kinetic analysis revealed that fluorescent mitochondria moved from central to subcortical areas of oocytes and were dispersed along plasma membranes. Such movement of mitochondria was inhibited by either cytochalasin B or cytochalasin D but not by colcemid, suggesting the involvement of microfilaments. This method of visualizing mitochondrial dynamics in live cells permits study of the pathophysiology of cytoskeleton-dependent intracellular traffic of mitochondria and associated energy metabolism during meiotic maturation of oocytes.
Assuntos
Espaço Intracelular/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Oócitos/metabolismo , Animais , Transporte Biológico , Citoesqueleto/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Cinética , Microscopia Confocal , Microtúbulos/metabolismo , Suínos , Imagem com Lapso de Tempo/métodosRESUMO
The purpose of performing pleural cauterization is developing heat denaturation, and we can induce pleural thickening and also reduce the bullae by shrinking the pleura It originates in a method of the cauterization whether there will be tissue damage. So a safe and reliable method of cauterization is required. Here, we investigated the indications for and effectiveness of cauterization techniques performed at our facility. We perform cauterization while dropping saline solution, so when using a Salient Monopolar Sealer, we can avoid excessive thermo-coagulation and more easily control cauterization. Furthermore, on the basis that only emphysematous pleura will turn white on cauterization, bullae can be distinguished, which is particularly effective in the case of lesions with unclear borders. In the case of a large emphysematous bulla, shrinkage of the bulla by cauterization can provide a sufficient surgical field, and a smaller lesion can then be stapled.
Assuntos
Vesícula/cirurgia , Cauterização/métodos , Enfisema Pulmonar/cirurgia , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BCL-6 gene alterations have been observed in 27-45% of diffuse large B-cell lymphomas (DLBs) with chromosomal translocations at 3q27. The deregulated expression of normal BCL-6 protein caused by this chromosomal translocation is believed to be responsible for lymphomagenesis. Recently, we demonstrated that BCL-6 is expressed at high levels in germinal center B-cells as a 92-98 kDa nuclear protein in a constitutively phosphorylated form. In this study, we show that BCL-6 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro at the sites phosphorylated in vivo. These numerous phosphorylation sites were found to be located in its serine- and proline-clustered (SPC) region (amino acids-250-483). BCL-6 phosphorylation significantly increased in Ramos cells following stimulation with 12-o-tetradecanoylphorbol-13-acetate (TPA) or BCL-6- and erk1-transfected COS-7 cells stimulated with epidermal growth factor (EGF), and the increase of phosphorylation was inhibited by MEK1 inhibitor, PD98059. Furthermore, we observed that BCL-6 was associated with MAPK in vivo and its SPC region was important for this association. These results suggest that the functions of BCL-6 are regulated by phosphorylation mediated by the MAPK signaling pathway.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The BCL-6 proto-oncogene encodes a 92- to 98-kDa transcriptional repressor containing the BTB/POZ domain at its N-terminal region and the zinc finger domain at its C-terminal region, respectively. In the present study, we examined the function of BCL-6 by using a recombinant adenovirus expressing BCL-6 (Ax1CA-BCL-6) and the lacZ reporter gene (Ax1CA-lacZ). Viability of CV-1 and HeLa cells infected with Ax1CA-BCL-6 was markedly reduced due to apoptosis, suggesting that BCL-6-overexpression induces apoptosis in CV-1 and HeLa cells. FACS analysis revealed that BCL-6-overexpressing cells are accumulated not only at the sub-G1 but also at G2/M phase. Induction of apoptosis by BCL-6 was preceded by down-regulation of apoptosis repressors BCL-2 and BCL-X(L). These results suggest that BCL-6 induces apoptosis by regulating the expression of these apoptosis-regulating genes.
Assuntos
Adenoviridae/genética , Apoptose/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Ciclo Celular/genética , Linhagem Celular , Células HeLa , Humanos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6 , Recombinação Genética , Transdução Genética , Proteína bcl-XRESUMO
We demonstrated in the present study that the BCL-6 transcripts were detectable not only in B cells, but also in circulating granulocytes and monocytes from normal individuals, and in human acute nonlymphocytic leukemia cells of certain subtypes (M3, M4, M5). Then, with an assumption that the BCL-6 gene expression may be related to the differentiation of myeloid cells, we analyzed the inducibility of BCL-6 gene expression along monocytic lineage differentiation in HL-60 and U-937 cells by treating them with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although the expression of BCL-6 transcripts was very low or undetectable in untreated HL-60 or U-937 cells, treatment of these cells with TPA to induce monocytic differentiation resulted in an apparent increase of BCL-6 mRNA, suggesting that BCL-6 gene expression is not limited to B cells and it is closely associated with monocytic lineage differentiation. The BCL-6 transcripts in TPA-treated U-937 cells were superinduced by the treatment with cycloheximide (CHX) and the half-life of the BCL-6 mRNA was apparently prolonged when TPA-treated U-937 cells were exposed to CHX in the presence of actinomycin D (ACD). Furthermore, the nuclear run-on assay revealed that the BCL-6 transcription signals were enhanced by TPA treatment. These results suggest that the increase of BCL-6 mRNA in U-937 cells stimulated with TPA to induce monocytic lineage differentiation is mediated by both transcriptional and post-transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação Neoplásica da Expressão Gênica , Células HL-60/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Fatores de Transcrição/biossíntese , Adulto , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas c-bcl-6 , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Previously, we found that the islet of pancreas stained with a antibody against the vascular permeability factor (VPF; also known as vascular endothelial growth factor, VEGF) protein. To determine how common this reaction was and whether it was a specific reaction for the islet, we examined its expression and specific cellular localization. Two different antibodies directed against VPF/VEGF peptide revealed an intense reaction for beta-cells in the human islets of Langerhans, and several human beta-cell tumors (insulinomas), but no reaction, were detectable in the vascular endothelium. In the fetal pancreas (second and third trimesters), the VPF/VEGF peptide was detected in immature islets. Northern blot analysis of cell lines derived from rodent insulinomas revealed expression of VPF/VEGF messenger ribonucleic acid. Western blot analysis of conditioned medium from one of these cell lines showed the presence of the released VPF/VEGF protein. These findings indicate that beta-cells have a specific role other than endocrine function in the pancreas. VPF/VEGF in beta-cells may be involved in the maintenance and control of permeability within the islet capillary system.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Linfocinas/metabolismo , Adulto , Sequência de Bases , Northern Blotting , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Sondas Moleculares/genética , Dados de Sequência Molecular , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The histological differentiation of thyroid carcinoma is known to correlate with prognosis. Ras oncogene mutations, which have been identified in various human cancers, have been suspected playing an important role in carcinogenesis and tumor progression. The purpose of this study was to clarify the mechanism of thyroid tumor progression, focusing on ras oncogenes. We examined ras mutations using nested polymerase chain reaction (PCR) and direct sequencing methods. The ras oncogene product was also examined immunohistochemically. Our results indicated that the incidence of ras mutations correlated with the histological differentiation of thyroid cancer. Three poorly differentiated carcinomas showed a higher rate of ras mutations than did 17 well-differentiated counterparts. Hot spots were not identified except for a relative accumulation of the N-ras gene at codon 61. There was a correlation between the immunoreactivity of the ras oncogene product and ras mutation, although the immunoreactivity of ras-p21 did not correlate with the histological differentiation. Mutation of the ras gene seemed to be one of the important events in the progression from well-differentiated carcinoma to poorly differentiated thyroid carcinoma.
Assuntos
Adenocarcinoma Folicular/genética , Adenoma/genética , Carcinoma Papilar/genética , Genes ras/genética , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias da Glândula Tireoide/genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Adenoma/química , Adenoma/patologia , Adolescente , Adulto , Idoso , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Primers do DNA/química , DNA de Neoplasias/análise , Progressão da Doença , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
ik3-1/Cables is associated with and phosphorylated by cdk3 in self-replicating cells. In postmitotic neurons, it may serve as an adaptor molecule, functionally connecting c-abl and cdk5, and supporting neurite growth. Here, we cloned cDNAs coding for mouse Trap (tudor repeat associator with Pctaire 2) to interact with ik3-1. ik3-1 interacts with a region of mouse Trap containing the C-terminal tudor repeat domains 4 and 5 (corresponding to amino acids 881-1086 of mouse Trap). Furthermore, the N-terminal 93-amino-acid domain of ik3-1 is essential for ik3-1 interaction with Trap. Moreover, ik3-1 is coimmunoprecipitated with Pctaire 2 from COS7 cells, although we could not clarify whether ik3-1 is directly associated with Pctaire 2 or indirectly associated with Pctaire 2 through Trap. In vitro kinase assay indicated that ik3-1 does not activate phosphorylation of myelin basic protein or histione H 1 by the Pctaire 2-mediated kinase. These findings led us to speculate that through ik3-1, the Pctaire family and Trap may be functionally connected with cdk3 or cdk5.
Assuntos
Proteínas de Transporte/química , Ciclinas/química , Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/química , Sequência de Aminoácidos , Animais , Células COS , Clonagem Molecular , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , DNA Complementar/metabolismo , Ativação Enzimática , Biblioteca Gênica , Immunoblotting , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteína Básica da Mielina/química , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
CD26/dipeptidyl peptidase IV (DPPIV) is a cell surface-bound ectopeptidase with important roles in T-cell activation and tumour biology. We now report that CD26/DPPIV enhances sensitivity to apoptosis induced by the antineoplastic agents doxorubicin and etoposide. In particular, CD26/DPPIV presence is associated with increased susceptibility to the mitochondrial pathway of apoptosis, documented by enhanced cleavage of poly (ADP ribose) polymerase (PARP), caspase-3 and caspase-9, Bcl-xl, and Apaf-1, as well as increased expression of death receptor 5 (DR5). We also show that the caspase-9-specific inhibitor z-LEHD-fmk inhibits drug-mediated apoptosis, leading to decreased PARP and caspase-3 cleavage, and reduced DR5 expression. Importantly, through detailed studies that demonstrate the association between topoisomerase II alpha expression and DPPIV activity, our data provide further evidence of the key role played by CD26 in biological processes.
Assuntos
Apoptose/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Dipeptidil Peptidase 4/fisiologia , Inibidores Enzimáticos/farmacologia , Anexina A5/metabolismo , Antígenos de Neoplasias , Antineoplásicos/farmacologia , Fator Apoptótico 1 Ativador de Proteases , Western Blotting , Caspases/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/enzimologia , Células Jurkat/patologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Propídio/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/metabolismo , Inibidores da Topoisomerase II , Transfecção , Proteína bcl-XRESUMO
CD26/dipeptidyl peptidase IV (DPPIV) is a surface antigen with multiple functions, including a role in T-cell activation and the development of certain human cancers. We previously demonstrated that CD26/DPPIV enhanced sensitivity of Jurkat cells to doxorubicin. We now show that expression of CD26/DPPIV enhanced sensitivity of CD26 Jurkat transfectants to G(2)-M arrest mediated by the antineoplastic agent etoposide. The increased sensitivity to etoposide-induced G(2)-M arrest was associated with disruption of cell cycle-related events, including hyperphosphorylation of p34(cdc2) kinase, change in cdc25C expression and phosphorylation, and alteration in cyclin B1 expression. CD26/DPPIV-associated enhancement of doxorubicin and etoposide-induced G(2)-M arrest was also observed in serum-free media, suggesting an effect of CD26 on cell-derived processes rather than serum-derived factors. Importantly, our work elucidated a potential mechanism for the enhanced susceptibility of CD26-expressing Jurkat cells to the topoisomerase II inhibitors by demonstrating that CD26/DPPIV surface expression was associated with increased topoisomerase II alpha levels and enhanced enzyme activity. Besides being the first to show a functional association between the multifaceted molecule CD26 and the key cellular protein topoisomerase II alpha, our studies provide additional evidence of a potential role for CD26 in the treatment of selected malignancies.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Fase G2/efeitos dos fármacos , Mitose/efeitos dos fármacos , Inibidores da Topoisomerase II , Antígenos de Neoplasias , Antineoplásicos Fitogênicos/farmacologia , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Ciclina B1 , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Interações Medicamentosas , Humanos , Células Jurkat , Transfecção , Fosfatases cdc25/metabolismoRESUMO
Intracellular redistribution of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early tumorigenic event in most colorectal tumours. In serrated adenoma (SA), a newly recognised subtype of colorectal adenoma, APC mutations are uncommon, and the contribution of beta-catenin to tumorigenesis remains unclear. We compared intracellular localisation of beta-catenin and presence of mutations in exon 3 of beta-catenin between 45 SAs, with 71 conventional adenomas (CADs), and eight carcinomas invading the submucosa (SCAs). Widespread or focal nuclear beta-catenin expression was demonstrated in 7% of SAs (three out of 45), 61% of CADs (43 out of 71), and 88% of SCAs (seven out of eight). Cytoplasmic immunostaining for beta-catenin was demonstrated in 16% of SAs (seven out of 45), 77% of CADs (55 out of 71), and 88% of SCAs (seven out of eight). No mutation in exon 3 of beta-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had beta-catenin mutations. No nuclear or cytoplasmic expression of beta-catenin was observed in the hyperplastic or conventionally adenomatous epithelium of mixed-type SAs. These findings suggest that beta-catenin mutation is unlikely to contribute to the tumorigenesis in SA, and that intracellular localisation of beta-catenin may not be associated with an early event of the tumour progression in most SAs.
Assuntos
Adenoma/genética , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/farmacologia , Regulação Neoplásica da Expressão Gênica , Transativadores/genética , Transativadores/farmacologia , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Caderinas , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Progressão da Doença , Feminino , Humanos , Líquido Intracelular/química , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , beta CateninaRESUMO
p70ik3-1 (a 70-kDa protein) contains a cyclin box, and binds to p35cdk3 in vivo and in vitro [Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun. 273, 442-447]. In spite of its structural similarity to cyclins, p70ik3-1 does not activate cyclin-dependent kinase 3 (cdk3)-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3-1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1 is upregulated. We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fosfoproteínas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 3 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Ciclinas/genética , DNA Complementar/genética , Humanos , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Especificidade por Substrato , TransfecçãoRESUMO
The BCL-6 gene is known to be located on chromosome 3q27, at the breakpoint of the 3q27-associated translocations that occur frequently in human non-Hodgkin's lymphomas (NHLs). To identify the BCL-6 protein, two antibodies that recognized distinct domains of this protein were raised in rabbits. Immunoprecipitation and immunoblotting of lysates of BCL-6-expressing cells using both antibodies showed a broad 92- to 98-kD band. Dephosphorylation of BCL-6 protein reduced the size of this band to 87 kD, suggesting that BCL-6 may be expressed in a phosphorylated form. Immunostaining with both antibodies showed that BCL-6 protein was localized in the nuclei of most of the germinal center B cells and a small number of marginal zone B cells. Furthermore, BCL-6 protein was expressed in follicular, Burkitt's, and diffuse large B-cell lymphomas. These results suggest that the BCL-6 protein, expressed in B cells of the germinal centers which are important in the maturation of immune responses, may play some physiological role(s) in the germinal center B cells.