RESUMO
The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA onto chromosome ends. Telomere length is maintained, by the presence of telomerase activity, in the vast majority of primary tumours and stem cells, suggesting that telomere maintenance is essential for cellular immortalization. Recently, the telomerase RNA component in human and mouse (TERC and Terc, respectively), a telomerase-associated protein TEP1/TLP1 (refs 6,7) and the human catalytic subunit protein TERT (refs 8,9) have been identified. To examine the role of telomerase in telomere maintenance and cellular viability, we established Terc-deficient embryonic stem (ES) cells. It is known that telomerase activity is absent in cells from Terc-knockout mice. Although the study showed that telomere shortening was observed in the Terc-deficient cells from first to six generation animals, whether telomerase-dependent telomere maintenance was essential for cellular viability remained to be elucidated. To address this issue, we examined Terc-deficient ES cells under long-term culture conditions. Accompanying the continual telomere shortening, the growth rate of Terc-deficient ES cells was gradually reduced after more than 300 divisions. An impaired growth rate was maintained to approximately 450 divisions, and then cell growth virtually stopped. These data clearly show that telomerase-dependent telomere maintenance is critical for the growth of mammalian cells.
Assuntos
RNA não Traduzido , RNA/fisiologia , Células-Tronco/citologia , Telomerase/fisiologia , Animais , Divisão Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Proteínas/metabolismo , RNA/genética , RNA Longo não Codificante , Mapeamento por Restrição , Telomerase/genética , Telômero/metabolismoRESUMO
Thymocytes mature through several stages of development, defined by cell surface markers such as CD3, CD4, and CD8, in response to environmental cues. Signal transduction resulting from lymphocyte-stromal cell interactions is likely to activate inducible transcription factors which in turn govern stage-specific gene expression. In this report we show that inducible transcription factors such as AP-1 and NF-AT are constitutively nuclear, in response to intrathymic signals, in freshly isolated thymocytes at all stages of maturation. In CD4+CD8+ double positive (DP), but not in the more immature CD4-CD8- double negative (DN) thymocytes, constant stimulus from the thymic environment is required to maintain nuclear AP-1. Thus, disruption of the thymus and incubation of thymocytes at 37 degrees C downregulates DNA binding by nuclear factors AP-1 and NF-AT. Similar treatment of thymocytes has previously been shown to downregulate CD3 zeta chain phosphorylation and increase T cell receptor CD3 expression on DP thymocytes, which is a feature of repertoire selection. Since mature T cells maintain inducible nuclear factors in an inactive form until an encounter with antigen, we propose that downregulation of nuclear DNA binding proteins may reflect another feature of this stage of T cell maturation.
Assuntos
Envelhecimento/imunologia , Núcleo Celular/metabolismo , Proteínas Nucleares , Linfócitos T/imunologia , Timo/imunologia , Fatores de Transcrição/metabolismo , Animais , Complexo CD3/biossíntese , Antígenos CD4/análise , Antígenos CD4/biossíntese , Antígenos CD8/análise , Antígenos CD8/biossíntese , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Fatores de Transcrição NFATC , Transdução de Sinais , Linfócitos T/metabolismo , Timo/crescimento & desenvolvimento , Timo/metabolismo , Fator de Transcrição AP-1/metabolismoRESUMO
The cytotoxic activity and pore-forming protein (PFP) expression of human peripheral blood (PB) gamma/delta T cells were examined. Fresh gamma/delta T cells isolated from PB lymphocytes by fluorescence-activated cell sorting exhibited a substantial natural killer-like cytotoxic activity against K562 target cells and had a high cytotoxic potential triggered by anti-CD3 monoclonal antibody (mAb) against P815 target cells bearing Fc gamma R. Immunocytochemical staining with an anti-PFP mAb revealed that virtually all PB gamma/delta T cells are granular lymphocytes with abundant PFP in their cytoplasmic granules. Constitutive expression of PFP in PB gamma/delta T cells was also demonstrated by Northern blot analysis. These observations support the proposed role of gamma/delta T cells in cytolytic immune surveillance in vivo.
Assuntos
Citotoxicidade Imunológica , Glicoproteínas de Membrana , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Anticorpos Monoclonais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos CD8 , Células Cultivadas , Humanos , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Proteínas de Membrana/análise , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMO
As a consequence of positive selection in the thymus, immature CD4(+)8(+) double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4(+) or CD8(+) T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)-CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or zeta chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.
Assuntos
Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Linhagem da Célula , CamundongosRESUMO
Our studies have analyzed pore-forming protein (PFP) mRNA expression in resting and stimulated human peripheral blood CD3- large granular lymphocytes (LGL), CD3+ T cells, and their CD4+ or CD8+ subsets. Signals that stimulate T cells to develop cytotoxic activity (i.e., IL-2 or OKT-3 mAb) led to the induction of PFP mRNA in T cells. The data indicated that IL-2 directly increased PFP mRNA in the CD8+ subset of T cells, in the absence of new DNA or protein synthesis. Abrogation of IL-2-induced PFP mRNA expression and cytotoxic potential of T cells by the anti-p75 IL-2 receptor mAb suggested that low numbers of p75 IL-2 receptors on CD8+ T cells were capable of transducing signals responsible for these IL-2-induced effects. The induction of T cell PFP mRNA via CD3, using OKT-3 mAb, was less rapid but greater than that caused by IL-2; however, a combination of PMA and ionomycin, which bypasses crosslinking of the TCR/CD3 complex, could not mimic this increase in PFP mRNA levels in T cells. The role of second messenger systems in regulating PFP mRNA expression remains to be determined. In contrast, high constitutive PFP mRNA expression was observed in CD3- LGL and these mRNA levels could not be enhanced by stimulation with IL-2. The cytotoxic potential of peripheral blood T cells and LGL induced in response to IL-2 correlated with IL-2-induced PFP mRNA levels in these cells and was consistent with PFP being one of several important molecules involved in the effector function of cytotoxic lymphocytes.
Assuntos
Expressão Gênica/efeitos dos fármacos , Interleucina-2/farmacologia , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Linfócitos T Citotóxicos/metabolismo , Anticorpos Monoclonais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3 , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Interferon gama/biossíntese , Cinética , Ativação Linfocitária , Perforina , Proteínas Citotóxicas Formadoras de Poros , RNA/sangue , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/análise , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
We have recently identified a dominant wave of CD4-CD8- (double-negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III-CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double-positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination-activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement-competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.
Assuntos
Antígenos de Diferenciação de Linfócitos T/biossíntese , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de IgG/biossíntese , Receptores Imunológicos/biossíntese , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Envelhecimento/imunologia , Animais , Sequência de Bases , Antígenos CD2 , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Ciclo Celular , Separação Celular , DNA , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/embriologia , Timo/crescimento & desenvolvimentoRESUMO
OBJECTIVES: Visceral fat accumulation is detrimental for brain health and is associated with cognitive impairment in older adults. The objectives of the present study were to examine the association between visceral fat accumulation and prevalence of mild cognitive impairment and its subtypes. DESIGN: a cross-sectional study. PARTICIPANTS: This study enrolled 6,109 community-dwelling older adults, including 3,434 women (mean age: 74.4 years) and 2,675 men (mean age: 74.3 years). Individuals with dementia, Parkinson's disease, stroke, Mini-Mental State Examination scores ≤23, and who could not perform basic activities of daily living independently were excluded. MEASUREMENTS: Participants underwent neurocognitive assessments to assess mild cognitive impairment (MCI) and its subtypes. Visceral fat area (VFA) was measured using abdominal bioelectrical impedance analysis. Participants were divided into quartile groups by VFA. RESULTS: There were 731 (21.3%) women and 562 (21.0%) men with MCI, and the median VFA values were 63.3 cm2 and 96.3 cm2, respectively. Women participants in the second (adjusted odds ratios [aOR], 0.71; 95% confidence interval [95% CI], 0.54-0.94), third (aOR, 0.66; 95% CI, 0.47-0.92), and fourth quartiles of VFA (aOR, 0.62; 95% CI, 0.41-0.93) had a significantly lower risk of MCI than those in the first quartile. Higher VFA quartiles in women were associated with lower risk of non-amnestic MCI. There were no significant differences in men between quartiles. CONCLUSIONS: Visceral fat accumulation was associated with MCI, especially non-amnestic MCI, in community-dwelling older Japanese women. These results suggest that visceral fat accumulation is partially protective against cognitive impairment.
Assuntos
Disfunção Cognitiva/etiologia , Gordura Intra-Abdominal/fisiopatologia , Idoso , Estudos Transversais , Feminino , Humanos , Vida Independente , JapãoRESUMO
Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.
Assuntos
Complexo CD3/genética , Proteínas de Ligação a DNA , Proteínas/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Sequência de Bases , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Membrana Celular/imunologia , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Timo/imunologiaRESUMO
The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.
Assuntos
Linfócitos B/imunologia , Proteínas Proto-Oncogênicas/fisiologia , Linfócitos T/imunologia , Animais , Apoptose , Linfócitos B/citologia , Sequência de Bases , Medula Óssea/imunologia , Células da Medula Óssea , Complexo CD3/imunologia , Linhagem Celular , Quimera , Homozigoto , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proto-Oncogenes , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologiaRESUMO
Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasu's arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasu's arteritis. We also examined the expression of a 65-kD heat-shock protein (HSP-65), human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 in the aortic tissue. Immunohistochemical studies showed that the infiltrating cells mainly consisted of gamma delta T lymphocytes, natural killer cells, macrophages, cytotoxic T lymphocytes and T helper cells, and that perforin was expressed in gamma delta T lymphocytes, natural killer cells, and cytotoxic T lymphocytes. In situ hybridization analysis also revealed expression of perforin mRNA in the infiltrating cells. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly onto the surface of arterial vascular cells. We also found that expression of HSP-65, human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 was strongly induced in the aortic tissue and might facilitate the recognition, adhesion and cytotoxicity of the infiltrating killer lymphocytes. These findings provide the first direct evidence that the infiltrating cells in the aortic tissue mainly consist of killer cells, and strongly suggest that these killer cells, especially gamma delta T lymphocytes, may recognize HSP-65 and play a critical role in the vascular cell injury of Takayasu's arteritis by releasing perforin.
Assuntos
Aorta/metabolismo , Aorta/patologia , Proteínas de Choque Térmico/biossíntese , Glicoproteínas de Membrana/biossíntese , Arterite de Takayasu/metabolismo , Arterite de Takayasu/patologia , Adulto , Antígenos CD/análise , Antígenos CD/biossíntese , Biomarcadores/análise , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/biossíntese , Feminino , Antígenos HLA-D/análise , Antígenos HLA-D/biossíntese , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Imuno-Histoquímica , Hibridização In Situ , Molécula 1 de Adesão Intercelular , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T Citotóxicos/metabolismoRESUMO
Inactivation of recombination activating gene (RAG)-1 or RAG-2 in mice results in the inability of developing lymphocytes to initiate V(D)J recombination, leading to the arrest of lymphocyte differentiation at a very early stage. Introduction of functionally assembled antigen-receptor genes or other potentially relevant genes into the RAG-deficient background can bypass the V(D)J recombination block and promote differentiation of the lymphocytes of RAG-deficient mice to various stages. This approach offers new means for analyzing the control of lymphocyte differentiation. In addition, generation of somatic chimeric mice by injecting mutant embryonic stem cells into the RAG-2-deficient blastocysts has also provided a powerful new method for assaying the potential roles of genes or regulatory elements in lymphocyte development or function.
Assuntos
Proteínas de Ligação a DNA , Genes RAG-1 , Sistema Imunitário/fisiologia , Proteínas/genética , Animais , Linfócitos B/fisiologia , Diferenciação Celular/genética , Sistema Imunitário/crescimento & desenvolvimento , Camundongos , Recombinação Genética/genética , Linfócitos T/fisiologiaRESUMO
Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.
Assuntos
Telomerase/fisiologia , Telômero/fisiologia , Animais , Sequência de Bases , Sobrevivência Celular , Clonagem Molecular , DNA , DNA Complementar , Camundongos , Dados de Sequência Molecular , Células-Tronco , Telomerase/genéticaRESUMO
Epigenetic gene regulation linked to oncogenic pathways is an important focus of cancer research. KDM3A, a histone H3 lysine 9 (H3K9) demethylase, is known to have a pro-tumorigenic function. Here, we showed that KDM3A contributes to liver tumor formation through the phosphatidylinositol 3-kinase (PI3K) pathway, which is often activated in hepatocellular carcinoma. Loss of Kdm3a attenuated tumor formation in Pik3ca transgenic (Tg) mouse livers. Transcriptome analysis of pre-cancerous liver tissues revealed that the expression of activator protein 1 (AP-1) target genes was induced by PI3K activation, but blunted upon Kdm3a ablation. Particularly, the expression of Cd44, a liver cancer stem marker, was regulated by AP-1 in a Kdm3a-dependent manner. We identified Cd44-positive hepatocytes with epithelial-mesenchymal transition-related expression profiles in the Pik3ca Tg liver and confirmed their in vivo tumorigenic capacity. Notably, the number and tumor-initiating capacity of Cd44-positive hepatocytes were governed by Kdm3a. As a mechanism in Kdm3a-dependent AP-1 transcription, Kdm3a recruited c-Jun to the AP-1 binding sites of Cd44, Mmp7 and Pdgfrb without affecting c-Jun expression. Moreover, Brg1, a component of the SWI/SNF chromatin remodeling complex, interacted with c-Jun in a Kdm3a-dependent manner and was bound to the AP-1 binding site of these genes. Finally, KDM3A and c-JUN were co-expressed in 33% of human premalignant lesions with PI3K activation. Our data suggest a critical role for KDM3A in the PI3K/AP-1 oncogenic axis and propose a novel strategy for inhibition of KDM3A against liver tumor development under PI3K pathway activation.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Epigênese Genética , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Transdução de SinaisRESUMO
Telomerase, a ribonucleoprotein complex, adds hexameric repeats called telomeres to the growing ends of chromosomal DNA. The enzyme telomerase activity is present in a vast majority of tumors but is repressed in most normal tissues. Recently, two groups have reported the molecular cloning of the putative catalytic subunit (hEST2/hTRT) of the telomerase gene. We investigated the expression of this gene in diverse tumor-derived cell lines and tumors as well as in various normal tissues. The expression of hEST2/hTRT was detectable in tumor-derived cell lines, primary breast tumors, pancreatic tumors, and kidney tumors. Furthermore, the expression of hEST2/hTRT was down-regulated in response to a differentiation inducer. However, several normal tissues also expressed varying levels of hEST2/hTRT. Early passage cultures of endothelial fibroblasts and some epithelial cells also expressed the telomerase gene, albeit at low levels. In contrast, the expression of TLP1/TP1, the human homologue of Tetrahymena p80 telomerase subunit, was similar in all of these samples. Our results indicate that the differences in expression of hEST2/hTRT in tumor versus normal cells are relative and are not absolute.
Assuntos
Neoplasias/enzimologia , Telomerase/genética , Neoplasias da Mama/enzimologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Expressão Gênica , Células HL-60 , Humanos , Reação em Cadeia da Polimerase , Distribuição Tecidual , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The results of well-characterized two-site enzyme immunoassays showed that the crude leptomeninges (consisting of the pia matter, arachnoid matter, and leptomeningeal vessels [LV]) from aged control brains and brains affected by Alzheimer disease (AD) contain very high levels of amyloid beta-protein (A beta). To learn about the source of A beta, we carefully dissected out both leptomeninges (LM) and LV under a dissecting microscope and determined the levels of soluble A beta in each. The purity of these dissected tissues was confirmed by the absence or presence of alpha-smooth muscle actin representing LV by Western blotting. Surprisingly, the amounts of A beta in each dissected sample were nearly equivalent on a weight basis. In each compartment from aged controls the level of A beta 1-42 was comparable to that of A beta 1-40, while in AD brain A beta 1-40 was a predominant species in both LM and LV. In some cases careful immunocytochemical examination revealed the presence of A beta deposits that were immunolabeled by several A beta monoclonal antibodies in leptomeningeal layers (most often in the arachnoid matter). The extent of A beta deposition in LM appeared to be much less than that explained by the soluble A beta levels, suggesting that immunocytochemically undetectable A beta can accumulate in LM. These observations indicate that leptomeninges are a large reservoir of A beta in normal aged individuals and in AD patients.
Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Aracnoide-Máter/metabolismo , Pia-Máter/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aracnoide-Máter/irrigação sanguínea , Aracnoide-Máter/citologia , Vasos Sanguíneos/metabolismo , Células Cultivadas , Humanos , Imuno-Histoquímica , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patologia , Meningioma/metabolismo , Meningioma/patologia , Pessoa de Meia-Idade , Pia-Máter/irrigação sanguínea , Pia-Máter/citologia , Testes de Precipitina , SolubilidadeRESUMO
Immunization of C57BL/6 mice with syngeneic tumor cells, MBL-2, resulted in the generation of antitumor effector cells in vivo. The immunized C57BL/6 mice permanently rejected viable MBL-2 lymphoma cells, but not B16 melanoma cells. Cytotoxic T cells obtained from MBL-2-immunized mouse peritoneal cells (PEC) showed specific cytotoxicity against MBL-2, but not to YAC-1, RDM-4 and Meth A cells. By sorting with FACStar, the specific CTL were characterized as TCR alpha beta+ CD8+ T cells. Moreover, the cytoplasm of in vivo-induced CTL was stained with a monoclonal antibody against perforin. The localization of perforin in cytoplasmic granules of CTL was demonstrated by electron microscope analysis. This experiment presented the first evidence that in vivo-induced CD8+ CTL against syngeneic tumor cells expressed significant amounts of perforin.
Assuntos
Glicoproteínas de Membrana , Proteínas de Membrana/metabolismo , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos CD8 , Citoplasma/metabolismo , Citotoxicidade Imunológica , Feminino , Imunização , Linfoma de Células T/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas/imunologiaRESUMO
The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.