RESUMO
The aim of this study was to establish a novel efficient cancer DNA vaccine approach. Many tumor-associated antigens (TAAs) have been reported; however, there is little information of the efficiency of each TAA. Normal cells barely undergo mitosis, whereas cancer cells divide frequently and grow well. Thus, G2/M-related antigens are cancer cell-specific and are regarded to be suitable candidates as targets of cancer immunotherapy. In this study, we compared the efficiencies of G2/M-related antigens including Birc5, Aurka, Nke2 and Plk1 by using a DNA vaccination model. Mice that had been immunized with G2/M-related antigens coding plasmid were challenged with CT26 colon cancer cells. Interestingly, Birc5- and Aurka-immunized mice showed an anti-tumor effect, whereas Nek2- and Plk1-immunized mice did not show any anti-tumor effect. We investigated the expression of G2/M-related antigens in cancer stem-like cell (CSC)/cancer-initiating cell (CIC) population to verify the difference in the anti-tumor effect. CSCs/CICs were isolated as side population (SP) cells using Hoechst 33342 dye from CT 26 cells. It was found that Birc5 and Aurka are expressed in both CSCs/CICs and non-CSCs/CICs (shared antigens), whereas Nek2 and Plk1 are expressed preferentially in non-CSCs/CICs (non-CSC antigens). Therefore, antigen expression in the CSC/CIC population might be related to the anti-tumor efficiency of cancer immunotherapy. Furthermore, we established a heat shock protein (Hsp90)-fused Birc5 plasmid to improve anti-cancer immunity. Birc5 fused to the N-terminal region of Hsp90 showed a stronger anti-tumor effect, whereas Birc5 fused to the C-terminal region of Hsp90 did not show enhancement compared with Birc5. These observations indicate that expression in the CSC/CIC population is essential to achieve tumor regression and that fusing antigens to the N-terminal region of Hsp90 enhances the anti-tumor effect.
Assuntos
Antígenos de Neoplasias/imunologia , Proteínas de Choque Térmico HSP90/imunologia , Proteínas Inibidoras de Apoptose/imunologia , Células-Tronco Neoplásicas/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Repressoras/imunologia , Vacinas de DNA , Animais , Aurora Quinase A , Aurora Quinases , Proteínas de Ciclo Celular/imunologia , Divisão Celular/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Fase G2/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Quinases Relacionadas a NIMA , Proteínas Proto-Oncogênicas/imunologia , Proteínas Recombinantes de Fusão , Células da Side Population , Survivina , Vacinação , Quinase 1 Polo-LikeRESUMO
The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inibidor de Quinase Dependente de Ciclina p21/imunologia , Memória Imunológica , Canal de Potássio Kv1.3/imunologia , Transdução de Sinais/imunologia , Proteína Smad3/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Cultivadas , Ciclina B1/genética , Ciclina B1/imunologia , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fase G2/genética , Fase G2/imunologia , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
PPARalpha ligands are medications used clinically to prevent cardiovascular events, however studies have shown that these agents are also anti-inflammatory. Our previous studies have shown that PPARalpha ligands induce lymphocyte depletion. PPARalpha ligands also potently upregulate TRB3, a protein that has been associated with cell cycle arrest. Therefore the following studies were undertaken to determine the mechanisms associated with lymphocyte depletion. Our studies demonstrate that WY14,643, a PPARalpha ligand, decreases the amount of lymphocytes recovered after stimulation and reduces cellular divisions. Cells treated with WY14,643 also accumulate in the G2/S phase of the cell cycle. TRB3 has been shown to inhibit the phosphorylation of AKT/Protein Kinase B, and reduced activation of AKT has been associated with decreased cellular divisions and survival. However in lymphocytes, TRB3 did not reduce the phosphorylation of AKT, and WY14,643 treatment was associated with enhanced activation of AKT. Drosophila tribbles (TRB3 homolog) causes G2 arrest by decreasing the expression of a Cdc25c homolog. Lymphocytes stimulated and treated with WY14,643 have reduced expression of Cdc25c, however this is not associated with enhanced expression of phosphorylated-Cdc2 which induces G2 arrest. Instead we observed that WY14,643 consistently reduces the protein and mRNA expression of Cyclin B1. Moreover, TRB3 inhibits activation of a Cyclin B1 promoter construct. In summary, we propose that PPARalpha ligands may reduce cellular number by augmenting TRB3 expression, which in turn induces cell cycle arrest by reducing the expression of Cyclin B1. Reduced cellular divisions and cell cycle arrest may be responsible for some of the immunomodulatory effects of these agents that have been consistently observed in human trials.
Assuntos
Proteínas de Ciclo Celular/imunologia , Fase G2/efeitos dos fármacos , Linfócitos/imunologia , PPAR alfa/agonistas , PPAR alfa/imunologia , Proliferadores de Peroxissomos/farmacologia , Pirimidinas/farmacologia , Fase S/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Ciclina B/biossíntese , Ciclina B/imunologia , Ciclina B1 , Fase G2/imunologia , Humanos , Ligantes , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , PPAR alfa/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Regiões Promotoras Genéticas/imunologia , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/imunologia , Fase S/imunologia , Regulação para Cima/imunologia , Fosfatases cdc25/imunologiaRESUMO
Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan fluorescence microscopy. The levels of TK1 increased 6-7h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified.
Assuntos
Fase G2/fisiologia , Timidina Quinase/biossíntese , Animais , Anticorpos Monoclonais , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Galinhas , Feminino , Fase G2/genética , Fase G2/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Fosforilação , Timidina Quinase/imunologia , Timidina Quinase/metabolismoRESUMO
The hallmark of autoimmunity is the activation and proliferation of autoreactive lymphocytes. Therefore, one potential strategy to treat autoimmunity is to target the proliferating autoreactive lymphocytes with antimitotic drugs. Paclitaxel and peloruside are two microtubule-stabilizing drugs that halt cell proliferation by stabilizing microtubules in the G(2)/M phase of the cell cycle. C57BL/6 mice treated for 5 consecutive days with paclitaxel or peloruside had a reduced incidence and significantly delayed development of EAE, a mouse model of MS. Although paclitaxel and peloruside were effective at inhibiting T cell proliferation in vitro, paclitaxel was shown to be ineffective at preventing the proliferation of autoreactive T cells in vivo during the 5-day treatment period. However, after the 5-day treatment, the ability of splenocytes or LN cells to proliferate in vitro was reduced significantly, suggesting that drug treatment targeted late but not early proliferative events in the animal. Moreover, in paclitaxel-treated, MOG-immunized mice, there was a complete inhibition of the recruitment of myeloid cells (especially macrophages) to the peripheral lymphoid organs. These results indicate that microtubule-stabilizing drugs are effective at reducing disease but require a prolonged exposure to paclitaxel in vivo to alter proliferation in the myeloid and lymphoid cell compartments.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Divisão Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fase G2/efeitos dos fármacos , Lactonas/farmacologia , Macrófagos/imunologia , Microtúbulos/imunologia , Esclerose Múltipla/tratamento farmacológico , Paclitaxel/farmacologia , Linfócitos T/imunologia , Moduladores de Tubulina/farmacologia , Animais , Divisão Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fase G2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Esclerose Múltipla/imunologia , Fatores de TempoRESUMO
IA-2 is a major autoantigen in type 1 diabetes and autoantibodies to it have become important diagnostic and predictive markers. IA-2 also is an intrinsic transmembrane component of dense core secretory vesicles and knock-out studies showed that IA-2 is a regulator of insulin secretion. Here we show that overexpression of IA-2 puts mouse insulinoma MIN-6 beta cells into a pre-apoptotic state and that exposure to high glucose results in G2/M arrest and apoptosis. Molecular study revealed a decrease in phosphoinositide-dependent kinase (PDK)-1 and Akt/protein kinase B (PKB) phosphorylation. Treatment of IA-2-transfected cells with IA-2 siRNA prevented both G2/M arrest and apoptosis and increased Akt/PKB phosphorylation. A search for IA-2 interacting proteins revealed that IA-2 interacts with sorting nexin (SNX)19 and that SNX19, but not IA-2, inhibits the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 and thereby suppresses the phosphorylation of proteins in the Akt signalling pathway resulting in apoptosis. We conclude that IA-2 acts through SNX19 to initiate the pre-apoptotic state. Our findings point to the possibility that in autoimmune diseases, tissue destruction may be autoantigen-induced, but not necessarily immunologically mediated.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/metabolismo , Células Secretoras de Insulina/imunologia , Animais , Apoptose/imunologia , Autoanticorpos/genética , Autoantígenos/imunologia , Autoimunidade , Proteínas de Transporte/metabolismo , Divisão Celular/imunologia , Fragmentação do DNA , Fase G2/imunologia , Células Secretoras de Insulina/patologia , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , RNA Interferente Pequeno/genética , Transdução de Sinais/imunologia , Nexinas de Classificação , Transfecção , Células Tumorais Cultivadas , Proteínas de Transporte Vesicular/metabolismoRESUMO
BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. RESULTS: Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. CONCLUSION: Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival.
Assuntos
Fase G2/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T/citologia , Proteínas do Core Viral/fisiologia , Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Fase G2/imunologia , Genes pX , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Transdução Genética , Transfecção , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Fosfatases cdc25/metabolismoRESUMO
Delay of cell cycle progression in response to double-strand DNA breaks (DSBs) is critical to allow time for DNA repair and prevent cellular transformation. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is activated in immature thymocytes along with TcRbeta gene V(D)J recombination. Active p38 MAP kinase promotes a G2/M cell cycle checkpoint through the phosphorylation and activation of p53 in these cells in vivo. Inactivation of p38 MAP kinase and p53 is required for DN3 thymocytes to exit the G2/M checkpoint, progress through mitosis and further differentiate. We propose that p38 MAP kinase is activated by V(D)J-mediated DSBs and induces a p53-mediated G2/M checkpoint to allow DNA repair and prevent cellular transformation.
Assuntos
Dano ao DNA/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Divisão Celular/imunologia , Reparo do DNA/fisiologia , Fase G2/imunologia , Camundongos , Camundongos Knockout , Camundongos SCID , Linfócitos T/citologia , Timo/citologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
APEX2/APE2 is a secondary mammalian apurinic/apyrimidinic endonuclease that associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. To determine the biologic significance of APEX2, we established APEX2-null mice. These mice were about 80% the size of their wild-type littermates and exhibited a moderate dyshematopoiesis and a relatively severe defect in lymphopoiesis. A significant accumulation of both thymocytes and mitogen-stimulated splenocytes in G(2)/M phase was seen in APEX2-null mice compared with the wild type, indicating that APEX2 is required for proper cell cycle progression of proliferating lymphocytes. Although APEX2-null mice exhibited an attenuated immune response against ovalbumin in comparison with wild-type mice, they produced both antiovalbumin immunoglobulin M (IgM) and IgG, indicating that class switch recombination can occur even in the absence of APEX2.
Assuntos
Divisão Celular/imunologia , Endonucleases/deficiência , Fase G2/imunologia , Crescimento/imunologia , Linfopoese/genética , Animais , Animais Recém-Nascidos , Atrofia , Primers do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Endonucleases/genética , Endonucleases/fisiologia , Hematopoese/genética , Hematopoese/imunologia , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Camundongos Knockout , Enzimas Multifuncionais , Reação em Cadeia da Polimerase , Baço/imunologia , Timo/patologiaRESUMO
CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present, among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot, immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase. Our data indicated that the PSTAIRE CDK- like protein has a direct bearing on the mitosis.
Assuntos
Nucléolo Celular/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/enzimologia , Mitose , Physarum polycephalum/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Anticorpos Monoclonais , Nucléolo Celular/ultraestrutura , Cromossomos , Citoplasma/ultraestrutura , Fase G2/imunologia , Mitose/imunologia , Physarum polycephalum/citologia , Physarum polycephalum/imunologia , Fase S/imunologiaRESUMO
The primary age-related loss in B cell progenitors is thought to be at the pro- to pre-B cell transition. However, we show that the frequencies and absolute numbers of all progenitor populations for the B cell lineage, including B-lineage-committed pro-B cells and multipotent B-lymphoid progenitors, decline in aged C57BL/6 mice. Moreover, when derived from aged mice, lymphoid progenitors within every population examined exhibited suboptimal IL-7 responsiveness, demonstrating that age-associated suboptimal IL-7R signaling is a general property of all early B-lineage precursors. Collectively, these data indicate that aging results in a previously unappreciated decline in the earliest stages of B cell development.
Assuntos
Envelhecimento/imunologia , Subpopulações de Linfócitos B/classificação , Subpopulações de Linfócitos B/citologia , Regulação para Baixo/imunologia , Células-Tronco Hematopoéticas/classificação , Células-Tronco Hematopoéticas/citologia , Receptores de Hialuronatos , Linfopoese/imunologia , Glicoproteínas de Membrana , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Células da Medula Óssea/classificação , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem da Célula/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Fase G1/imunologia , Fase G2/imunologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Interleucina-7/fisiologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Mitocondriais , Receptores de Complemento/biossínteseRESUMO
CD4(+) T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4(+) T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Regulação Viral da Expressão Gênica/imunologia , HIV-1/genética , Linhagem Celular Transformada , Fase G2/genética , Fase G2/imunologia , Proteínas de Fluorescência Verde , HIV-1/metabolismo , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Contagem de Linfócitos , Mitose/genética , Mitose/imunologia , Transcrição Gênica/imunologia , Vírion/metabolismoRESUMO
We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several Gram-negative bacteria. Moreover, we have shown that CdtB impairs lymphocyte function by inducing G(2) arrest of the cell cycle. We now report that both CdtB as well as an extract prepared from an Escherichia coli strain that expresses all three of the A. actinomycetemcomitans cdt genes (rCdtABC) induce apoptosis. Pretreatment of lymphocytes with either CdtB or rCdtABC leads to DNA fragmentation in activated lymphocytes at 72 and 96 h. No DNA fragmentation was induced in nonactivated cells. Flow cytometric analysis of the Cdt-treated lymphocytes demonstrates a reduction in cell size and an increase in nuclear condensation. Mitochondrial function was also perturbed in cells pretreated with either CdtB or rCdtABC. An increase in the expression of the mitochondria Ag, Apo 2.7, was observed along with evidence of the development of a mitochondrial permeability transition state; this includes a decrease in the transmembrane potential and elevated generation of reactive oxygen species. Activation of the caspase cascade, which is an important biochemical feature of the apoptotic process, was also observed in Cdt-treated lymphocytes. Overexpression of the bcl-2 gene in the human B lymphoblastoid cell line, JY, led to a decrease in Cdt-induced apoptosis. Interestingly, Bcl-2 overexpression did not block Cdt-induced G(2) arrest. The implications of our results with respect to the immunosuppressive functions of Cdt proteins are discussed.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Apoptose/imunologia , Toxinas Bacterianas/farmacologia , Fase G2/imunologia , Imunossupressores/farmacologia , Linfócitos T/citologia , Apoptose/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Linhagem Celular Transformada , Células Cultivadas , Fragmentação do DNA/imunologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Fase G2/efeitos dos fármacos , Humanos , Cinética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/microbiologia , TransfecçãoRESUMO
TGF-beta is a potent inducer of apoptosis in many Burkitt's lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-beta treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-beta treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-beta-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-beta-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-beta treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-beta-mediated apoptosis is not dependent on Fas ligand, TNF-alpha, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-beta induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.
Assuntos
Apoptose/imunologia , Linfoma de Burkitt/imunologia , Caspases/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Antígenos CD/fisiologia , Proteínas Reguladoras de Apoptose , Linfoma de Burkitt/enzimologia , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Linhagem Celular Transformada , Ativação Enzimática/imunologia , Fase G1/imunologia , Fase G2/imunologia , Humanos , Hidrólise , Ligantes , Glicoproteínas de Membrana/fisiologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Membro 25 de Receptores de Fatores de Necrose Tumoral , Receptores Tipo I de Fatores de Necrose Tumoral , Proteína do Retinoblastoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologiaRESUMO
We have previously shown that Actinobacillus actinomycetemcomitans produces an immunosuppressive factor that is encoded by the cdtB gene, which is homologous to a family of cytolethal distending toxins (Cdt) expressed by several gram-negative bacteria. In this study, we report that the cdt locus in A. actinomycetemcomitans is composed of five open reading frames, designated orf1, orf2, cdtA, cdtB, and cdtC. The deduced amino acid sequences of the five open reading frames are highly conserved among A. actinomycetemcomitans strains 652, Y4, 29522, and HK1651. There is also strong homology with the Cdt proteins of Haemophilus ducreyi (87-91%), but only partial homology with that of Campylobacter jejuni and Escherichia coli (29-48%). Analysis of A. actinomycetemcomitans mRNA by RT-PCR suggests that the two small open reading frames upstream of cdtA are coexpressed with cdtA, cdtB, and cdtC. We next utilized a series of plasmids that express various combinations of the cdt genes to determine their requirement for expression of immunoinhibitory activity. Cell extracts of E. coli transformed with each of the plasmids were tested for their capacity to induce G2 arrest in the cell cycle of PHA-activated human T cells. These experiments suggest that expression of cdtB alone is sufficient to induce G2 arrest in human T cells, but do not exclude the possibility that cdtC also contributes to cell cycle arrest. The implications of our results with respect to the function of the individual Cdt proteins are discussed.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Toxinas Bacterianas/genética , Fase G2/imunologia , Regulação Bacteriana da Expressão Gênica/imunologia , Genes Bacterianos/imunologia , Inibidores do Crescimento/genética , Óperon/imunologia , Linfócitos T/imunologia , Aggregatibacter actinomycetemcomitans/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Células Cultivadas , Fase G2/genética , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/imunologia , Inibidores do Crescimento/toxicidade , Humanos , Imunossupressores/imunologia , Imunossupressores/toxicidade , Dados de Sequência Molecular , Família Multigênica/imunologia , Linfócitos T/citologia , Linfócitos T/microbiologiaRESUMO
Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with myelin basic protein (MBP) and adjuvants. Rats were treated with second daily injections of saline or cyclosporin A (CsA) from the day of inoculation. Saline-treated rats had an acute episode of disease followed by clinical recovery. Rats treated with CsA 16 or 32 mg/kg had minimal signs of EAE at the usual time after inoculation, but developed signs of disease after treatment was ceased. Rats treated with CsA 8 mg/kg had a delayed first episode of disease and then developed a relapsing or a chronic persistent course of disease. CsA 4 mg/kg delayed the onset of disease. To study the effects of CsA on the inflammatory infiltrate, cells were extracted from the spinal cords of rats with EAE, 16 h after a single injection of CsA or saline. Extracted cells were labelled with antibodies to T cells, CD11b/c (macrophages/microglia), CD95 (Fas) and Fas ligand. CsA 4 mg/kg did not alter the composition of the inflammatory infiltrate. Treatment with higher single doses of CsA caused a dose-dependent decline in the percentage of T cell receptor (TCR) alphabeta+ cells in the inflammatory infiltrate. All doses of CsA caused a significant increase in the number and percentage of cells that were apoptotic. CsA treatment caused an increase in the percentages of CD5+ and TCR alphabeta+ cells that were apoptotic. There was a decline in the percentage of apoptotic T cells that were Vbeta8.2+, compared to the percentage of non-apoptotic T cells that were Vbeta8.2+, in CsA treated rats compared to saline-treated controls. This suggests that, while CsA treatment caused a non-specific increase in the overall level of T cell apoptosis in the spinal cord, it abrogated the selective apoptosis of Vbeta8.2+ encephalitogenic T cells that normally occurs during spontaneous recovery from acute EAE.
Assuntos
Apoptose/efeitos dos fármacos , Ciclosporina/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Imunossupressores/farmacologia , Proteína Básica da Mielina/imunologia , Doença Aguda , Animais , Apoptose/imunologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Fase G1/imunologia , Fase G2/imunologia , Imunização , Masculino , Mitose/imunologia , Proteína Básica da Mielina/farmacologia , Ratos , Ratos Endogâmicos Lew , Fase de Repouso do Ciclo Celular/imunologia , Fase S/imunologia , Medula Espinal/química , Medula Espinal/citologia , Medula Espinal/imunologia , Linfócitos T/imunologiaRESUMO
We have previously shown that Actinobacillus actinomycetecomitans produces an immunosuppressive factor (ISF) capable of impairing human lymphocyte function by perturbing cell cycle progression. We now report that ISF is the product of the cdtB gene, one of three genes encoding the family of cytolethal distending toxins (Cdt). The ISF polypeptide exhibits >/=95% identity with Hemophilus ducreyi CdtB protein and =60% homology with Escherichia coli or Campylobacter jejuni CdtB. Pretreatment of PHA-activated lymphocytes with 5-25 ng ISF results in G2 arrest of CD4+ and CD8+ T cells. Similarly, treatment of HeLa cells results in G2 arrest and cell elongation and distension. However, lymphocytes are at least 5 times more sensitive to ISF than HeLa cells and do not undergo the elongation and distension that characterizes interactions of Cdts with cell lines. ISF-treated lymphocytes express normal cyclin A and B1 levels, but contain reduced levels of cell cycle-dependent kinase-1 (Cdk1). Additionally, the majority of Cdk1 is in the hyperphosphorylated, inactive, form. In contrast, PHA-induced G2 cells contain elevated levels of the hypophosphorylated, active Cdk1. Failure of ISF-treated cells to dephosphorylate Cdk1 is not associated with decreased availability of Cdc25. These studies suggest that the CdtB protein alone is capable of inducing G2 arrest in lymphocytes and cell cycle arrest, elongation, and distension of HeLa cells. Our studies also suggest that lymphocytes may be primary targets for A. actinomycetemcomitans CdtB (ISF) and possibly for other Cdt family members as well. Thus, Cdts may function to impair host immunity and contribute to the pathogenesis of disease associated with Cdt-producing organisms.
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Toxinas Bacterianas/química , Fase G2/imunologia , Imunossupressores/química , Linfócitos T/citologia , Sequência de Aminoácidos , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Sequência de Bases , Proteína Quinase CDC2/biossíntese , Células HeLa , Humanos , Imunossupressores/isolamento & purificação , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
In vitro proliferation assays are widely used in biomedical research. We describe the immunoenzymatic (ELISA) detection of a recently described proliferation associated protein (p100) by means of a new monoclonal mouse IgG1 antibody (Ki-S2). P100 is a 100 kDa nuclear protein that is specifically detected during the cell cycle phases S, G2 and M. Comparative studies on lectin-stimulated leukocytes using 3H-thymidine labelling and Ki-67 antibodies revealed a statistically significant positive correlation. Since p100 is absent in GO and G1 cells, its detection permits the precise and specific measurement of actual cell cycle events under culture conditions.
Assuntos
Proteínas de Ciclo Celular/imunologia , Ciclo Celular , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Anticorpos Monoclonais/análise , Ciclo Celular/imunologia , Fase G2/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mitose/imunologia , Proteínas Nucleares/imunologia , Fase S/imunologia , Células U937RESUMO
Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging.
Assuntos
Envelhecimento/imunologia , Proteína Quinase CDC2/metabolismo , Ciclo Celular/imunologia , Linfócitos T/citologia , Linfócitos T/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Proteína Quinase CDC2/biossíntese , Ciclina B/biossíntese , Ciclina B1 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Fase G2/imunologia , Humanos , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Substâncias Macromoleculares , Mitose/imunologia , Fosforilação , Linfócitos T/imunologiaRESUMO
Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.