RESUMO
BACKGROUND: Here, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer. METHODS: We examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs. RESULTS: Concentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib. CONCLUSIONS: These data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer.
Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dasatinibe , Feminino , Citometria de Fluxo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-yes/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-yes/metabolismo , Pirimidinas/administração & dosagem , Receptor EphA2/antagonistas & inibidores , Receptor EphA2/metabolismo , Tiazóis/administração & dosagem , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
AIMS/HYPOTHESIS: In obesity, beta cells activate compensatory mechanisms to adapt to the higher insulin demand. Interleukin-1 receptor antagonist (IL-1Ra) prevents obesity-induced hyperglycaemia and is a potent target for the treatment of diabetes, but the mechanisms of its secretion and regulation in obesity are unknown. In the present study, we hypothesise the regulation of IL-1Ra secretion by purinergic P2X(7) receptors in islets. METHODS: Production and regulation of P2X(7) were studied in pancreatic sections from lean and obese diabetic patients, non-diabetic controls and in isolated islets. IL-1Ra, IL-1beta and insulin secretion, glucose tolerance and beta cell mass were studied in P2x7 (also known as P2Rx7)-knockout mice. RESULTS: P2X(7) levels were elevated in beta cells of obese patients, but downregulated in patients with type 2 diabetes mellitus. Elevated glucose and non-esterified fatty acids rapidly activated P2X(7) and IL-1Ra secretion in human islets, and this was inhibited by P2X(7) blockade. In line with our results in vitro, P2x7-knockout mice had a lower capacity to secrete IL-1Ra. They exhibited severe and rapid hyperglycaemia, glucose intolerance and impaired beta cell function in response to a high-fat/high-sucrose diet, were unable to compensate by increasing their beta cell mass in response to the diet and showed increased beta cell apoptosis. CONCLUSIONS/INTERPRETATION: Our study shows a tight correlation of P2X(7) activation, IL-1Ra secretion and regulation of beta cell mass and function. The increase in P2X(7) production is one mechanism that may explain how beta cells compensate by adapting to the higher insulin demand. Disturbances within that system may result in the progression of diabetes.
Assuntos
Células Secretoras de Insulina/fisiologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Receptores Purinérgicos P2/fisiologia , Animais , Sobrevivência Celular , Cruzamentos Genéticos , Complicações do Diabetes/fisiopatologia , Teste de Tolerância a Glucose , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/patologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/fisiopatologia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Valores de Referência , Tubulina (Proteína)/genéticaRESUMO
Inhibition of the 26S proteasome reduces the severity of several immune-mediated diseases. Here, we report that the proteasome also regulates transfer-induced diabetes in nonobese mice. Treatment of recipient mice with the proteasome inhibitor N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (MG132) resulted in a 76% reduction in transfer-induced diabetes. The closely related inhibitor carbobenzoxy-l-leucyl-l-leucinal that inhibits calpains but not the proteasome had no protective effect, suggesting that MG132 acted via inhibition of the proteasome. MG132 decreased proliferation of transferred T cells in the pancreatic lymph nodes in vivo and prevented their expansion in a dose-dependent manner in vitro, consistent with a direct effect by MG132 on the T cells. MG132 did not prevent migration of transferred T cells into the islets but reduced the number of mice with severe infiltration. We suggest that MG132 prevents transfer-induced diabetes by directly targeting the autoreactive T cells and lowering their diabetogenic potential.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Complexos Multienzimáticos/antagonistas & inibidores , Linfócitos T/imunologia , Transferência Adotiva , Animais , Divisão Celular/imunologia , Cisteína Endopeptidases/imunologia , Inibidores de Cisteína Proteinase/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Citometria de Fluxo , Glicosúria , Ilhotas Pancreáticas/imunologia , Leupeptinas/farmacologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Complexos Multienzimáticos/imunologia , Complexo de Endopeptidases do Proteassoma , Linfócitos T/citologiaRESUMO
Psychological and immunological responses of cancer patients to a psychosocial intervention program will be assessed over time. Previously it has been proposed that there are two large divisions in cancer histology (type I and II) and that the psychobiology of patients will vary accordingly.
Assuntos
Neoplasias/imunologia , Neoplasias/psicologia , Humanos , Neoplasias/classificaçãoRESUMO
Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Peptídeo Hidrolases/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Caspases/metabolismo , Cisteína Endopeptidases/isolamento & purificação , Citosol/enzimologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Linfoma , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Tripsina/metabolismo , Células Tumorais CultivadasRESUMO
The ubiquitin/proteasome-dependent proteolytic pathway is an attractive target for therapeutics because of its critical involvement in cell cycle progression and antigen presentation. However, dissection of the pathway and development of modulators are hampered by the complexity of the system and the lack of easily detectable authentic substrates. We have developed a convenient reporter system by producing N-end rule and ubiquitin fusion degradation (UFD)-targeted green fluorescent proteins that allow quantification of ubiquitin/proteasome-dependent proteolysis in living cells. Accumulation of these reporters serves as an early predictor of G2/M arrest and apoptosis in cells treated with proteasome inhibitors. Comparison of reporter accumulation and cleavage of fluorogenic substrates demonstrates that the rate-limiting chymotrypsin-like activity of the proteasome can be substantially curtailed without significant effect on ubiquitin-dependent proteolysis. These reporters provide a new powerful tool for elucidation of the ubiquitin/proteasome pathway and for high throughput screening of compounds that selectively modify proteolysis in vivo.
Assuntos
Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Genes Reporter , Proteínas Luminescentes/metabolismo , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Ubiquitinas/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Leupeptinas/farmacologia , Proteínas Luminescentes/genética , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Sulfonas/farmacologia , Transfecção , Ubiquitinas/genéticaRESUMO
Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.
Assuntos
Doenças dos Bovinos/virologia , DNA Viral/análise , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/genética , Sêmen/virologia , Animais , Bovinos , Dexametasona/farmacologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/isolamento & purificação , Injeções Intradérmicas , Masculino , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon gamma in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I-deficient tumor cells were approximately 10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I-deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize.
Assuntos
Antígenos de Histocompatibilidade Classe I/fisiologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Antígenos/análise , Antígenos de Superfície , Linhagem Celular , Interferon gama/genética , Interleucina-12/biossíntese , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos CBA , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Fenótipo , Proteínas/análise , Fator de Necrose Tumoral alfa/biossínteseRESUMO
MHC class I molecules can prevent NK cell-mediated cytotoxicity by interacting with inhibitory receptors on the effector cells. Different conclusions have been reached regarding possible peptide selectivity of these receptors. To address whether peptide selectivity is an exclusive feature of human or immunoglobulin-superfamily receptors, we have studied a system based on the murine NK receptor Ly-49C in the lectin-superfamily. Loading of TAP-deficient RMA-S cells with the H-2Kb-restricted, ovalbumin-derived peptide OVA(257 - 264) (pOVA) induced their ability to bind Ly-49C-transfected reporter cells, and also protected them from killing by Ly-49C+ NK cells. Other peptides that bound and stabilized H-2Kb equally well differed in their NK protective capacity. Comparison of the MHC class I peptide complexes (crystal structures and molecular models) revealed a conformational motif encompassing the C-terminal parts of the alpha1 helix (73 - 77) and the bound peptide that was common for the protective complexes. Substitution analysis of pOVA suggested that position 7 in the peptide may be critical for optimal protection as well as for the conformational motif at position 73 - 77. In conclusion, protection mediated by the murine C-type lectin receptor Ly-49C is peptide dependent and selective.
Assuntos
Antígenos Ly/metabolismo , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Humanos , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Complexo Principal de Histocompatibilidade/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Receptores Imunológicos/imunologia , Receptores Semelhantes a Lectina de Células NK , Células Tumorais CultivadasRESUMO
In order to deduce the ancestral genome arrangement in the karyotypically diverse marsupial family Macropodidae, and to assess chromosome change in this family, chromosome-specific paints from the tammar wallaby (2n = 16) were hybridized to metaphase spreads from the two species proposed to represent the 2n = 22 ancestral karyotype, as well as species with derived 2n = 20 and 2n = 14 karyotypes. Identical patterns were observed in the two 2n = 22 species, from which the rearrangements to form the three derived karyotypes may be easily deduced to be 1, 3 and 4 different fusions, respectively. The identical Thylogale and Dorcopsis genomes may both be used to represent the pleisiomorphic macropodid chromosome complement. Variation in the X chromosome was also investigated by hybridizing an X-Y shared tammar wallaby 12-kb repeat element to chromosomes from the other four macropodid species, finding that it hybridized only to the most closely related species, and therefore is of recent origin.
Assuntos
Macropodidae/genética , Filogenia , Animais , Coloração Cromossômica , Feminino , Cariotipagem , Macropodidae/classificação , MasculinoRESUMO
Triggering of a T cell requires interaction between its specific receptor (TCR) and a peptide antigen presented by a self-major histocompatibility complex (MHC) molecule. TCR recognition of self-MHC by itself falls below the threshold of detection in most systems due to low affinity. To study this interaction, we have used a read-out system in which antigen-specific effector T cells are confronted with targets expressing high levels of MHC compared with the selecting and priming environment. More specifically, the system is based on CD8(+) T cells selected in an environment with subnormal levels of MHC class I in the absence of beta2-microglobulin. We observe that the MHC restriction element can trigger viral peptide-specific T cells independently of the peptide ligand, provided there is an increase in self-MHC density. Peptide-independent triggering required at least four times the natural in vivo level of MHC expression. Furthermore, recognition of the restriction element at expression levels below this threshold was still enough to compensate for lack of affinity to peptides carrying alanine substitutions in major TCR contact residues. Thus, the specificity in TCR recognition and T cell activation is fine tuned by the avidity for self-MHC, and TCR avidities for peptide and MHC may substitute for each other. These results demonstrate a functional role for TCR avidity for self-MHC in tuning of T cell specificity, and support a role for cross-reactivity on "self" during T cell selection and activation.
Assuntos
Antígenos H-2/fisiologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Citotóxicos/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Linhagem Celular , Antígeno de Histocompatibilidade H-2D , Ativação Linfocitária , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Microglobulina beta-2/fisiologiaRESUMO
We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb -/- mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although beta2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb -/- mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by beta2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb -/- mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb -/- mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb -/- animals also have natural killer cells that retain their cytotoxic potential.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos H-2/genética , Células Matadoras Naturais/imunologia , Animais , Cruzamentos Genéticos , Citotoxicidade Imunológica , Camundongos , Camundongos Knockout , Recombinação GenéticaRESUMO
Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.
Assuntos
Doenças dos Bovinos , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1/fisiologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais , Ativação Viral , Latência Viral , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Especificidade de Anticorpos , Bovinos , DNA Viral/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Herpesvirus Bovino 1/crescimento & desenvolvimento , Herpesvirus Bovino 1/isolamento & purificação , Imunidade Materno-Adquirida , Reação em Cadeia da Polimerase , Proteínas ViraisRESUMO
Proteolysis is essential for the execution of many cellular functions. These include removal of incorrectly folded or damaged proteins, the activation of transcription factors, the ordered degradation of proteins involved in cell cycle control, and the generation of peptides destined for presentation by class I molecules of the major histocompatibility complex. A multisubunit protease complex, the proteasome, accomplishes these tasks. Here we show that in mammalian cells inactivation of the proteasome by covalent inhibitors allows the outgrowth of inhibitor-resistant cells. The growth of such adapted cells is apparently maintained by the induction of other proteolytic systems that compensate for the loss of proteasomal activity.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Adaptação Fisiológica , Animais , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Hidrólise , Camundongos , Complexo de Endopeptidases do Proteassoma , Sulfonas/farmacologia , Thermoplasma/enzimologia , Células Tumorais Cultivadas , Ubiquitinas/metabolismoRESUMO
Mice lacking beta2-microglobulin (beta2m- mice) express greatly reduced levels of MHC class I molecules, and cells from beta2m- mice are therefore highly sensitive to NK cells. However, NK cells from beta2m- mice fail to kill beta2m- normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of beta2m- NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to beta2m- NK cells, we made the following observations: First, beta2m- NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to beta2m- concanavalin A (Con A)-activated T cell blasts. Secondly, beta2m- NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than beta2m+ NK cells. Thirdly beta2m- NK cells were not only tolerant to beta2m- Con A blasts but also to Con A blasts from H-2Kb-/Db- double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of beta2m- NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in beta2m- mice, which may instead operate through mechanisms involving triggering pathways.