Assuntos
Doença Crônica/prevenção & controle , Saúde Global , Comportamentos Relacionados com a Saúde , Estilo de Vida , Nações Unidas , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/economia , Consumo de Bebidas Alcoólicas/prevenção & controle , Causas de Morte/tendências , Doença Crônica/economia , Doença Crônica/mortalidade , Redução de Custos/tendências , Estudos Transversais , Países Desenvolvidos/economia , Países em Desenvolvimento/economia , Dieta com Restrição de Carboidratos/economia , Dieta com Restrição de Gorduras/economia , Dieta Hipossódica/economia , Quimioterapia Combinada/economia , Medicamentos Genéricos/economia , Medicamentos Genéricos/uso terapêutico , Previsões , Saúde Global/economia , Saúde Global/tendências , Custos de Cuidados de Saúde/tendências , Humanos , Atividade Motora , Fatores de Risco , Fumar/efeitos adversos , Fumar/economia , Abandono do Uso de Tabaco/economia , Estados UnidosRESUMO
Signals that control the fine balance between cell death and cell survival are altered during tumorigenesis. Understanding the mechanisms by which this balance is perturbed, leading to excessive cell survival, is important for designing effective therapies. Proteins belonging to the B-cell lymphoma (BCL) family are known to regulate death responses to apoptotic signals, especially those originating within cells. A subset of BCL family members capable of inhibiting cell death is known to contribute to tumorigenesis; however, it is not known whether all six antiapoptotic BCL family members play a causal role in tumor development. Using a mouse model of MYC-driven leukemia, we showed that, in addition to the well characterized BCL2 and BCLxl (BCL2L1), the other four family members -- BCLw (BCL2L2), BCLb (BCL2L10), BFL1 (BCL2A1) and MCL1 -- also cooperate with MYC to accelerate leukemogenesis. In addition, high levels of each family member are found in either solid human tumors or cell lines derived from human leukemias or lymphomas.
Assuntos
Apoptose/genética , Genes myc , Leucemia Mieloide/genética , Proteínas Oncogênicas/fisiologia , Humanos , Leucemia Mieloide/patologiaRESUMO
Mutations of proto-oncogenes are common events in the pathogenesis of cancers, as shown in a wide range of studies during the 30 years since the discovery of these genes. The benefits of novel therapies that target the products of mutant alleles in human cancers, and the demonstrated dependence of cancers in mouse models on continued expression of initiating oncogenes, are especially promising signs that revolutionary improvements in cancer care are possible. Full realization of the promise of targeted therapies, however, will require better definitions of the genotypes of human cancers, new approaches to interrupt the biochemical consequences of oncogenic mutations, and a greater understanding of drug resistance and tumor progression. In this paper, we summarize recent efforts toward these goals in our laboratory and others.
Assuntos
Neoplasias/genética , Oncogenes , Adenocarcinoma/genética , Animais , Resistencia a Medicamentos Antineoplásicos , Genes erbB-1 , Genótipo , Humanos , Neoplasias Pulmonares/genética , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias Experimentais/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proto-OncogenesRESUMO
To investigate the role of an activated K-Ras gene in the initiation and maintenance of lung adenocarcinomas, we developed transgenic mice that express murine K-Ras4b(G12D) under the control of doxycycline in type II pneumocytes. Focal proliferative lesions of alveolar type II pneumocytes were observed as early as seven days after induction with doxycycline; after two months of induction, the lungs contained adenomas and adenocarcinomas, with focal invasion of the pleura at later stages. Removal of doxycycline caused a rapid fall in levels of mutant K-Ras RNA and concomitant apoptotic regression of both the early proliferative lesions and the tumors. Tumor burden was dramatically decreased by three days after withdrawal, and tumors were undetectable after one month. When similar experiments were performed with animals deficient in either the p53 gene or the Ink4A/Arf locus, tumors arose more quickly (within one month of exposure to doxycycline) and displayed more obvious histological features of malignancy; nevertheless, these tumors also regressed rapidly when the inducer was removed, implying that continued production of mutant K-Ras is necessary to maintain the viability of tumor cells in the absence as well as the presence of tumor suppressor genes. We also show that the appearance and regression of these pulmonary tumors can be readily monitored in anesthetized transgenic animals by magnetic resonance imaging.
Assuntos
Adenocarcinoma/genética , Apoptose , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Genes ras/genética , Neoplasias Pulmonares/genética , Transgenes/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Bromodesoxiuridina , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Primers do DNA/química , Genótipo , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Recidiva Local de Neoplasia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 x 10(3) infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10(5) IU/ml and can be concentrated by ultracentrifugation to titers of 10(7) IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the beta-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.
Assuntos
Alpharetrovirus/genética , Vetores Genéticos , Lentivirus/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , TransfecçãoAssuntos
Biotecnologia , Editoração , Pesquisa , Academias e Institutos/economia , Academias e Institutos/organização & administração , Biotecnologia/economia , Propriedade Intelectual , Internet , Investimentos em Saúde , Cidade de Nova Iorque , Pesquisa/economia , Pesquisa/organização & administraçãoRESUMO
BACKGROUND: The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. RESULTS: We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. CONCLUSIONS: Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation.
Assuntos
Microdomínios da Membrana/enzimologia , Linfócitos T/enzimologia , Linhagem Celular , Ativação Enzimática , Humanos , Células Jurkat , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Linfócitos T/imunologia , Tirosina/metabolismoRESUMO
BACKGROUND: Germline mutations in the tumor suppressor PTEN predispose human beings to breast cancer, and genetic and epigenetic alterations of PTEN are also detected in sporadic human breast cancer. Germline Pten mutations in mice lead to the development of a variety of tumors, but mammary carcinomas are infrequently found, especially in mice under the age of six months. RESULTS: To better understand the role of PTEN in breast tumor development, we have crossed Pten heterozygous mice to MMTV-Wnt-1 transgenic mice that routinely develop ductal carcinomas in the mammary gland. Female Wnt-1 transgenics heterozygous for Pten developed mammary tumors earlier than Wnt-1 transgenics that were wild type for Pten. In most tumors arising in Pten heterozygotes, the Pten wild-type allele was lost, suggesting that cells lacking Pten function have a growth advantage over cells retaining a wild type allele. Tumors with LOH contained high levels of activated AKT/PKB, a downstream target of the PTEN/PI3K pathway. CONCLUSIONS: An animal model has been developed in which the absence of Pten collaborates with Wnt-1 to induce ductal carcinoma in the mammary gland. This animal model may be useful for testing therapies specific for tumors deregulated in the PTEN/PI3K/AKT pathway.
RESUMO
Mutant src(-/-) mice have osteopetrosis resulting from defective osteoclasts, the cells that resorb bone. However, signaling pathways involving Src family members in osteoclasts remain unclear. We demonstrate that expression of a truncated Src molecule, Src251, lacking the kinase domain, induces osteopetrosis in wild-type and src(+/-) mice and worsens osteopetrosis in src(-/-) mice by a novel mechanism, increased osteoclast apoptosis. Induction of apoptosis by Src251 requires a functional SH2, but not an SH3, domain and is associated with reduced AKT kinase activity. Expression of Src251 dramatically reduces osteoclast survival in response to RANKL/TRANCE/OPGL, providing evidence that Src family kinases are required in vivo for survival signaling pathways downstream from TNF family receptors.
Assuntos
Proteínas Serina-Treonina Quinases , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Animais , Apoptose , Sequência de Bases , Sobrevivência Celular , Galinhas , Primers do DNA/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/patologia , Osteopetrose/genética , Osteopetrose/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Domínios de Homologia de src , Quinases da Família src/químicaRESUMO
He may have stepped away from the hottest seat in biomedical research, but Nobel Laureate Harold Varmus shows no signs of withdrawing from the frontline of the biomedical research community, and he still displays the inimitable combination of political astuteness and scientific expertise that made his reign as director of the United States National Institutes of Health so successful. Varmus spoke to Nature Medicine for the first in a series of profiles that the journal will run on scientists that make a difference to biomedical research.
Assuntos
Diretores Médicos , Institutos de Câncer , Internet , Liderança , Manobras Políticas , National Institutes of Health (U.S.) , Editoração , Estados UnidosRESUMO
Vertebrates have two Armadillo-like proteins, beta-catenin and plakoglobin. Mutant forms of beta-catenin with oncogenic activity are found in many human tumors, but plakoglobin mutations are not commonly found. In fact, plakoglobin has been proposed to suppress tumorigenesis. To assess differences between beta-catenin and plakoglobin, we compared several of their biochemical properties. After transient transfection of 293T cells with an expression vector encoding either of the two proteins, soluble wild type beta-catenin does not significantly accumulate, whereas soluble wild type plakoglobin is readily detected. As anticipated, beta-catenin is stabilized by the oncogenic mutation S37A; however, the analogous mutation in plakoglobin (S28A) does not alter its half-life. S37A-beta-catenin activates a TCF/LEF-dependent reporter 20-fold more potently than wild type beta-catenin, and approximately 5-fold more potently than wild type or S28A plakoglobin. These differences may be attributable to an enhanced affinity of S37A beta-catenin for LEF1 and TCF4, as observed here by immunoprecipitation assays. We show that the carboxyl-terminal domain is largely responsible for the difference in signaling and that the Armadillo repeats account for the remainder of the difference. The relatively weak signaling by plakoglobin and the failure of the S28A mutation to enhance its stability, may explain why plakoglobin mutations are infrequent in malignancies.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Transdução de Sinais/fisiologia , Transativadores , Proteínas de Peixe-Zebra , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desmoplaquinas , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteínas Wnt , beta Catenina , gama CateninaRESUMO
Wnt-1 was first identified as a protooncogene activated by viral insertion in mouse mammary tumors. Transgenic expression of this gene using a mouse mammary tumor virus LTR enhancer causes extensive ductal hyperplasia early in life and mammary adenocarcinomas in approximately 50% of the female transgenic (TG) mice by 6 months of age. Metastasis to the lung and proximal lymph nodes is rare at the time tumors are detected but frequent after the removal of the primary neoplasm. The potent mitogenic effect mediated by Wnt-1 expression does not require estrogen stimulation; tumors form after an increased latency in estrogen receptor alpha-null mice. Several genetic lesions, including inactivation of p53 and over-expression of Fgf-3, collaborate with Wnt-1 in leading to mammary tumors, but loss of Sky and inactivation of one allele of Rb do not affect the rate of tumor formation in Wnt-1 TG mice.
Assuntos
Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas de Peixe-Zebra , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Hormônios/metabolismo , Hiperplasia/genética , Neoplasias Mamárias Experimentais/virologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Wnt , Proteína Wnt1RESUMO
Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17 beta. Ectopic expression of XSox17 beta ventralizes embryos by inhibiting the Wnt pathway downstream of beta-catenin but upstream of the Wnt-responsive gene Siamois. XSox17 beta also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and beta-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses beta-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17 beta maps to a region C-terminal to the HMG box; this region of XSox17 beta physically interacts with the Armadillo repeats of beta-catenin. Two additional Sox proteins, XSox17 alpha and XSox3, likewise bind to beta-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transativadores , Fatores de Transcrição , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Proteínas de Ligação a DNA/genética , Endossomos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/genética , Histocitoquímica , Proteínas de Homeodomínio/genética , Microinjeções , Ligação Proteica , Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1 , Fatores de Transcrição SOXF , Proteínas Wnt , Xenopus/embriologia , beta CateninaRESUMO
In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras , Transdução de Sinais , Transativadores , Proteínas de Peixe-Zebra , Animais , Proteína Axina , Caderinas/metabolismo , Linhagem Celular , Conexinas/genética , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Imunofluorescência , Genes Reporter , Proteínas de Homeodomínio/genética , Humanos , Membranas Intracelulares/metabolismo , Modelos Moleculares , Plasmídeos , Proteínas/metabolismo , Deleção de Sequência , Fatores de Transcrição/genética , Proteínas Wnt , Proteína Wnt1 , Xenopus , Proteínas de Xenopus , beta Catenina , gama CateninaRESUMO
To develop models of human cancer we have expressed the avian retroviral receptor, TVA, under a variety of mammalian promoters in transgenic mice, thus rendering mice susceptible to infection with avian leukosis virus-derived gene vectors. TVA-based retroviral gene transfer offers advantages over current murine models of human cancer. A single transgenic mouse line can be used to evaluate multiple genetic lesions, individually and in combination. Furthermore, mutant genes are introduced somatically into animals, as occurs in the majority of naturally occurring tumors. Because the avian viral vectors replicate only in avian cells, the viral receptor in infected transgenic mouse cells remains available for multiple rounds of infection with different ASLV vectors. We discuss the theoretical and practical aspects of using recombinant avian retroviruses with TVA transgenic mice to generate cancer models.
Assuntos
Modelos Animais de Doenças , Técnicas de Transferência de Genes , Neoplasias/genética , Alpharetrovirus/genética , Animais , Proteínas Aviárias , Humanos , Camundongos , Camundongos Transgênicos , Receptores Virais/genéticaRESUMO
Estrogens have important functions in mammary gland development and carcinogenesis. To better define these roles, we have used two previously characterized lines of genetically altered mice: estrogen receptor-alpha (ER alpha) knockout (ERKO) mice, which lack the gene encoding ER alpha, and mouse mammary virus tumor (MMTV)-Wnt-1 transgenic mice (Wnt-1 TG), which develop mammary hyperplasia and neoplasia due to ectopic production of the Wnt-1 secretory glycoprotein. We have crossed these lines to ascertain the effects of ER alpha deficiency on mammary gland development and carcinogenesis in mice expressing the Wnt-1 transgene. Introduction of the Wnt-1 transgene into the ERKO background stimulates proliferation of alveolar-like epithelium, indicating that Wnt-1 protein can promote mitogenesis in the absence of an ER alpha-mediated response. The hyperplastic glandular tissue remains confined to the nipple region, implying that the requirement for ER alpha in ductal expansion is not overcome by ectopic Wnt-1. Tumors were detected in virgin ERKO females expressing the Wnt-1 transgene at an average age (48 weeks) that is twice that seen in virgin Wnt-1 TG mice (24 weeks) competent to produce ER alpha. Prepubertal ovariectomy of Wnt-1 TG mice also extended tumor latency to 42 weeks. However, pregnancy did not appear to accelerate the appearance of tumors in Wnt-1 TG mice, and tumor growth rates were not measurably affected by late ovariectomy. Small hyperplastic mammary glands were observed in Wnt-1 TG males, regardless of ER alpha gene status; the glands were similar in appearance to those found in ERKO/Wnt-1 TG females. Mammary tumors also occurred in Wnt-1 TG males; latency tended to be longer in the heterozygous ER alpha and ERKO males (86 to 100 weeks) than in wild-type ER alpha mice (ca. 75 weeks). We conclude that ectopic expression of the Wnt-1 proto-oncogene can induce mammary hyperplasia and tumorigenesis in the absence of ER alpha in female and male mice. The delayed time of tumor appearance may depend on the number of cells at risk of secondary events in the hyperplastic glands, on the carcinogenesis-promoting effects of ER alpha signaling, or on both.
Assuntos
Mama/patologia , Neoplasias Mamárias Animais/genética , Vírus do Tumor Mamário do Camundongo/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Peixe-Zebra , Animais , Transformação Celular Viral , Receptor alfa de Estrogênio , Estrogênios/metabolismo , Feminino , Deleção de Genes , Técnicas de Transferência de Genes , Hiperplasia , Masculino , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transformação Genética , Proteínas Wnt , Proteína Wnt1RESUMO
Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.
Assuntos
Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/enzimologia , Códon de Iniciação/genética , Cisteína/química , Citoplasma/enzimologia , Primers do DNA/genética , Ativação Enzimática , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Células Jurkat , Camundongos , Ácidos Palmíticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Mutação Puntual , Proteínas Tirosina Quinases/química , Linfócitos T/enzimologiaRESUMO
Nearly all human gliomas exhibit alterations in one of three genetic loci governing G1 arrest: INK4a-ARF, CDK4, or RB. To discern the roles of CDK4 amplification and INK4a-ARF loss in gliomagenesis, we compared the behavior of astrocytes lacking a functional INK4a-ARF locus with astrocytes overexpressing CDK4. Either a deficiency of p16(INK4a) and p19(ARF) or an increase in Cdk4 allows cultured astrocytes to grow without senescence. Astrocytes overexpressing CDK4 grow more slowly than INK4a-ARF-deficient astrocytes and convert to a tetraploid state at high efficiency; in contrast, INK4a-ARF-deficient cells remain pseudodiploid, consistent with properties observed in human gliomas with corresponding lesions in these genes.
Assuntos
Astrócitos/citologia , Quinases Ciclina-Dependentes/biossíntese , Fase G1 , Glioma/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas , Animais , Células Cultivadas , Quinase 4 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Humanos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Ploidias , Proteínas/genética , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/genéticaRESUMO
The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%-50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a-ARF locus, which encodes two gene products (p16(INK4a) and p19(ARF)) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a-ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.