RESUMEN
Foamy virus (FV) vectors are promising for hematopoietic stem cell (HSC) gene therapy but preclinical data on the clonal composition of FV vector-transduced human repopulating cells is needed. Human CD34(+) human cord blood cells were transduced with an FV vector encoding a methylguanine methyltransferase (MGMT)P140K transgene, transplanted into immunodeficient NOD/SCID IL2Rγ(null) mice, and selected in vivo for gene-modified cells. The retroviral insertion site profile of repopulating clones was examined using modified genomic sequencing PCR. We observed polyclonal repopulation with no evidence of clonal dominance even with the use of a strong internal spleen focus forming virus promoter known to be genotoxic. Our data supports the use of FV vectors with MGMTP140K for HSC gene therapy but also suggests additional safety features should be developed and evaluated.
Asunto(s)
Inmunodeficiencia Combinada Grave/genética , Spumavirus/genética , Integración Viral/genética , Animales , Pruebas Genéticas/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas , Humanos , Ratones Endogámicos NOD , Ratones SCID , Acondicionamiento PretrasplanteRESUMEN
The accumulation of genetic and epigenetic alterations mediates colorectal cancer (CRC) formation by deregulating key signaling pathways in cancer cells. In CRC, one of the most commonly inactivated signaling pathways is the transforming growth factor-beta (TGF-ß) signaling pathway, which is often inactivated by mutations of TGF-ß type II receptor (TGFBR2). Another commonly deregulated pathway in CRC is the phosphoinositide-3-kinase (PI3K)-AKT pathway. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of PI3K-AKT signaling and is silenced in â¼30% of CRC. The combination of TGFBR2 inactivation and loss of PTEN is particularly common in microsatellite-unstable CRCs. Consequently, we determined in vivo if deregulation of these two pathways cooperates to affect CRC formation by analyzing tumors arising in mice that lack Tgfbr2 and/or Pten specifically in the intestinal epithelium. We found that lack of Tgfbr2 (Tgfbr2(IEKO)) alone is not sufficient for intestinal tumor formation and lack of Pten (Pten(IEKO)) alone had a weak effect on intestinal tumor induction. However, the combination of Tgfbr2 inactivation with Pten loss (Pten(IEKO);Tgfbr2(IEKO)) led to malignant tumors in both the small intestine and colon in 86% of the mice and to metastases in 8% of the tumor-bearing mice. Moreover, these tumors arose via a ß-catenin-independent mechanism. Inactivation of TGF-ß signaling and loss of Pten in the tumors led to increased cell proliferation, decreased apoptosis and decreased expression of cyclin-dependent kinase inhibitors. Thus, inactivation of TGF-ß signaling and loss of PTEN cooperate to drive intestinal cancer formation and progression by suppressing cell cycle inhibitors.
Asunto(s)
Neoplasias del Colon/patología , Fosfohidrolasa PTEN/deficiencia , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Mutación , Fosfohidrolasa PTEN/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
To investigate the signaling function of the Src-family protein tyrosine kinase Lck in mature T cells, we generated transgenic mice that expressed Lck in thymocytes but not in peripheral lymphocytes. We compared the phenotype and signaling capacity of Lck-deficient T cells with T cells from mice expressing a dominant inhibitory form of Lck and found that both mouse strains have diminished numbers of mature CD8(+) T cells and respond poorly to CD28 costimulation. However, while T cells that lack Lck fail to mobilize Ca(2+) after stimulation, those expressing the dominant negative protein do so normally. Our data demonstrate that Lck plays several unique roles in mature lymphocyte signaling.
Asunto(s)
Antígenos CD28/fisiología , Calcio/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , Linfocitos T/fisiología , Animales , Interleucina-2/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Considerable evidence supports a role for the Src family protein tyrosine kinase Lck in regulating multiple aspects of thymocyte development. In this report, we establish that early events in T lymphopoiesis are restored to Lck-deficient mice by provision of a transgene encoding a version of Lck that cannot interact with the coreceptors CD4 and CD8. In addition, we demonstrate that later events in thymocyte development, specifically, the processes of positive and negative selection, are compromised in mice where the only Lck available cannot associate with either CD4 or CD8. We conclude that not only is Lck activity required for positive and negative selection, but that that activity must be coupled to the CD4 and CD8 coreceptors.
Asunto(s)
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/enzimología , Timo/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/deficiencia , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología , Linfocitos T/enzimología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Transgenes/inmunologíaRESUMEN
Thymic stromal lymphopoietin (TSLP) is a newly identified cytokine that uniquely promotes B lymphopoiesis to the B220+/IgM+ immature B cell stage. In addition, TSLP shares many biological properties with the related cytokine IL-7. This can be explained by the finding that the receptor complexes for TSLP and IL-7 both contain the IL-7R alpha-chain; IL-7Ralpha is paired with the common gamma-chain (gammac) in the IL-7 receptor complex and the unique TSLP-R chain in the TSLP receptor complex. Although TSLP and IL-7 both induce tyrosine phosphorylation of the transcription factor Stat5, only IL-7-mediated signal transduction could be associated with activation of Janus family kinases (Jaks). Because Stat5 phosphorylation following cytokine stimulation is generally mediated by Jaks, the lack of Jak activation after TSLP treatment suggested the possibility that tyrosine-phosphorylated Stat5 may be nonfunctional. Herein, we demonstrate that TSLP induces a functional Stat5 transcription factor in that TSLP stimulation results in Stat5-DNA complex formation and transcription of the Stat5-responsive gene CIS. We also show that the TSLP receptor complex is functionally reconstituted using TSLP-R and IL-7Ralpha and that TSLP-mediated signal transduction requires Stat5. Moreover, TSLP-mediated signaling is inhibited by suppressor of cytokine signaling (SOCS)-1 and a kinase-deficient version of Tec but not by kinase-deficient forms of Jak1 and Jak2.
Asunto(s)
Linfocitos B/inmunología , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Leche , Transactivadores/metabolismo , Subgrupos de Linfocitos B/inmunología , Oncostatina M , Péptidos , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Receptores de Citocinas/metabolismo , Factor de Transcripción STAT5 , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas , Proteínas Supresoras de Tumor , Linfopoyetina del Estroma TímicoRESUMEN
7-Amino-4-methyl-6-sulfocoumarin-3-acetic acid (AMCA-S, also called Alexa 350) 2 was synthesized as a new water-soluble blue fluorescent dye for protein labeling. Compared with its nonsulfonated counterpart (AMCA) 1 the new dye gave significantly higher fluorescence quantum yields on proteins.
Asunto(s)
Acetatos/síntesis química , Cromonas/síntesis química , Colorantes Fluorescentes/síntesis química , Proteínas/análisis , Acetatos/química , Cromonas/química , Colorantes Fluorescentes/química , Solubilidad , Espectrometría de Fluorescencia , AguaRESUMEN
We have previously shown that protein prenylation occurs in the Trypanosomatids Trypanosoma brucei (T. brucei), Trypanosoma cruzi, and Leishmania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect (procyclic) form T. brucei. A PFT that transfers the farnesyl group from farnesyl pyrophosphate to a cysteine that is 4 residues upstream of the C terminus of the Ras GTP-binding protein RAS1-CVIM has now been purified 60,000-fold to near homogeneity from procyclic T. brucei. By screening a mixture of hexapeptides SSCALX (X is 20 different amino acids), it was found that SSCALM binds to T. brucei PFT with sub-micromolar affinity, and affinity chromatography using this peptide was a key step in the purification of this enzyme. On SDS-polyacrylamide gel electrophoresis, the enzyme migrates as a pair of bands with apparent molecular masses of 61 and 65 kDa, and thus its subunits are approximately 30% larger than those of the mammalian homolog. The 61-kDa band was identified as the putative beta-subunit by photoaffinity labeling with a 32P-labeled analog of farnesyl pyrophosphate. Mimetics of the C-terminal tetrapeptide of prenyl acceptors have been previously shown to inhibit mammalian PFT, and these compounds also inhibit T. brucei PFT with affinities in the nanomolar to micromolar range, although the structure-activity relationship is very different for parasite versus mammalian enzyme. Unlike mammalian cells, the growth of bloodstream T. brucei is completely inhibited by low micromolar concentrations of two of the PFT inhibitors, and these compounds also block protein farnesylation in cultured parasites. These compounds also potently block the growth of the intracellular (amastigote) form of T. cruzi grown in fibroblast host cells. The results suggest that protein farnesylation is a target for the development of anti-trypanosomatid chemotherapeutics.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Trypanosoma brucei brucei/enzimología , Tripanosomiasis/tratamiento farmacológico , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/aislamiento & purificación , Animales , Antiprotozoarios/química , Antiprotozoarios/uso terapéutico , Dimerización , Diseño de Fármacos , Etiquetas de Fotoafinidad , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Attachment of the prenyl groups farnesyl and geranylgeranyl to specific eukaryotic cell proteins by protein prenyltransferases is required for the functioning of a number of cellular processes including signal transduction. In this study it was found that previously reported inhibitors of mammalian protein farnesyltransferase (PFT) [those that mimic the substrate farnesyl pyrophosphate and those that mimic the protein acceptor of the farnesyl group (CaaX mimetic)] inhibit in vitro farnesylation catalyzed by partially purified Trypanosoma brucei (T. brucei) PFT. The most potent PFT inhibitors at concentrations of 3-10 microM inhibit the growth of insect (procyclic) and bloodstream forms of T. brucei. One of the PFT inhibitors was found to block the incorporation of radiolabeled mevalonic acid (the precursor of prenyl groups) into specific T. brucei proteins. This study also shows that protein prenylation occurs in the protozoan parasites Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana). The growth of T. cruzi intracellular form (amastigote) is also sensitive to PFT inhibitors, whereas the insect form (epimastigote) is considerably more resistant to inhibition of protein farnesylation. On the other hand, growth of 3T3 fibroblast cells (host cells for amastigote growth) was not affected by up to 100 microM PFT inhibitors. The growth of L. mexicana insect form (promastigote) is modestly inhibited by protein farnesyltransferase inhibitors. These results suggest the potential for the development of PFT inhibitors for treating trypanosomiasis and leishmaniasis.