RESUMEN
Highly malignant tumors express high levels of the minichromosome maintenance 2 (MCM2) protein, which is associated with advanced tumor grade, advanced stage, and poor prognosis. In a previous study, we showed that Friend leukemia virus (FLV) envelope protein gp70 bound MCM2, impaired its nuclear translocation, and enhanced DNA-damage-induced apoptosis in FLV-infected hematopoietic cells when the cells expressed high levels of MCM2. Here, we show that MCM2 is highly expressed in clinical samples of invasive carcinoma of the breast, especially triple-negative breast cancer (TNBC), and in cancer stem cell (CSC) marker-positive breast cancer cells. To generate a cancer therapy model using gp70, we introduced the gp70 protein into the cytoplasm of murine breast cancer cells that express high levels of MCM2 by conjugating the protein transduction domain (PTD) of Hph-1 to gp70 (Hph-1-gp70). Hph-1-gp70 was successfully transduced into the cytoplasm of breast cancer cells. The transduced protein enhanced the DNA damage-induced apoptosis of cancer cells in vitro and in vivo. Therefore, an MCM2-targeted strategy using Hph-1-gp70 treatment to induce DNA damage might be a successful therapy for highly malignant breast cancers such as TNBC and for the eradication of CSC-like cells from breast cancer tissue.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/biosíntesis , Adulto , Anciano , Animales , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Complejo Represivo Polycomb 1/farmacología , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes de Fusión/farmacología , Proteínas Oncogénicas de Retroviridae/farmacología , Transducción Genética , Transfección , Proteínas del Envoltorio Viral/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
At 18,954 nucleotides, the J paramyxovirus (JPV) genome is one of the largest in the family Paramyxoviridae, consisting of eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. To study the function of novel paramyxovirus genes in JPV, a plasmid containing a full-length cDNA clone of the genome of JPV was constructed. In this study, the function of the small hydrophobic (SH) protein of JPV was examined by generating a recombinant JPV lacking the coding sequence of the SH protein (rJPVΔSH). rJPVΔSH was viable and had no growth defect in tissue culture cells. However, more tumor necrosis factor alpha (TNF-α) was produced during rJPVΔSH infection, suggesting that SH plays a role in inhibiting TNF-α production. rJPVΔSH induced more apoptosis in tissue culture cells than rJPV. Virus-induced apoptosis was inhibited by neutralizing antibody against TNF-α, suggesting that TNF-α contributes to JPV-induced apoptosis in vitro. The expression of JPV SH protein inhibited TNF-α-induced NF-κB activation in a reporter gene assay, suggesting that JPV SH protein can inhibit TNF-α signaling in vitro. Furthermore, infection of mice with rJPVΔSH induced more TNF-α expression, indicating that SH plays a role in blocking TNF-α expression in vivo.
Asunto(s)
FN-kappa B/efectos de los fármacos , Infecciones por Paramyxoviridae/virología , Paramyxovirinae/patogenicidad , Proteínas Oncogénicas de Retroviridae/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Apoptosis , Línea Celular , Chlorocebus aethiops , Células L , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , FN-kappa B/metabolismo , Infecciones por Paramyxoviridae/metabolismo , Paramyxovirinae/genética , Paramyxovirinae/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células Vero , Ensayo de Placa ViralRESUMEN
Receptors of the EGF receptor or ErbB family of growth factor receptor tyrosine kinases are frequently overexpressed in a variety of solid tumours, and the aberrant activation of their tyrosine kinase activities is thought to contribute to tumour growth and progression. Much effort has been put into developing inhibitors of ErbB receptors, and both antibody and small-molecule approaches have exhibited clinical success. Recently, a number of endogenous negative regulatory proteins have been identified that suppress the signalling activity of ErbB receptors in cells. These include intracellular RING finger E3 ubiquitin ligases such as cbl and Nrdp1 that mediate ErbB receptor degradation, and may include a wide variety of secreted and transmembrane proteins that suppress receptor activation by growth factor ligands. It will be of interest to determine the extent to which tumour cells suppress these pathways to promote their progression, and whether restoration of endogenous receptor-negative regulatory pathways may be exploited for therapeutic benefit.
Asunto(s)
Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , Genes erbB/fisiología , Neoplasias/fisiopatología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Progresión de la Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias/genética , Proteína Oncogénica v-cbl , Proteínas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteínas Oncogénicas de Retroviridae/farmacología , Transducción de Señal , Ubiquitina-Proteína LigasasRESUMEN
The adaptor protein Crk has been reported to associate with focal adhesions and is thought to be involved in integrin-mediated signaling pathway. However, the precise mechanism of Crk-dependent regulation of cytoskeleton still remains under investigation. In this study, we have established a v-Crk-inducible cell line in rat fibroblasts 3Y1 cells and found that v-Crk activated Rho and induced actin stress fiber formation. In addition to the induction of tyrosine-phosphorylation of p130(Cas) and paxillin, we demonstrated that v-Crk induced threonine-phosphorylated bands sized at 72/78 kDa found specifically in 3Y1 cells. Both of the inhibitors of Rho and Rho-associated kinase, C3 and Y27632, respectively, inhibited these v-Crk-induced biochemical effects. Although v-Crk-induced cells exhibited a decrease of cell motility, integrin stimulation recovered the suppression of motility. Furthermore, v-Crk enhanced motility in chemotactic assay toward fibronectin with additional activation of Rho and the increase of levels of CD44 cleavage. These results suggest that v-Crk activated Rho and induced actin stress fiber formation and CD44 cleavage leading to the regulation of cell motility.
Asunto(s)
Proteínas de Fase Aguda/metabolismo , Movimiento Celular , Fibroblastos/metabolismo , Proteínas Oncogénicas de Retroviridae/farmacología , Transducción de Señal , Animales , Línea Celular , Células Cultivadas , Proteína Oncogénica v-crk , Fosforilación , Fosfotreonina/metabolismo , Ratas , Ratas Endogámicas , Proteínas Oncogénicas de Retroviridae/genética , Treonina/metabolismoRESUMEN
Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly characterized cell surface receptor, thereby promoting the gp21-dependent fusion of the viral and cellular membranes. Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion with Molt-4 target cells. It has been suggested that this peptide acts by competitively binding to Hsc70, a putative cellular receptor for HTLV-1. We now demonstrate that P-197 inhibits membrane fusion among diverse HTLV-1-permissive target cells. Importantly, most of these cells lack detectable levels of Hsc70, indicating that P-197 inhibits membrane fusion by a mechanism that is Hsc70 independent. We now suggest that competition for primary receptor binding is unlikely to account for the inhibitory activity of P-197. Understanding the mechanism by which P-197 functions may reveal concepts of general relevance to antiretroviral chemotherapy.
Asunto(s)
Productos del Gen env/farmacología , Proteínas HSP70 de Choque Térmico/fisiología , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Oncogénicas de Retroviridae/farmacología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Proteínas del Choque Térmico HSC70 , Células HeLa , Humanos , Datos de Secuencia MolecularRESUMEN
Efficient entry into, and infection of, human cells by human T-cell leukaemia virus type-1 (HTLV-1) is mediated by the viral envelope glycoproteins, gp46 and gp21. The gp46 surface glycoprotein binds to an as yet unidentified cell surface receptor, thereby, allowing the gp21 transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. In the absence of membrane fusion viral penetration and entry into the host cell cannot occur. The envelope glycoproteins are also a major target for neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and represent ideal constituents for a recombinant HTLV-1 vaccine. Given the importance of the envelope proteins in HTLV-1 pathogenesis there is increasing interest in obtaining sufficient quantities of these proteins for biochemical, biophysical and biological analyses. We have now developed a system for production of large amounts of a glycosylated and functional form of soluble recombinant gp46 (sRgp46), and have used this recombinant material for analysis of envelope function and receptor binding activity. We find that, the sRgp46 molecules expressed in our system are immunologically indistinguishable from the native virally expressed surface glycoproteins; that sRgp46 binds to T-cells in a dose dependent and saturable manner; and that cell surface binding by sRgp46 can be inhibited by neutralising antibodies. Importantly, we demonstrate that these sRgp46 molecules potently inhibit syncytia formation and viral infection of target cells, and that regions outwith the SU domain of envelope are not required for binding to target cells or for inhibiting membrane fusion. The sRgp46 produced in our study will provide new opportunities to investigate envelope-receptor interactions, and will be of utility in defining the conformationally sensitive antigenic determinants of the HTLV-1 surface glycoprotein.
Asunto(s)
Productos del Gen env/farmacología , Células Gigantes/efectos de los fármacos , Antígenos HTLV-I/farmacología , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas Oncogénicas de Retroviridae/farmacología , Animales , Unión Competitiva , Células Cultivadas , Relación Dosis-Respuesta a Droga , Drosophila , Células Gigantes/virología , Antígenos HTLV-I/biosíntesis , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Células Jurkat , Pruebas de Neutralización , Unión Proteica , Receptores Virales/metabolismo , Proteínas Recombinantes/farmacología , Linfocitos T/metabolismoRESUMEN
Interleukin-1 (IL-1) mediates numerous host responses through rapid activation of nuclear factor-kappaB (NF-kappaB), but signal pathways leading to the NF-kappaB activation appear to be complicated and multiplex. We propose a novel regulatory system for NF-kappaB activation by the extracellular signal-related kinase (ERK) pathway. In a human glioblastoma cell line, T98G, IL-1-induced NF-kappaB activation was significantly augmented by the pretreatment of a specific MEK inhibitor, PD98059. In contrast, ectopic expression of a constitutive activated form of Raf (v-Raf) reduced IL-1-induced NF-kappaB activation, and this inhibition was completely reversed by PD98059. Interestingly, PD98059 sustained IL-1-induced NF-kappaB DNA binding activity by an electrophoretic mobility shift assay and also IkappaBalpha degradation, presumably by augmenting and sustaining the proteasome activation. Concomitantly, two NF-kappaB dependent genes, A20 and IkappaBalpha expression were prolonged with PD98059. These data suggested that MEK-ERK pathway exerts a regulatory effect on NF-kappaB activation, providing a novel insight on the role of MEK-ERK pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Flavonoides/farmacología , Proteínas I-kappa B , Interleucina-1/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , FN-kappa B/metabolismo , Unión Competitiva/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glioblastoma/metabolismo , Humanos , Quinasa I-kappa B , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Complejos Multienzimáticos/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Proteínas Nucleares , Proteínas Oncogénicas v-raf , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/biosíntesis , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 6 Asociado a Receptor de TNF , Factor de Transcripción AP-1/metabolismo , Transfección , Células Tumorales Cultivadas , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
The v-Crk oncogene encodes an adaptor protein containing an SH2 domain and an SH3 domain. v-Crk-transformed fibroblast cells display enhanced tyrosine phosphorylation levels, and the v-Crk protein localizes in focal adhesions, suggesting that transformation may be due to enhanced focal complex signaling. Here we investigated the mechanism of transformation and found that v-Crk-transformed NIH 3T3 cells display growth rates and serum requirements similar to control cells. However, v-Crk enhanced survival in conditions of serum starvation. Both an intact SH2 and SH3 domain are required; moreover, SH2 mutants displayed dominant interfering properties, enhancing cell death. Using other cell death-inducing stimuli, it appeared that v-Crk in general inhibits apoptosis and enhances cell survival. In search of the signaling pathways involved, we found that v-Crk-transformed cells show constitutively higher levels of phospho-protein kinase B (PKB)/Akt and PKB/Akt activity, especially in conditions of serum starvation. These data strongly suggest involvement of the phosphatidylinositol 3-kinase/PKB survival pathway in the v-Crk-induced protection against apoptosis. In accordance, inhibition of this pathway by wortmannin or LY924002 reduced protection against starvation-induced apoptosis. In addition to the phosphatidylinositol 3-kinase/PKB pathway, a MEK-dependent pathway and an unknown additional pathway are also implicated in resistance against apoptosis. Activation of survival pathways may be the most important function of v-Crk in its oncogenic properties.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Oncogénicas de Retroviridae/farmacología , Células 3T3 , Androstadienos/farmacología , Animales , Apoptosis/efectos de los fármacos , Células COS , Cricetinae , Activación Enzimática , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones , Proteína Oncogénica v-crk , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Transfección , Wortmanina , Dominios Homologos srcRESUMEN
To investigate the roles of human T-cell leukemia virus type 1 (HTLV-1) envelope (Env) proteins gp46 and gp21 in the early steps of infection, the effects of the 23 synthetic peptides covering the entire Env proteins on transmission of cell-free HTLV-1 were examined by PCR and by the plaque assay using a pseudotype of vesicular stomatis virus (VSV) bearing the Env of HTLV-1 [VSV(HTLV-1)]. The synthetic peptide corresponding to amino acids 400 to 429 of the gp21 Env protein (gp21 peptide 400-429, Cys-Arg-Phe-Pro-Asn-Ile-Thr-Asn-Ser-His-Val-Pro-Ile-Leu-Gln-Glu-Arg-P ro-Pro-Leu-Glu-Asn-Arg-Val-Leu-Thr-Gly-Trp-Gly-Leu) strongly inhibited infection of cell-free HTLV-1. By using the mutant peptide, Asn407, Ser408, and Leu413, -419, -424, and -429 were confirmed to be important amino acids for neutralizing activity of the gp21 peptide 400-429. Addition of this peptide before or during adsorption of HTLV-1 at 4 degrees C did not affect its entry. However, HTLV-1 infection was inhibited about 60% when the gp21 peptide 400-429 was added even 30 min after adsorption of HTLV-1 to cells, indicating that the amino acid sequence 400 to 429 on the gp21 Env protein plays an important role at the postbinding step of HTLV-1 infection. In contrast, a monoclonal antibody reported to recognize the gp46 191-196 peptide inhibited the infection of HTLV-1 at the binding step.
Asunto(s)
Productos del Gen env/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Péptidos/farmacología , Proteínas Oncogénicas de Retroviridae/farmacología , Secuencia de Aminoácidos , Animales , Gatos , Línea Celular , Productos del Gen env/síntesis química , Productos del Gen env/química , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Proteínas Oncogénicas de Retroviridae/síntesis química , Proteínas Oncogénicas de Retroviridae/química , Transcripción Genética , Proteínas del Envoltorio Viral/química , Ensayo de Placa Viral , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
In response to hormones and growth factors, cultured neonatal ventricular myocytes increase in profile, exhibit myofibrillogenesis, and re-express genes whose expression is normally restricted to the fetal stage of ventricular development. These include atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM)-alpha-actin. By using luciferase reporter plasmids, we examined whether oncogenes that activate the extracellular signal-regulated kinase cascade (srcF527, Ha-rasV12, and v-raf) increased expression of "fetal" genes. Transfection of myocytes with srcF527 stimulated expression of ANF, SkM-alpha-actin, and beta-MHC by 62-, 6.7-, and 50-fold, respectively, but did not induce DNA synthesis. Stimulation of ANF expression by srcF527 was greater than by Ha-rasV12, which in turn was greater than by v-raf. General gene expression was also increased but to a lesser extent. The response to srcF527 was inhibited by dominant-negative Ha-rasN17. Myocyte area was increased by srcF527, Ha-rasV12, and v-raf, and although it altered myocyte morphology by causing a pseudopodial appearance, srcF527 did not detectably increase myofibrillogenesis either alone or in combination with Ha-rasV12. A kinase-dead src mutant increased myocyte size to a much lesser extent than srcF527 and also did not inhibit ANF-luciferase expression in response to phenylephrine. We conclude that members of the Src family of tyrosine kinases may be important in mediating the transcriptional changes occurring during cardiac myocyte hypertrophy and that Ras and Raf may be downstream effectors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genes ras , Genes src , Corazón/crecimiento & desarrollo , Miocardio/citología , Proteínas Proto-Oncogénicas c-raf/genética , Actinas/metabolismo , Animales , Factor Natriurético Atrial/farmacología , Tamaño de la Célula , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Genisteína/farmacología , Inhibidores de Crecimiento/farmacología , Luciferasas/genética , Cadenas Pesadas de Miosina/farmacología , Proteínas Oncogénicas v-raf , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas Oncogénicas de Retroviridae/farmacología , Transfección , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoAsunto(s)
Productos del Gen nef/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Péptidos/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Productos del Gen nef/síntesis química , Productos del Gen nef/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/antagonistas & inhibidores , Interleucina-2/antagonistas & inhibidores , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , ARN Mensajero , Proteínas Oncogénicas de Retroviridae/síntesis química , Proteínas Oncogénicas de Retroviridae/farmacología , Transducción de Señal , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Membrane anchorage of Ras oncoproteins, required for transforming activity, depends on their carboxy-terminal farnesylcysteine. We previously showed that S-trans,trans-farnesylthiosalicylic acid (FTS), a synthetic farnesylcysteine mimetic, inhibits growth of ErbB2- and Ras-transformed cells, but not of v-Raf-transformed cells, suggesting that FTS interferes specifically with Ras functions. Here we demonstrate that FTS dislodges Ras from membranes of H-Ras-transformed (EJ) cells, facilitating its degradation and decreasing total cellular Ras. The dislodged Ras that was transiently present in the cytosol was degraded relatively rapidly, causing a decrease of up to 80% in total cellular Ras. The half-life of Ras was 10 +/- 4 h in FTS-treated EJ cells and 27 +/- 4 h in controls. The dislodgment of membrane Ras and decrease in total cellular Ras were dose-dependent: 50% of the effects occurred at 10-15 microM, comparable to concentrations (7-10 microM) required for 50% growth inhibition in EJ cells. Higher concentrations of FTS (25-50 microM) were required to dislodge Ras from Rat-1 cell membranes expressing normal Ras, suggesting some selectivity of FTS toward oncogenic Ras. Membrane localization of the prenylated G beta gamma of heterotrimeric G proteins was not affected by FTS in EJ cells. An FTS-related compound, N-acetyl-S-farnesyl-L-cysteine, which does not inhibit EJ cell growth, did not affect Ras. FTS did not inhibit growth of Rat-1 cells transformed by N-myristylated H-Ras and did not reduce the total amount of this Ras isoform. The results suggest that FTS affects docking of Ras in the cell membrane in a rather specific manner, rendering the protein susceptible to proteolytic degradation.
Asunto(s)
Proteínas ras/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Farnesol/análogos & derivados , Farnesol/farmacología , Inhibidores de Crecimiento/farmacología , Metilación/efectos de los fármacos , Proteínas Oncogénicas v-raf , Ratas , Receptor ErbB-2/farmacología , Proteínas Oncogénicas de Retroviridae/farmacología , Salicilatos/farmacología , Proteínas ras/antagonistas & inhibidores , Proteínas ras/efectos de los fármacosRESUMEN
The v-Rel oncoprotein is a member of the Rel/NF-kappaB family of transcription factors. v-Rel induces oncogenic transformation and inhibits apoptosis. To identify aberrantly expressed cellular genes in v-Rel transformed cells, gene expression patterns in normal and v-Rel transformed cells were compared by mRNA differential display. Northern blotting analysis with radiolabeled cDNAs from differential display confirmed the reproducible differential expression of 10 transcripts in v-Rel transformed cells. One of the identified genes, termed ch-IAP1, encodes a chicken homolog of the inhibitor-of-apoptosis protein (IAP) family. ch-IAP1 contains N-terminal baculovirus IAP repeats (BIR), the hallmark of IAPs, and has a C-terminal RING finger motif commonly present in the other IAPs. Like other IAPs, ch-IAP1 is expressed predominantly in the cytoplasm of cells. ch-IAP1 is highly expressed in v-Rel transformed fibroblasts, B- and T-cell lines, and spleen cell lines. In contrast, ch-IAP1 expression levels were low in chicken cell lines transformed by several other unrelated tumor viruses. Additionally, ch-IAP1 expression is downregulated in temperature-sensitive (ts) v-Rel transformed spleen cells at the nonpermissive temperature. Overexpression of the full-length ch-IAP1 suppresses mammalian cell apoptosis induced by the interleukin-1-converting enzyme (ICE), a member of the mammalian caspase family of cysteine proteases. Furthermore, expression of exogenous ch-IAP1 inhibits apoptosis of ts v-Rel transformed spleen cells at the nonpermissive temperature.
Asunto(s)
Apoptosis/genética , Clonación Molecular/métodos , Proteínas Oncogénicas de Retroviridae/genética , Animales , Virus del Sarcoma Aviar/genética , Línea Celular , Embrión de Pollo , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Neoplasias/genética , Proteínas Oncogénicas v-rel , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Proteínas Oncogénicas de Retroviridae/farmacología , Temperatura , Transfección/genética , Transformación Genética/genéticaRESUMEN
Rap1 was identified as gene whose overexpression suppressed transformation by ras. Rap1 belongs to the Ras family. The amino acid sequences of Rap1 and Ras show 55% identity to each other. Due to this high sequence similarity, Rap1 binds to effector molecules of Ras, however, Rap1 does not activate them. Thus, Rap1 functions are antagonistic to Ras in the cells. C3G was identified as a Crk SH3-binding guanine nucleotide exchange factor. Biochemical and cell biological analyses revealed that C3G is a Rap1 activator. Since it has been considered that Crk transduces signals from tyrosine kinases, this finding suggests that the activity of Rap1 is also under the control of tyrosine kinases. Overexpression of C3G in ras-transformed cells caused the morphology of the cells to revert to that of normal cells. Moreover, a mutant cell line that was resistant to EGF-dependent transformation was isolated. In the cell line a mutation was found in crk gene that was the cause of the resistance. These findings suggest that Crk-C3G-Rap1 pathway may function as an anti-transformation machinery.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Genes ras , Proteínas ras/antagonistas & inhibidores , Proteínas ras/fisiología , Animales , Transformación Celular Neoplásica , Factores de Intercambio de Guanina Nucleótido , Humanos , Proteína Oncogénica v-crk , Proteínas/fisiología , Proteínas Oncogénicas de Retroviridae/farmacología , Transducción de Señal , Células Tumorales Cultivadas , Proteínas de Unión al GTP rap , Factores de Intercambio de Guanina Nucleótido ras , Proteínas ras/químicaRESUMEN
We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.
Asunto(s)
Vectores Genéticos/genética , Glutatión Transferasa/metabolismo , Oocitos/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Embrión no Mamífero/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/farmacología , Microinyecciones , Datos de Secuencia Molecular , Mutación , Proteínas Oncogénicas v-raf , Oocitos/efectos de los fármacos , ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Proteínas Oncogénicas de Retroviridae/farmacología , Transcripción GenéticaRESUMEN
The v-Rel oncoprotein belongs to the Rel/NF-kappaB family of transcription factors. It transforms chicken lymphoid cells in vitro and induces fatal lymphomas in vivo. In this study, we used a tetracycline-regulated system to characterize the role of v-Rel in cell transformation. We show that the continued expression of v-Rel is necessary to maintain the viability of transformed lymphoid cells and enables primary spleen cells to escape apoptosis in vitro culture. In agreement with a possible role for v-Rel in the inhibition of programmed cell death, its inducible expression in HeLa cells prevented TNFalpha-induced apoptosis. While the repression of v-Rel was accompanied by the rapid degradation of IkappaBalpha, changes in the steady-state levels of the apoptosis inhibitors Bcl-2 and Bcl-X(L) were only observed following the onset of cell death in transformed lymphoid cells. This suggests that the anti-apoptotic activity of v-Rel may affect other apoptosis inhibitors or other factors in the death pathway. Together, these findings demonstrate that v-Rel blocks apoptosis and suggest that this activity may be an important component of its transforming function.
Asunto(s)
Apoptosis , Proteínas Oncogénicas de Retroviridae/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , División Celular , Transformación Celular Neoplásica , Células Cultivadas , Pollos , Proteínas Oncogénicas v-rel , Proteínas Oncogénicas de Retroviridae/farmacología , Bazo/metabolismo , Tetraciclina/farmacología , Transactivadores/farmacologíaRESUMEN
Studies of the effects of retroviruses on the immune system, which date back through thirty years of investigations, are reviewed. In the earliest published studies in the 1960s, it was demonstrated that mice infected with oncogenic viruses were immunosuppressed. Since then, numerous articles have been published describing profound immunodeficiencies observed in vivo in humans infected with human immunodeficiency virus and in animals such as cats infected with the feline immunodeficiency virus. In vitro investigations have shown that inactivated retroviruses or transmembrane envelope protein p15E as well as a synthetic 17-amino acid peptide (CKS-17) impressively conserved within the transmembrane envelope protein of several animal or human retroviruses are highly immunosuppressive. More recently, dysfunction of cytokines produced by CKS-17 at both a cellular and molecular level have been found to mimic influences observed in vivo in patients infected with the human immunodeficiency virus. CKS-17 has also been shown to induce cAMP in vitro. The significance of these observations to understanding the immunological disturbances observed in malignancy, cytokine biosynthesis, and modulations of immune functions through cAMP is discussed.
Asunto(s)
Síndromes de Inmunodeficiencia/etiología , Síndromes de Inmunodeficiencia/virología , Inmunosupresores/farmacología , Infecciones por Retroviridae/etiología , Infecciones por Retroviridae/inmunología , Proteínas Oncogénicas de Retroviridae/farmacología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Infecciones por Retroviridae/patologíaRESUMEN
The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/metabolismo , Proteínas de la Leche , Proteína Oncogénica pp60(v-src)/farmacología , Transactivadores/metabolismo , Dominios Homologos src , Proteínas de Fase Aguda/metabolismo , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Oncogénicas de Retroviridae/farmacología , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Transducción de Señal , Relación Estructura-ActividadRESUMEN
In humans and animals, retroviruses have been implicated in nervous system disease. Our objective was to characterize the neurotoxicity of a peptide sequence derived from an animal retrovirus, the feline leukemia virus (FeLV). Using a peptide sequence from the subtype FeLV-C envelope protein variable region 5 (VR5), cytotoxicity was demonstrated in studies that evaluated neuronal survival, neurite outgrowth, and alterations in intracellular calcium ion concentration. The FeLV subtype isolate FeLV-CSarma possesses an envelope protein VR5 amino acid sequence that varies by four amino acids from the VR5 amino acid sequence of subtype FeLV-AGlasgow. The polypeptide representing the VR5 of FeLV-CSarma (FeLV-CVR5) is significantly more neurotoxic than the polypeptide sequence representing the VR5 of FeLV-AGlasgow (FeLV-AVR5). FeLV-CVR5 (> or = 3 microM) exposure resulted in significant dose-dependent neurotoxicity. Antibodies to FeLV-CVR5 blocked this effect. Neurite outgrowth was significantly reduced at all tested concentrations (3-12 microM) of FeLV-CVR5, with a 92% reduction in neurite length at 12 microM. FeLV-AVR5 was significantly less neurotoxic with respect to neurite outgrowth than was FeLV-CVR5. The significant reduction in neurotoxicity for FeLV-AVR5 illustrates the importance of the 4-amino-acid difference between it and FeLV-CVR5. Alterations in intracellular calcium ion concentration were associated with this neurotoxicity.
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Calcio/metabolismo , Neuronas/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/farmacología , Proteínas del Envoltorio Viral/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Ganglios Parasimpáticos/citología , Homeostasis , Neuronas/metabolismo , Nervio Oculomotor/citología , Péptidos/farmacología , Péptidos/toxicidad , Proteínas Oncogénicas de Retroviridae/química , Proteínas Oncogénicas de Retroviridae/toxicidad , Factores de Tiempo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/toxicidadRESUMEN
The Dbl oncogene is the putative exchange factor for two small GTP-binding proteins, RhoA and CDC42 which are involved in the polymerization of actin to produce stress fibers and filopodia, respectively. We report here that Dbl oncogene-transformed NIH3T3 cells show actin stress fibers only when cells are plated on fibronectin. Plating of cells on collagen I and IV as well as on poly-D-lysine and gelatin induces polymerization of actin to form filopodia, lamellipodia and membrane ruffles but not stress fibers. The putative collagen receptors, alpha1/beta1 and alpha2/beta1 integrins are expressed at reduced level in Dbl-transformed cells compared to untransformed NIH3T3 fibroblasts. Nevertheless, adhesion to collagens is not altered. Inhibitory monoclonal antibody to mouse integrin beta1 subunit blocked adhesion of both Dbl-transformed and untransformed NIH3T3 cells, demonstrating that adhesion to collagen I and IV is mediated by the beta1 family of integrins. Dbl product rapidly induces the depolymerization of actin stress fibers, rounding up of the cells, and formation of filopodia and lamellipodia when microinjected in NIH3T3 cells plated on gelatin. Thus, Dbl may exert its effect on actin cytoskeleton organization in response to extracellular proteins by altering integrin-mediated signalling pathways.