RESUMEN
Using recombinant DNA technology, we have generated Chinese hamster ovary (CHO) cell lines that synthesize latent transforming growth factor beta 1 (TGF-beta 1) to study immune regulation by TGF-beta 1. In vitro, latent TGF-beta 1 synthesized by transfectants or added exogenously as a purified complex after activation inhibited CTL generation to a similar extent as seen with acid-activated recombinant human (rHu) TGF-beta 1. In vivo, serum from nu/nu mice bearing CHO/TGF-beta 1 tumors contained significant levels of latent TGF-beta 1 in addition to depressed natural killer (NK) activity in spleens which paralleled that seen in C3H/HeJ mice treated with acid-activated rHuTGF-beta 1. rHuTGF-beta 1 treatment of mice receiving heart allografts resulted in significant enhancement of organ graft survival. Because of possible regulated tissue-specific activation, administration of latent rather than active TGF-beta may provide a better route to deliver this powerful immunosuppressive agent in vivo.
Asunto(s)
Transformación Celular Neoplásica , Células Asesinas Naturales/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Línea Celular , Cricetinae , Cricetulus , Femenino , Supervivencia de Injerto , Trasplante de Corazón/inmunología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ovario , Proteínas Recombinantes/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Transfección , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Single amino acid changes were introduced into normal (non-oncogenic) and activated forms of the human H-ras protein at a position (residue 116) proposed on structural grounds to represent a contact site with guanine nucleotides. Substitutions at this site could significantly reduce the ability of both forms to bind and hydrolyze guanosine 5'-triphosphate; these substitutions, however, did not necessarily diminish the transforming capacity of activated derivatives. One substitution that severely impairs these functions activated the transforming potential of the otherwise normal polypeptide.
Asunto(s)
Proteínas de Neoplasias/genética , Oncogenes , Secuencia de Aminoácidos , Transformación Celular Neoplásica/metabolismo , ADN/genética , Guanosina Trifosfato/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Proteína Oncogénica p21(ras)RESUMEN
Oncogenic Ras proteins transform animal cells to a malignant phenotype only when modified by farnesyl residues attached to cysteines near their carboxyl termini. The farnesyltransferase that catalyzes this reaction recognizes tetrapeptides of the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is a carboxyl-terminal methionine or serine. Replacement of the two aliphatic residues with a benzodiazepine-based mimic of a peptide turn generated potent inhibitors of farnesyltransferase [50 percent inhibitory concentration (IC50) < 1 nM]. Unlike tetrapeptides, the benzodiazepine peptidomimetics enter cells and block attachment of farnesyl to Ras, nuclear lamins, and several other proteins. At micromolar concentrations, these inhibitors restored a normal growth pattern to Ras-transformed cells. The benzodiazepine peptidomimetics may be useful in the design of treatments for tumors in which oncogenic Ras proteins contribute to abnormal growth, such as that of the colon, lung, and pancreas.
Asunto(s)
Transferasas Alquil y Aril , Antineoplásicos/farmacología , Benzodiazepinonas/farmacología , Proteínas Oncogénicas/metabolismo , Prenilación de Proteína/efectos de los fármacos , Transferasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Benzodiazepinonas/química , Células CHO , División Celular/efectos de los fármacos , Línea Celular Transformada , Transformación Celular Neoplásica/efectos de los fármacos , Cricetinae , Diseño de Fármacos , Farnesiltransferasa , Datos de Secuencia Molecular , Oligopéptidos/farmacologíaRESUMEN
Transforming growth factor beta 1 (TGF-beta 1) is a key regulator of cell growth and differentiation. Under normal physiological conditions, it is made as a biologically latent complex whose significance is unknown. Previous work has indicated that active TGF-beta 1 has a very short plasma half-life in rats (Coffey, R. J., L. J. Kost, R. M. Lyons, H. L. Moses, and N. F. La-Russo. 1987. J. Clin. Invest. 80:750-757). We have investigated the possibility that latent complex formation may extend the plasma half-life of TGF-beta 1 and alter its organ distribution. Radiolabeled latent TGF-beta 1 was formed by noncovalent association of 125I-TGF-beta 1 with the TGF-beta 1 precursor "pro" region from recombinant sources. TGF-beta 1 in this latent complex had a greatly extended plasma half-life (greater than 100 min) in rats compared with active TGF-beta 1 (2-3 min). Whereas active TGF-beta 1 was rapidly taken up by the liver, kidneys, lungs, and spleen and degraded, TGF-beta 1 in the latent complex was largely confined to the circulation, and was less than 5% degraded after 90 min. The pharmacokinetics of TGF-beta 1 in the latent complex were shown to be critically dependent on the degree of sialylation of the complex. The results suggest that formation of latent complexes may switch endogenous TGF-beta 1 from an autocrine/paracrine mode of action to a more endocrine mode involving target organs distant from the site of synthesis.
Asunto(s)
Factor de Crecimiento Transformador beta/farmacocinética , Animales , Autorradiografía , Tasa de Depuración Metabólica , Peso Molecular , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Ratas , Proteínas Recombinantes/farmacocinética , Ácidos Siálicos/fisiología , Relación Estructura-Actividad , Distribución Tisular , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes.
Asunto(s)
División Celular/fisiología , Transformación Celular Neoplásica , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/farmacología , Linfocinas/biosíntesis , Linfocinas/farmacología , Transfección , Animales , Células CHO , División Celular/efectos de los fármacos , Células Clonales , Cricetinae , Factores de Crecimiento Endotelial/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Humanos , Hibridación in Situ , Cinética , Linfocinas/genética , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/ultraestructura , ARN Mensajero/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Factores de Tiempo , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.
Asunto(s)
Transformación Celular Neoplásica , Genes ras , Transcripción Genética , Animales , Línea Celular , Replicación del ADN , Amplificación de Genes , Cinética , Ratas , TransfecciónRESUMEN
We examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COS cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). We constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.
Asunto(s)
Quimera , Genes Virales , Genes , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Plásmidos , Virus 40 de los Simios/genética , Secuencia de Bases , Línea Celular , ADN/análisis , Enzimas de Restricción del ADN , Humanos , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , TransfecciónRESUMEN
We expressed normal and activated human cellular Ha-ras cDNAs which encode 21,000-dalton polypeptides (p21s) in Saccharomyces cerevisiae by their insertion into a 2 micron-based replicating plasmid vector under 3-phosphoglycerate kinase promoter control. We found that newly synthesized p21 in S. cerevisiae was produced as a soluble precursor (pro-p21) which matured into a form electrophoretically indistinguishable from the processed form (p21) observed in mammalian cells. Coincident with the processing event was translocation to a membrane component, suggesting a coupling of the two events. Using vectors that direct the synthesis of p21 variants possessing the ability to autophosphorylate in vitro, we found that processing of p21 did not significantly affect this autophosphorylation reaction. In contrast to Escherichia coli, marked phenotypic changes were observed in S. cerevisiae as a consequence of the synthesis of p21, including reduction in growth rate and induction of flocculation. Accompanying these phenotypic alterations was a significant elevation of adenylate cyclase activity.
Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas Proto-Oncogénicas/genética , Saccharomyces cerevisiae/genética , Adenilil Ciclasas/metabolismo , Agregación Celular , Compartimento Celular , Ciclo Celular , Clonación Molecular , Regulación de la Expresión Génica , Ingeniería Genética , Humanos , Peso Molecular , Fenotipo , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/citologíaRESUMEN
Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.
Asunto(s)
Empalme Alternativo , Genes ras , Hormona del Crecimiento/genética , Fosfotransferasas (Aceptor de Grupo Alcohol) , Fosfotransferasas/genética , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/metabolismo , Secuencia de Bases , Línea Celular , Desoxirribonucleasa I/genética , Expresión Génica , Genes , Vectores Genéticos , Humanos , Intrones , Kanamicina Quinasa , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN/genética , ARN/aislamiento & purificación , Precursores del ARN/genética , ARN Nuclear Pequeño/genética , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , TransfecciónRESUMEN
We introduced the gene encoding the hepatitis B virus surface antigen (HBsAg) into simian virus 40 (SV40)-based plasmids capable of autonomously replicating in both Escherichia coli and permissive monkey cells. After introduction into monkey cells by transfection, these plasmids directed the synthesis of high levels of HBsAg, as determined by immunofluorescence, radioimmunoassays, and identification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides comprising the antigen. Expression was dependent upon the presence of an SV40 promoter, with both the early and late promoters able to effectively initiate transcription. Using expression of HBsAg to assay promoter function, we demonstrated that an intact copy of the SV40 72-base pair repeat, which constitutes an essential element of the SV40 early promoter during the lytic SV40 cycle and which can enhance the transcriptional activity of heterologous promoters, was not required for HBsAg expression, suggesting that the hepatitis genome contains an enhancer element capable of complementing that provided by the 72-base pair repeat element of SV40. The antigen appears to be glycosylated after synthesis in transfected cells and is apparently secreted, as evidenced by the localization of [35S]cysteine-labeled antigen to the medium of transfected cultures. Using constructions in which the first ATG sequence appearing in HBsAg mRNA was that corresponding to the gene encoding the mature form of the antigen, we demonstrated that these post-translational events could occur without the involvement of a putative precursor peptide suggested by the DNA sequence of the viral genome. In view of the inability of hepatitis B virus to propagate in vitro, this strategy offers a convenient approach for further characterizing the biosynthesis of this antigen and may provide a means to identify additional polypeptides encoded by this virus.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/genética , Plásmidos , Animales , Células Cultivadas , Genes , Genes Virales , Haplorrinos , Virus de la Hepatitis B/genética , Operón , ARN Mensajero/genética , Virus 40 de los Simios/genética , Transcripción Genética , Proteínas Virales/aislamiento & purificaciónRESUMEN
Transforming growth factor alpha (TGF-alpha) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-alpha promoter and localized the 5' end of the TGF-alpha transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.
Asunto(s)
Genes Homeobox , Genes , Regiones Promotoras Genéticas , Transcripción Genética , Factores de Crecimiento Transformadores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Quimera , Humanos , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Mapeo RestrictivoRESUMEN
We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.
Asunto(s)
Transformación Celular Viral , Oncogenes , Proteínas Quinasas/genética , Proteínas Virales/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Compartimento Celular , Células Cultivadas , Mutación , Neoplasias Experimentales/patología , Proteína Oncogénica pp60(v-src) , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas , Ratas , Relación Estructura-Actividad , TransfecciónRESUMEN
We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential.
Asunto(s)
Mutación , Oncogenes , Proteínas Proto-Oncogénicas/genética , ADN/metabolismo , Escherichia coli/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Genes , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
We demonstrate an example of signal transduction by an integrin and have begun to define the pathway through which this signaling is achieved. We constructed a stably transfected derivative of 293 cells (ATCC 1573) that expresses the platelet integrin GPIIbIIIa (alpha IIb beta 3). This cell line, clone B, adheres to and spreads on fibrinogen, a ligand for alpha IIb beta 3, while the parent cell line does not. Stimulation of these cells either by adhesion to fibrinogen or with antiserum directed against alpha IIb beta 3 results in induction of calcium oscillations, followed by tyrosine phosphorylation of at least one protein of molecular weight approximately 125 kDa. We establish that this phosphorylation, as well as the morphological rearrangements, requires the mobilization of calcium.
Asunto(s)
Plaquetas/metabolismo , Calcio/metabolismo , Integrinas/metabolismo , Transducción de Señal , Tirosina/metabolismo , Adhesión Celular , Línea Celular , Tamaño de la Célula , Fibrinógeno , Periodicidad , Fosforilación , TransfecciónRESUMEN
It is of great interest in tumor immunology to know whether oncogene products could be used not only as tumor markers for cancer diagnosis, but also as immunogens for cancer therapy. BALB/c mice immunized with syngeneic fibroblasts, Escherichia coli cells producing p60v-src, or the purified p60v-src protein extracted from the E. coli producer cells showed transplantation resistance to a Rous sarcoma virus-induced tumor but not a Kirsten sarcoma virus-induced tumor. In contrast, mice immunized with cells not producing p60v-src or their derived proteins or with chicken ovalbumin did not show any significant resistance. These findings suggest that p60v-src can act as a specific transplantation rejection antigen in mice.
Asunto(s)
Proteínas de los Retroviridae/inmunología , Sarcoma Aviar/inmunología , Animales , Antígenos de Neoplasias/inmunología , Antígenos Virales de Tumores/inmunología , Antígenos de Histocompatibilidad/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Proteína Oncogénica pp60(v-src) , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Within the past three years there has been a rapid expansion in our knowledge of the role TGF-beta mediates in regulating immune responses in vitro. Whether the TGF-beta will be clinically useful to suppress immune responses to transplanted organs or autoimmune responses is unknown. However, now that highly purified quantities of TGF-beta are available through recombinant DNA technologies, questions concerning the in vivo immunosuppressive activities of TGF-beta can be answered.
Asunto(s)
Factores de Crecimiento Transformadores/fisiología , Animales , Rechazo de Injerto , Humanos , Activación de Linfocitos , Choque Séptico/fisiopatologíaRESUMEN
The genome of human hepatitis B virus (HBV) contains an enhancer element within a 360-base-pair transcribed region located between sequences encoding the virus surface and core antigens. Beyond the usual properties associated with enhancers (i.e., activity 5' or 3' of a heterologous promoter and relative orientation independence), DNA sequences encompassing this enhancer exhibited unexpected characteristics. Using gene expression assays in both stably and transiently transfected cells, we found that the HBV enhancer element, when located in a transcribed region of chimeric genes, dramatically increased expression levels of genes controlled by the simian virus 40 promoter/enhancer. This synergism was not observed, however, when the HBV enhancer was located outside of the transcribed region. When these transcribed sequences were reversed in orientation, expression levels decreased significantly. These data suggest that RNA stability and transcriptional activity may be affected by sequences associated with this DNA region. In contrast to the findings of others, we found that the HBV enhancer activated transcription in a relatively cell-type-independent manner when the enhancer was located either 5' of the promoter or in the 3' untranslated region of gene constructs. The implications of these and other properties of the HBV enhancer region on viral gene expression and its life cycle are discussed.
Asunto(s)
Elementos de Facilitación Genéticos , Genes Virales , Virus de la Hepatitis B/genética , Animales , Secuencia de Bases , Línea Celular , ADN Viral/genética , Regulación de la Expresión Génica , Vectores Genéticos , Humanos , Plásmidos , Regiones Promotoras Genéticas , ARN Viral/genética , Virus 40 de los Simios/genética , Transcripción GenéticaRESUMEN
The T24/EJ allele of the Ha-ras proto-oncogene owes its powerful oncogenic activity not merely to the well documented mutation that perturbs the structure of the encoded polypeptide, but in addition to a second single nucleotide alteration in an intron that causes a tenfold increase in expression. This effect on expression is maintained upon transfer of the surrounding DNA to a heterologous gene, and as such defines a novel regulatory element.