RESUMEN
Type II collagen and aggrecan are major components of the extracellular matrix of articular cartilage. Their biosynthesis and catabolism are regulated by chondrocytes. They may be used as markers of chondrocyte phenotype for cells cultured in vitro. Type II collagen gene expression was detected by amplification of type II collagen-specific sequences, using cDNA produced by reverse transcription of mRNA extracted from freshly isolated and cultured human articular chondrocytes by the polymerase chain reaction (PCR). The synthesis of gene product was confirmed by immunohistochemical localization of type II collagen in cartilage sections and in cultured chondrocytes. Aggrecan core protein was also immunolocalized in cartilage sections and in chondrocytes in culture. Expression of type II collagen or aggrecan was not detected immunohistochemically in skin or bone. These results demonstrate that human articular chondrocytes can be characterized in culture, by the combined application of PCR and immunohistochemistry. Interleukin-1beta (IL-1beta) may play an important role in the destruction of cartilage matrix in arthritis, whereas transforming growth factor-beta (TGFbeta) may have an opposing effect and their combined actions may modulate chondrocyte phenotype. The effect of rhIL-1beta and rhTGFbeta on the production of type II collagen by chondrocytes in culture was investigated. It was shown that TGFbeta enhanced the production of type II collagen, localized immunocytochemically, in cultured chondrocytes. IL-1beta inhibited expression of mRNA for type II collagen. The implications of this study, in terms of a better understanding of degenerative cartilage disease, are discussed.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Colágeno/metabolismo , Proteínas de la Matriz Extracelular , Interleucina-1/farmacología , Proteoglicanos/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Agrecanos , Células Cultivadas , Humanos , Inmunohistoquímica , Lectinas Tipo C , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismoRESUMEN
OBJECTIVE: To investigate the pattern of cytokine gene expression in human articular chondrocytes in culture in response to interleukin-1 beta (IL-1 beta). The effect of serum and variations in culture conditions was also studied. METHODS: Messenger RNA was extracted from cells, reverse-transcribed to complementary DNA, and amplified by the polymerase chain reaction (PCR), using specific oligonucleotide primers. The PCR products were validated by restriction analysis with specific enzymes and by Southern blot analysis. RESULTS: In cultured articular chondrocytes, IL-1 beta, IL-1 alpha, granulocyte colony-stimulating factor (CSF), and granulocyte-macrophage CSF cytokine genes were expressed only after induction by IL-1 beta. However, IL-6, IL-8, and macrophage CSF genes were expressed constitutively. The expression of IL-1 beta was dose and time dependent. CONCLUSION: Using PCR, it was possible to demonstrate gene expression for several cytokines in human articular chondrocytes in culture. It was evident that some cytokine genes were expressed constitutively and some were inducible by IL-1 beta.
Asunto(s)
Cartílago Articular/citología , Citocinas/genética , Interleucina-1/farmacología , Secuencia de Bases , Southern Blotting , Células Cultivadas , Electroforesis en Gel de Agar , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , ARN/genéticaRESUMEN
There are important interactions between prostatic tumours and bone. This study was designed to examine whether prostatic tissue can express bone inductive factors, in particular, the Bone Morphogenetic Proteins (BMPs). The polymerase chain reaction (PCR) has been used to screen for the expression of BMPs one to six in the prostatic tissue of patients with benign prostatic hyperplasia (BPH), non-metastatic prostatic adenocarcinoma and metastatic prostatic adenocarcinoma. BMPs were expressed in both benign and malignant prostate tissue and in the prostate tumour cell lines, PC3 and DU145. BMPs were also expressed in ocular melanoma tissue, a tissue which rarely metastasizes to bone. BMP-6 expression was detected in the prostate tissue of over 50% of patients with clinically defined metastatic prostate adenocarcinoma, but was not detected in non-metastatic or benign prostate samples or in ocular melanoma tissue. These findings suggest that the BMPs may play a role in the osteoinductive activity of prostate metastases and that the pattern of expression of BMPs may be important in the pathogenesis of osteoblastic metastases associated with prostate adenocarcinoma.
Asunto(s)
Adenocarcinoma/química , Proteínas de Neoplasias/análisis , Hiperplasia Prostática , Neoplasias de la Próstata/química , Proteínas/análisis , Proteína Morfogenética Ósea 3 , Proteínas Morfogenéticas Óseas , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
The Transforming Growth Factor-beta (TGF beta) family of polypeptides elicits diverse biological actions on a wide range of cell types. There are known to be several isoforms of TGF beta coded for by different genes, with possibly differential expression and potencies. We have demonstrated that there is constitutive expression of three forms of transforming growth factor beta in adult human articular chondrocytes. The presence of 10% fetal calf serum in the culture medium may influence expression. The addition of transforming growth factor beta or interleukin 1 beta to the culture medium does not appear to consistently influence the expression of TGF beta by the cells.
Asunto(s)
Cartílago Articular/fisiología , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/genética , Secuencia de Bases , Cartílago Articular/efectos de los fármacos , Humanos , Técnicas In Vitro , Interleucina-1/farmacología , Datos de Secuencia Molecular , Familia de Multigenes , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , Proteínas Recombinantes/farmacología , Mapeo Restrictivo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
We have transfected high-molecular-weight DNA from human thyroid carcinomas into murine 3T3 cells. As a result we identified several foci of morphologically distinct transformed cells in each of the tumour DNA transfected cultures. After a total of three rounds of transfection, the transformed cells were shown to form tumours in nude mice. Southern blot analysis of DNA prepared from third-round transfectants demonstrated the presence of human Alu repetitive sequences and, after hybridization with probes for known oncogenes, indicated the presence of the human H-RAS oncogene in 3T3 cells transfected with three out of four anaplastic carcinoma DNA samples. It appears therefore that activation of RAS genes may be an important event in the development of the anaplastic thyroid tumours.
Asunto(s)
Carcinoma/genética , Genes ras , Neoplasias de la Tiroides/genética , Animales , Carcinoma/patología , Línea Celular , Transformación Celular Neoplásica , ADN/análisis , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Neoplasias de la Tiroides/patología , TransfecciónRESUMEN
We have been studying the expression of a range of proto-oncogenes in human thyroid tumour tissue by using Northern blot analysis. We have demonstrated the expression of a MOS mRNA of 1 kb in all thyroid samples. Furthermore, in a medullary carcinoma sample we also observed additional mRNA species of 1.7 and 2.2 kb. Southern blot analysis of DNA prepared from the same tumour sample did not reveal a rearrangement of the gene. These findings are the first report of MOS expression in any human tissue, and indicate that MOS oncogene activation might be important in the development of some thyroid tumours.
Asunto(s)
Carcinoma/genética , Proto-Oncogenes , Proteínas de los Retroviridae/genética , Neoplasias de la Tiroides/genética , Northern Blotting , Southern Blotting , Humanos , Proteínas Oncogénicas v-mos , Proto-Oncogenes Mas , ARN Mensajero/análisisRESUMEN
We have cloned part of the ETS 1 proto-oncogene and demonstrated the presence of two polymorphic Sst I restriction sites. A probe derived from one of our clones revealed the presence of 8.3 kb, 9.5 kb and/or 11.5 kb fragments on Southern blots of human DNA samples. The relative frequencies of these alleles appear to be significantly different between Saudi and Western populations, but there are no apparent differences in these frequencies between Saudi non-leukemic and leukemic individuals.
Asunto(s)
Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Factores de Transcripción , Alelos , Américas/etnología , Southern Blotting , ADN/análisis , Europa (Continente)/etnología , Frecuencia de los Genes , Humanos , Hibridación de Ácido Nucleico , Proto-Oncogenes Mas , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Arabia SauditaRESUMEN
The antiviral properties of a herpex simplex virus type 1-specific 'helper' T cell clone were investigated. The clone was found to be deficient in interleukin 2 production, although it produced interleukin 3 and interferon-gamma upon stimulation with the virus in vitro. Supernatants containing these lymphokines were observed to increase the virocidal activity of macrophages in vitro and furthermore induced these cells to mediate cytotoxic activity against virus-infected target cells. Macrophage activation was linked to the presence of interferon-gamma in the clone supernatant. The implications of these results for protection against this virus in vivo are discussed.
Asunto(s)
Interferón gamma/inmunología , Macrófagos/inmunología , Simplexvirus/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Células Cultivadas , Células Clonales , Citotoxicidad Inmunológica , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Interleucina-3/biosíntesis , Interleucina-3/inmunología , Activación de Macrófagos , Macrófagos/microbiología , Ratones , Simplexvirus/crecimiento & desarrolloRESUMEN
The production of prostacyclin (PGI2) by cultured porcine aortic endothelial cells, in response to serum and the calcium ionophore A23187, was inhibited by TMB-8, an antagonist of intracellular calcium mobilization. The calcium-channel blocker methoxyverapamil (D600) inhibited serum-induced PGI2 production in but had little effect on A23187-induced PGI2 production. Calmodulin activity was detected in endothelial-cell lysates and was inhibited by the calmodulin antagonist W7, which also inhibited PGI2 production in response to both agonists. Calcium and calmodulin appear to play an important role in mediating PGI2 production by the vascular endothelium.
Asunto(s)
Aorta/metabolismo , Calcio/fisiología , Calmodulina/fisiología , Epoprostenol/biosíntesis , Animales , Aorta/efectos de los fármacos , Calcimicina/farmacología , Células Cultivadas , Endotelio/metabolismo , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Galopamilo/farmacología , Sulfonamidas/farmacología , PorcinosRESUMEN
1. The effect of plasma and serum from normal subjects on the production of prostacyclin by cultured porcine endothelial cells was investigated. 2. Both plasma and serum from all subjects studied significantly stimulated the production of prostacyclin by cultured endothelial cells, measured by the radioimmunoassay of its stable metabolite 6-oxoprostaglandin F1 alpha. 3. Serum caused a consistently greater stimulation than plasma from the same individual. The stimulation was dose-dependent and inhibited by indomethacin. Heparin added to serum also inhibited this response. 4. Extracts from isolated washed platelets were tested for their ability to increase prostacyclin production. Extracts from platelets which had been induced to aggregate and release their granule contents in response to thrombin, caused stimulation. 5. These results indicated the invariable presence in plasma and serum of factors that stimulate the production of prostacyclin by endothelial cells in vitro. At least one of these factors is derived from platelets. These factors may be involved in the regulation of prostacyclin production by the vascular endothelium under normal conditions and in disease states.
Asunto(s)
Aorta/citología , Plaquetas/metabolismo , Epoprostenol/biosíntesis , Prostaglandinas/biosíntesis , Prostaglandinas/sangre , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Aorta/metabolismo , Productos Biológicos/análisis , Células Cultivadas , Reacciones Cruzadas , Endotelio/citología , Endotelio/metabolismo , Heparina/farmacología , Humanos , Indometacina/farmacología , Plasma , Porcinos , Trombina/farmacologíaRESUMEN
We have investigated the effects of plasma and serum from normal subjects on the production of prostacyclin (PGI2) by cultured porcine aortic vascular endothelial cells, measured by radioimmunoassay of its stable metabolite 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha). Both plasma and serum caused significant stimulation of the production of 6-keto PGF1 alpha over basal levels. Serum caused consistently greater stimulation than plasma from the same individual. Washed platelet suspensions were induced to aggregate using thrombin and the supernatants stimulated the production of 6-keto PGF1 alpha by cultured endothelial cells. Preliminary studies also show that a stimulatory factor is released from cultured human leucocytes. Serum from patients with systemic lupus erythematosus (SLE) and severe systemic sclerosis (SS), two connective tissue diseases with autoimmune features and vascular complications, showed significantly reduced levels of stimulation when compared with a control group.