RESUMO
A expressao constitutiva do oncogene EJ-ras conferiu resposta atenuada a glicocorticoide em fibroblastos de camundongo da linhagem NIH3T3 (NIH3T3ras). A proteina e o mRNA do receptor de glicocorticoide (GR) em celulas NIH3T3ras diminuiram para 20 a 25//do total apresentado pelas celulas NIH3T3 nao transformadas. Analise do DNA genomico sugeriu que nao existem rearranjos ou delecoes substanciais no gene de GR apos transformacao celular com EJ-ras. Ensaios de "run-on" mostraram que a transcricao do gene de GR em celulas NIH3T3ras foi 50 a 60//daquela observada na celula parenteral NIH3T3. Alem disso, a atividade transcricional do promotor do gene GR em celulas NIH3T3ras, avaliados por ensaios de transfeccao transitoria, foi 50//da quantificada para NIH3T3. A diminuicao da expressao de GR apos a transformacao com EJ-ras nao reflete caracteristicas celulares especificas, ja que, 88//dos clones de NIH3T3ras e 100//dos clones de fibroblastos de camundongo da linhagem L929 (L929ras), gerados por transfecao com EJ-ras, apresentaram esta mesma caracteristica. Notou-se tambem que a baixa expressao de GR foi acompanhada por um aumento na expressao de c-jun em 87//dos clones NIH3T3ras e 100//dos L929ras. A atividade transcricional do promotor do gene de GR, medida em estudos de transfeccao transitoria, foi inibida em 75//apos a expressao de v-jun, enquanto que v-fos levou a um aumento de aproximadamente duas vezes. Nossos resultados sugerem que a diminuicao da resposta celular a glicocorticoide, apos transformacao com EJ-ras, e mediada, pelo menos em parte, por uma menor expressao do GR. Alem disso, o aumento na razao Jun/Fos parece ser um dos responsaveis pela transcricao diminuida do gene de GR nestas celulas
Assuntos
Camundongos , Animais , Fibroblastos/análise , Oncogenes , Receptores de Glucocorticoides/fisiologia , Regulação da Expressão Gênica , Fatores de TranscriçãoRESUMO
The distribution of the beta-subunit of platelet-derived growth factor receptor (PDGFR-beta) was assessed by a sensitive immunoalkaline phosphatase technique using the monoclonal antibody PR7212. Frozen tissue sections of several nonneoplastic human tissues were stained along with 42 soft tissue sarcomas, 16 benign soft tissue proliferations, and 7 epithelial tumors. In all nonneoplastic tissue, there was intense labeling of cell processes of perivascular fibroblasts or pericytes in and about the walls of muscular blood vessels and of fibroblast cell processes around some glandular and ductal epithelia. No PDGFR-beta was found in the endothelial cells of muscular arteries and veins, but cells of uncertain identity within some capillaries were immunoreactive and the possibility that endothelial cells of some small capillaries express PDGFR-beta could not be excluded. In kidney there was strong labeling of glomerular mesangial cells and interstitial fibroblasts. Some histological types of soft tissue sarcomas were uniformly and strongly labeled with anti-PDGFR-beta, but other types were infrequently labeled or unreactive. The order of decreasing frequency and strength of labeling of the various types of benign and malignant soft tissue proliferations was as follows: benign fibromatosis and neurofibroma greater than malignant fibrous histiocytoma greater than liposarcoma greater than leiomyosarcoma greater than rhabdomyosarcoma. No tumor cell labeling was detected in epithelioid, synovial or clear cell sarcomas, leiomyomas, or carcinomas, but there was usually strong labeling of fibroblast and/or pericyte cell processes within tumor, especially around blood vessels. We conclude that PDGFR-beta is strongly expressed by vascular and stromal tissues of most tumors and normal organs and by tumor cells of several types of soft tissue tumors and proliferations, most notably those of fibroblastic origin.
Assuntos
Vasos Sanguíneos/análise , Receptores de Superfície Celular/análise , Neoplasias de Tecidos Moles/análise , Membrana Celular/análise , Feminino , Fibroblastos/análise , Humanos , Masculino , Músculo Liso Vascular/análise , Receptores do Fator de Crescimento Derivado de PlaquetasRESUMO
A simple method was developed to isolate low abundance hormone receptor poly(A)+ RNA from cells in tissue culture. Adherent cells in tissue culture plates were directly released with proteinase K and solubilized in SDS. Oligo(dT) cellulose was directly added to the lysate to obtain poly(A)+ RNA. Yields and purity of the poly(A)+ RNA were comparable to other more lengthy methods. IGF-I receptor and insulin receptor mRNA could be detected on Northern blot without any degradation.
Assuntos
Hibridização de Ácido Nucleico , Poli A/isolamento & purificação , RNA/isolamento & purificação , Receptor de Insulina/genética , Receptores de Superfície Celular/genética , Animais , Neoplasias da Mama/análise , Linhagem Celular , Sondas de DNA , Fibroblastos/análise , Humanos , Camundongos , Músculos/análise , RNA Mensageiro , Receptores de Somatomedina , Células Tumorais CultivadasRESUMO
In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin-binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin-binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin-binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin-binding protein, along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin-binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.
Assuntos
Proteínas de Ligação a Calmodulina/análise , Núcleo Celular/análise , Cricetinae/embriologia , Cricetulus/embriologia , Replicação do DNA/fisiologia , Fibroblastos/análise , Animais , Batroxobina/análise , Batroxobina/metabolismo , Calmodulina/análise , Calmodulina/metabolismo , Calmodulina/fisiologia , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ligação a Calmodulina/fisiologia , Núcleo Celular/ultraestrutura , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Insulina/farmacologia , Interfase/fisiologia , Isoleucina/metabolismo , Isoleucina/fisiologiaRESUMO
We have studied the protein-DNA interactions of human papillomavirus types 16 and 18 constitutive enhancer elements using DNasel footprinting experiments with nuclear extracts from four cervical carcinoma cell lines (C33A, HeLa, SiHa, and CaSki) and one fibroblast cell line (143B). Among nine footprints for the HPV 16 enhancer region, six footprints contain nuclear factor 1 (NF1) binding GCCAA motif. In vitro competition experiments suggest that the same factors are shared by all six of these motifs. Two other sequence motifs have consensus sequences for transcription factor AP1. Another sequence motif, for which uv crosslinking studies reveal interaction with four protein molecules, is a strong positive modulator of HPV 16 enhancer function in vivo and shares 100% homology to a sequence motif, GTTTTAA, in the tissue-specific enhancer of the c-mos oncogene. Footprints on the HPV 18 enhancer show five protected regions with homologies to NF1, AP1 and EFII transcription factor binding motifs. One sequence motif of the HPV 18 enhancer has three repeats of a TTTTA sequence contained within the c-mos sequence motif and interacts with at least four different individual polypeptides, as judged by uv crosslinking experiments.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Papillomaviridae/genética , Sequência de Bases , Ligação Competitiva , Desoxirribonuclease I , Epitélio/análise , Fibroblastos/análise , Humanos , Dados de Sequência Molecular , Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
We investigated the role of extracellular magnesium on capillary endothelial cell migration and proliferation, components of endothelial cell function that play an important role in angiogenesis and wound healing. Cell migration and proliferation were tested in six different MgSO4 concentrations and in various culture conditions. The Boyden chamber procedure was used to evaluate migration of bovine adrenal cortex capillary endothelial cells. We found that low magnesium concentration inhibited cell migration, but a dose-dependent increase in migration was observed when magnesium level was increased beyond the normal serum concentration (up to 2.4 mM magnesium; p less than 0.0001). Cell proliferation was also inhibited by very low magnesium concentration, an effect observed under all conditions studied. When cell proliferation was stimulated by acidic or basic fibroblast growth factors, it appeared that a ceiling was reached, an increasing magnesium concentration had no additional stimulatory effect. However, a dose-dependent increase in proliferation (p less than 0.005) was observed when magnesium concentration was increased above the normal serum level (0.8 mM) in culture conditions that did not cause marked cell proliferation. Thus, magnesium has an important role in endothelial cell migration and proliferation: very low extracellular magnesium concentrations inhibit and supranormal levels enhance both migration and proliferation. These results suggest that magnesium deficiency might adversely influence the healing and reendothelialization of vascular injuries and the healing of myocardial infarction and might also result in delayed or inadequate angiogenesis, effects potentially leading to infarct expansion and inadequate collateral development.
Assuntos
Capilares/citologia , Divisão Celular , Movimento Celular , Endotélio Vascular/citologia , Magnésio/fisiologia , Neovascularização Patológica , Cicatrização , Animais , Capilares/metabolismo , Bovinos , Contagem de Células , Células Cultivadas , Endotélio Vascular/metabolismo , Fibroblastos/análise , Substâncias de Crescimento/análise , Magnésio/sangue , Magnésio/metabolismo , Deficiência de Magnésio/complicaçõesRESUMO
The present study shows that homocysteine depleted cystine from cystinotic fibroblasts in vitro. No toxic effects were noted as judged by morphology and growth patterns. Efflux of radioactivity from cystinotic cells prelabeled with [35S]cystine was greater in homocysteine-treated cystinotic cells than in untreated controls. This radioactivity was found, by high voltage electrophoresis separation of effluxed products, to consist mainly of [35S]cystine, along with smaller amounts of [35S]homocysteine-cysteine mixed disulfide. When homocysteine and cysteamine were presented together to cystinotic cells at dose levels individually ineffective in removing cystine from these cells, a marked synergistic effect was observed and cystine content fell to 10% of that seen in untreated cystinotic fibroblasts. Similarly, synergistic effects of cystine depletion from cystinotic cells were demonstrated when cells were treated with a combination of cysteamine and dithiothreitol or glutathione. Incubation of cystinotic cells with homocysteine, dithiothreitol, or cysteamine in combination with vitamin C did not yield synergistic effects. The above findings suggest a novel way to probe metabolic processes in these mutant cells. Exploration of these synergistic effects may lead to more efficacious therapeutic protocols for cystinosis.
Assuntos
Cisteamina/farmacologia , Cistina/análise , Cistinose/metabolismo , Homocisteína/farmacologia , Ácido Ascórbico/farmacologia , Células Cultivadas , Cistina/metabolismo , Fibroblastos/análise , Glutationa/farmacologia , HumanosRESUMO
Dissociated cell culture preparations from rat and guinea-pig atria and interatrial septum, and from rat ventricles were studied using a combined autoradiographic and immunocytochemical approach. Alpha-atrial natriuretic peptide (125I-ANP1-128) bindings sites were confined to subpopulations of identified non-neuronal cells in each type of culture preparation, and had distinct patterns of labelling. The density of ANP1-28 binding sites was substantially greater in guinea-pig cultures than in rat cultures and was least in rat ventricular cultures. ANP1-28-labelled subpopulations of S-100-like immunoreactive glial cells were only seen in guinea-pig cultures. Von Willebrand factor (vWF)-like immunoreactive endothelial cells and vWF-negative endothelioid cells expressed ANP1-28 binding sites in both the guinea-pig and rat atrial cultures, but were unlabelled in rat ventricular cultures. In contrast, labelled subpopulations of fibronectin-like immunoreactive fibroblasts were present in all of the three types of culture preparation studied. ANP-like immunoreactive myocytes were present in both atrial and ventricular cultures. These cells did not, however, express ANP1-28 binding sites.
Assuntos
Fator Natriurético Atrial/metabolismo , Miocárdio/citologia , Receptores de Superfície Celular/análise , Animais , Autorradiografia , Células Cultivadas , Endocárdio/ultraestrutura , Fibroblastos/análise , Cobaias , Átrios do Coração , Septos Cardíacos/citologia , Ventrículos do Coração , Ratos , Receptores do Fator Natriurético Atrial , Especificidade da Espécie , Fator de von Willebrand/análiseRESUMO
The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 x 10(10) Swiss 3T3 fibroblasts. Partial amino acid sequence data were obtained from five peptides generated by S. aureus V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic 80-87 kDa PKC substrates from both actively proliferating A431 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80-87 kDa PKC substrates detected in fully differentiated neural tissue.
Assuntos
Fosfoproteínas/isolamento & purificação , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fibroblastos/análise , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Fragmentos de Peptídeos/análiseRESUMO
Cobalt-chromium-based alloys are widely used in oral and orthopedic implantology. Although they are relatively well tolerated, biological complications could occur which sometimes are due to the insufficient biocompatibility of the alloy. This study shows the effects of an alloy (Co (base), 28% Cr, 5.5% Mo, 1% Ni, 0.95% Si, 0.7% Fe, 0.65% Mn, 0.25% C), on differentiated human cells derived from an oral implantation site, specifically alveolar bone osteoblasts and gingival fibroblasts. The cytocompatibility of the alloy is determined by the study of cell proliferation, determination of total cell protein and intracellular alkaline phosphatase contents, cytoskeleton, and cell morphology. The alloy is presented to the cells in four different surface states: rough cast, specular polished, microbead blasted, and RF sputtered. The results demonstrate that the same material has different effects on the basal and specific cellular functions, according to its surface state. For this alloy we can classify its cytocompatibility according to its surface state in such an order: Microbead blasted much greater than specular polished greater than RF sputtered greater than rough cast.
Assuntos
Ligas de Cromo , Fibroblastos/citologia , Teste de Materiais , Osteoblastos/citologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ligas de Cromo/toxicidade , Fibroblastos/análise , Fibroblastos/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Osteoblastos/análise , Osteoblastos/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Numerous studies have shown that intact cancer cells and cell extracts have the capacity to lyse erythrocytes in vitro. The transformation of NIH-3T3 fibroblasts by ras oncogenes has recently been demonstrated to result in tumour cells releasing a haemolytic factor. The purpose of this study has been to purify and further characterise the soluble tumour haemolytic factor (sTHF) produced by mouse fibroblasts transformed by T24 human bladder cancer DNA and by the cloned Harvey murine sarcoma viral oncogene. To this end, transformed fibroblasts were cultivated in serum-free medium. The cell-free supernatant was treated with ammonium sulphate and the precipitate achieved at 60-100% saturation was dialysed and applied to a gel filtration column. A haemolytic factor was eluted with an Mr between 65,000 and 75,000. Zinc chelate and strong anion exchange column chromatography resulted in greater than 3,000-fold enrichment of sTHF. SDS-PAGE of sTHF resulted in a single protein band of 66,000 Da. Soluble THF had no immunological cross-reactivity with known cytokines produced by lymphocytes and macrophages. The pathophysiological role of sTHF in cancer remains to be determined.
Assuntos
Fatores Biológicos/isolamento & purificação , Fibroblastos/análise , Animais , Western Blotting , Linhagem Celular , Transformação Celular Neoplásica , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Genes ras , Humanos , Ratos , Ratos EndogâmicosRESUMO
BACKGROUND: Indirect-immunofluorescence studies of skin and cultured dermal fibroblasts from patients with the Marfan syndrome demonstrate apparent deficiency of one element of connective tissue--the microfibrillar-fiber system--in assays using specific antibodies against fibrillin, a major microfibrillar protein. This study was designed to test whether these immunostaining abnormalities are consistent and diagnostic features of the disease. METHODS: We studied patients with either the Marfan syndrome or various other inherited connective-tissue disorders and normal subjects according to a single-blind protocol in which coded samples of skin, fibroblast cultures, or both were analyzed without knowledge of the clinical diagnosis and classified as "Marfan" or "non-Marfan" before the sample codes were broken. RESULTS: Of the 27 patients with the Marfan syndrome, 24 were correctly identified by the decreased content of microfibrillar fibers in their skin, cultured fibroblasts, or both; in contrast, 19 of 25 patients with other heritable disorders of connective tissue and all 13 normal subjects were correctly classified as "non-Marfan" by these assays (P less than 0.001). CONCLUSIONS: These results document consistent, relatively specific abnormalities of microfibrillar fibers in the Marfan syndrome. The biomechanical incompetence of these structural elements, due to quantitative or qualitative abnormalities, may account for the pleiotropic clinical manifestations of the disease. Therefore, various defects in the expression, structure, assembly, or degradation of the constituent structural glycoprotein (or glycoproteins) of microfibrils may be implicated in the causation of the Marfan syndrome.
Assuntos
Citoesqueleto de Actina/análise , Citoesqueleto/análise , Síndrome de Marfan/metabolismo , Proteínas dos Microfilamentos/análise , Citoesqueleto de Actina/ultraestrutura , Adolescente , Adulto , Anticorpos Monoclonais , Criança , Pré-Escolar , Doenças do Tecido Conjuntivo/metabolismo , Feminino , Fibrilinas , Fibroblastos/análise , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/patologia , Pele/análiseRESUMO
Human embryonic lung diploid fibroblast, IMR-90 cells secreted a tumor cytotoxic factor. The fibroblast-derived tumor cytotoxic factor (F-TCF) has a cytotoxic activity to Sarcoma 180 and a cytostatic and degenerative activities to KB cells. F-TCF has been purified about 540,000-fold with 23.3% recovery from 75 liters of the conditioned medium containing 5% newborn calf serum. The purified F-TCF is a basic glycoprotein with isoelectric point values of 7.4 to 8.6. It was stable in the pH range from 6.0 to 9.0 and was stable at the heating temperature of 60 degrees C for 10 min, but completely inactivated by reducing it with 2-mercaptoethanol. F-TCF has molecular weight of 76 to 80 kD on SDS-PAGE under non-reducing conditions and is a heterodimer consisting of a large alpha subunit with 52 to 56 kD and a small beta subunit with 30 to 34 kD. F-TCF was identified as one of human hepatocyte growth factors by the physicochemical properties including N terminal and a few internal amino acid sequences. We have confirmed that F-TCF has an ability to dramatically stimulate DNA synthesis in adult rat hepatocytes in the low dose range of 1 to 10 ng/ml.
Assuntos
Fibroblastos/análise , Glicoproteínas/isolamento & purificação , Substâncias de Crescimento/isolamento & purificação , Sequência de Aminoácidos , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Glicoproteínas/farmacologia , Substâncias de Crescimento/farmacologia , Fator de Crescimento de Hepatócito , Humanos , Células KB , Dados de Sequência Molecular , Ratos , Sarcoma Experimental/patologia , Células Tumorais CultivadasRESUMO
Type I collagen is the most abundant collagen type in soft tissues and the only type found in mineralized bone. We established a rapid equilibrium radioimmunoassay for the carboxyterminal propeptide of human type I procollagen (PICP), to be used as an indicator of the synthesis of type I collagen. We isolated type I procollagen from the medium of primary cultures of human skin fibroblasts, digested the protein with highly purified bacterial collagenase, and purified PICP by lectin-affinity chromatography, gel filtration, and ion-exchange separation on HPLC. The purity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N-terminal amino acid sequencing of its component chains. The final radioimmunoassay was established with polyclonal rabbit antibodies. Material antigenically related to PICP is readily detected in human serum. There is only one form of the serum antigen, its molecular size and affinity to the antibodies being similar to those of the isolated propeptide. Intra- and interassay CVs are 3% and 5%, respectively. Preliminary reference intervals for healthy adults (18 to 61 years of age) are 38-202 micrograms/L for men and 50-170 micrograms/L for women: in men the concentration is inversely related to age. The serum antigen is stable during storage and after repeated thawing.
Assuntos
Fragmentos de Peptídeos/análise , Pró-Colágeno/análise , Adolescente , Adulto , Fatores Etários , Sequência de Aminoácidos , Osso e Ossos/metabolismo , Células Cultivadas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Feminino , Fibroblastos/análise , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Pró-Colágeno/isolamento & purificação , Radioimunoensaio , Fatores SexuaisRESUMO
We used the results of skin fibroblast type I collagen analysis to improve the accuracy of diagnosis and genetic counseling for six patients with osteogenesis imperfecta. The fibroblasts of two patients with osteogenesis imperfecta type I synthesized a reduced quantity of qualitatively normal type I procollagen. Another patient with osteogenesis imperfecta type I had two populations of type I collagen molecules, one apparently normal and the other with a substitution of cysteine for glycine in the triple helical domain. Three sporadic cases with osteogenesis imperfecta types II, III, and IV were studied; in each proband a normal and an abnormal overmodified population of type I collagen molecules were demonstrated, and parental collagens were normal in the two available patients. These results indicated that the probands were heterozygous for new dominant mutations and assisted our genetic counseling, especially in osteogenesis imperfecta types II and III, which were formerly believed to be inherited in an autosomal recessive fashion. The results could not exclude parental germ line mosaicism for a new dominant mutation, which has resulted in recurrence in siblings of some patients with osteogenesis imperfecta, so prenatal diagnosis was therefore offered for future pregnancies. Analysis of chorionic villus cell collagen may facilitate antenatal diagnosis in selected cases, and the study of a larger number of patients may allow correlation of the biochemical defects with the natural history and prognosis.
Assuntos
Colágeno/análise , Osteogênese Imperfeita/metabolismo , Adulto , Células Cultivadas , Pré-Escolar , Feminino , Fibroblastos/análise , Aconselhamento Genético , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/patologia , Diagnóstico Pré-Natal , Prognóstico , Pele/patologiaRESUMO
Secreted phosphoprotein I (SPP), also known as 2ar, osteopontin, 44-kDa bone phosphoprotein, bone sialoprotein I, and transformation-related phosphoprotein, is a 41.5-kDa glycosylated phosphoprotein secreted by many mammalian cell lines and expressed in a limited set of tissues. Using a cDNA probe, we found that SPP mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen. This contrasts with the transient induction we had previously observed after a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In a set of 5 independently isolated T24-H-ras-transfected mouse C3H 10T1/2 cell lines, the levels of SPP mRNA correlated well with ras mRNA levels and with both experimental and spontaneous metastatic ability. SPP mRNA expression was also elevated in a derivative of mouse LTA cells transfected with genomic DNA from B16F1 melanoma cells and selected for increased experimental metastatic ability in the chick embryo. This apparent association of SPP expression with invasion, progression and metastasis, along with the presence of a functional ArgGlyAsp (RGD) cell adhesion site in SPP (osteopontin), leads us to propose that SPP may act as an autocrine adhesion factor for tumor cells.
Assuntos
Fosfoproteínas/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Sialoglicoproteínas/biossíntese , Neoplasias Cutâneas/metabolismo , Animais , Northern Blotting , Carcinoma de Células Escamosas/análise , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Feminino , Fibroblastos/análise , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos , Metástase Neoplásica , Osteopontina , Papiloma/análise , Papiloma/metabolismo , Fosfoproteínas/análise , RNA Mensageiro/análise , RNA Neoplásico/análise , Sialoglicoproteínas/análise , Neoplasias Cutâneas/análiseRESUMO
The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.
Assuntos
Isoenzimas/genética , Músculos/enzimologia , Fosfofrutoquinase-1/genética , Splicing de RNA , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/genética , Sondas de DNA , Éxons , Fibroblastos/análise , Humanos , Íntrons , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
S-Adenosylmethionine decarboxylase (AdoMetDC) activity was elevated 18.8-fold in Swiss 3T3 fibroblasts which were depleted of cellular polyamines by using the inhibitor difluoromethylornithine (DFMO). Although the cellular level of AdoMetDC mRNA and the half-life of active AdoMetDC protein were also increased (4.3- and 1.5-fold respectively), together they could not account for the magnitude of the increase in AdoMetDC activity. These data suggested that the translation of AdoMetDC mRNA must be increased in the polyamine-depleted cells to account fully for the elevation in activity. The cellular distribution of AdoMetDC mRNA was examined in the polyamine-depleted cells, and it was found almost exclusively associated with large polysomes. In contrast, AdoMetDC mRNA in untreated controls was very heterogeneous, with the proportion associated with monosomes equal to that associated with large polysomes. The shift of the AdoMetDC message into large polysomes occurred within 18 h after addition of DFMO to the cultures and could be reversed by adding exogenous putrescine. The effect of polyamine depletion on AdoMetDC translation was specific, since there was no change in the distribution in polysomes of either actin mRNA or the translationally controlled mRNA encoding ribosomal protein S16 in the DFMO-inhibited cells. Thus the translational efficiency of AdoMetDC mRNA in vivo is regulated either directly or indirectly by the concentration of intracellular polyamines through a mechanism involving translational initiation, which results in a change in the number of ribosomes associated with this mRNA.
Assuntos
Adenosilmetionina Descarboxilase/genética , Poliaminas Biogênicas/fisiologia , Carboxiliases/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Adenosilmetionina Descarboxilase/metabolismo , Animais , Células Cultivadas , Citosol/análise , Eflornitina/farmacologia , Fibroblastos/análise , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Putrescina/farmacologia , RNA Ribossômico 16S/metabolismoRESUMO
A 14-yr-old Turkish girl presented with serum cholesterol levels of 15-20 mmol/l, skin and tendon xanthomata, and anginal attacks. A coronary angiography demonstrated severe coronary atherosclerosis including a 70% stenosis at the origin of the left coronary artery. The clinical diagnosis, homozygous familial hypercholesterolaemia, was confirmed by: (1) investigation of the family revealing hypercholesterolaemia in both her parents and siblings; (2) fibroblast association studies, in which the specific association of low density lipoprotein (LDL) was 35% of normal; and (3) LDL turnover study, in which the fractional catabolic rate of LDL was decreased to 0.213 pools/day. Treatment with cholestyramine or simvastatin had little effect on serum cholesterol levels. After coronary artery bypass grafting, the patient was treated with selective LDL apheresis using columns containing dextran-sulphate bound to cellulose. These columns bind apolipoprotein B containing lipoproteins but not high density lipoproteins. After 2 yr of therapy, the level of serum cholesterol has declined by 56%. Skin xanthomata have disappeared and there is no recurrence of angina pectoris. On repeated coronary angiography, two of the three bypasses are patent and there is no progression of atherosclerotic lesions. We conclude that LDL apheresis is an efficient procedure to lower serum cholesterol in patients who do not respond to pharmacological treatment of hypercholesterolaemia.
Assuntos
Remoção de Componentes Sanguíneos , LDL-Colesterol , Homozigoto , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/terapia , Adolescente , Adulto , Anticolesterolemiantes/uso terapêutico , Arteriosclerose/complicações , Colesterol/sangue , LDL-Colesterol/sangue , Ponte de Artéria Coronária , Estudos de Avaliação como Assunto , Feminino , Fibroblastos/análise , Humanos , Hiperlipoproteinemia Tipo II/genética , Masculino , Pessoa de Meia-Idade , Receptores de LDL/análise , Fatores de TempoRESUMO
Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ([Ca2+]i) in entherocytes, thymocytes and fibroblasts. In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in [Ca2+]i. The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions. Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.