摘要:

AbstractThe role of growth hormone (GH) in the differentiation process of Obi 771 mouse preadipocyte cells has been studied under culture conditions that were serum-free and hormone-supplemented and which were previously shown to lead to terminal differentiation. In the absence of GH, a dramatic decrease in the adipogenic activity of the culture medium could be observed, as indicated 12 days after confluence by the low levels of glycerol-3-phosphate dehydrogenase activity and the sharp reduction of the number of triacylglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel loss of the mitogenic potency of the culture medium. Determination of the half-maximal and maximal concentrations of GH required for the restoration of growth and differentiation were identical, 0.5 and 2 nM, respectively. Despite the presence of insulin-like growth factor-I (IGF-I) to substitute for supraphysiological concentrations of insulin and to saturate IGF-I receptor, GH was still required to induce terminal differentiation of a maximal number of cells. However, protein kinase C activators such as prostaglandin F2a, phorbol esters and diacylglycerol were able to mimic GH in promoting a maximal mitogenic-adipogenic response, indicating that the ability of GH to induce diacylglycerol production (Doglio et al., 1989; Catalioto et al., 1990) plays a prominent role in this process. Furthermore, in agreement with the fact that the mitoses which precede terminal differentiation of Obi 771 preadipocytes are strictly controlled by cAMP and only modulated by protein kinase C., terminal differentiation of Ob 1771 preadipocytes occured in the absence of GH upon supplementation with high concentrations of carbaprostacyclin, added as a cAMP-elevating agent or with 8-Br-cAMP, added as a cAMP analogue. It is concluded that the control exerted by GH on terminal differentiation of mouse preadipocytes corresponds to a modulating mitogenic effect mediated through protein kinase C activation and leading to a potentiation of the cAMP and IGF-I mitogenic signalling pathways.

ISSN:0897-7194    

DOI:10.3109/08977199209021538

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractTGF-β1 stimulates thymidine incorporation and the growth rate of mouse NIH3T3 fibroblasts and of those cells transformed by the EJ-H-ras oncogene (TR15 cells), in the presence and the absence of serum. Thymidine incorporation, in serum-deprived cells, is stimulated to a higher degree by 0.1-1 ng/ml of TGF-βin NIH3T3 than in TR15 cells, which have a 10-fold higher basal level of incorporation. In both cell types TGF-β1 is as active, or more active than other mitogens (TGF-α, PDGF-AB, bFGF) at the same concentration. The growth rate of NIH3T3 cells, in low serum or serum-free (S−) medium, is stimulated by only 10 picograms/ml of TGF-β1, and that of TR15 cells, in S−medium, by only 1 picogram/m1. In contrast, TGF-β1 inhibits mitogenically unestablished mouse embryo fibroblasts and these fibroblasts immortalized spontaneously and able to grow in S−medium. It also inhibits the anchorage-independent growth of TR15 cells. NIH3T3 and TR15 cells respond, similarly, to TGF-βactivated by acification of their culture medium. The kinetics of thymidine incorporation and of activation of thec-mycproto-oncogene, observed already after 1 hr, in treated NIH3T3 and TR15 cells, suggests a direct mitogenic stimulation. The level of activatedc-mycRNA is 2-fold higher at 2 hr, and subsequently decreases relatively less in the TR15 cells.

ISSN:0897-7194    

DOI:10.3109/08977199209021539

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractBasic fibroblast growth factor (basic FGF) is a mitogen for isolated epiphyseal growth plate chondrocytes. To determine whether basic FGF might function as an autocrine stimulus to longitudinal skeletal growthin utero, we investigated the synthesisand release of basic FGF by isolated growth plate chondrocytes from the ovine fetus, the expression of mRNA for a high affinity basic FGF receptor by these cells, and the contribution of endogenous basic FGF to the DNA synthetic rate of the cellsin vitro. Chondrocytes were isolated from the proximal tibial growth plate of the lamb fetuses between 35 and 132 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. Viability was confirmed over the duration of the experiments by the exclusion of trypan blue, and an absence of lactate dehydrogenase accumulation in conditioned medium. Immunocytochemistry of chondrocyte monolayers showed immunoreactive basic FGF to be present in the cytoplasm of approximately 80% of sub-confluent cells which was accompanied by pronounced nuclear staining in approximately 30% of cells. Serum-free, conditioned culture medium, extracellular matrix and chondrocyte cytoplasm contained 52±2 pM/μg DNA, 66±2 pM/μg DNA and 22±3 pM/μg DNA basic FGF, respectively (mean±S.E.M.,n=8 fetuses), for cells obtained from animals of 35-40 days' gestation when assessed by radioimmunoassay. Chondrocyte-conditioned medium increased endothelial cell proliferationin vitro(a specific bio-assay for basic FGF and related peptides); and the mitogenic activity was removed from conditioned medium by incubation with heparin-Sepharose demonstrating that this was due to heparin-binding protein(s). Western blot analysis of conditioned medium using a specific basic FGF antibody revealed a single immunoreactive protein of approximately 18 kDa molecular size. The appearance of radiommunoassayable basic FGF in conditioned medium, extracellular matrix, and chondrocyte cytoplasm observed during culture was blocked by co-incubation with cycloheximide. The levels of immunoreactive basic FGF present in each compartment decreased with gestational age as did basal DNA synthetic rate assessed by the incorporation of PH] thymidine. Incubation of chondrocytes with transforming growth factorβ, resulted in a significant increase while exposure to insulin-like growth factors or insulin caused a decrease, in the content and release of basic FGF. Basic FGF presence was unaltered when medium was supplemented with varying amounts of glucose (2.7-16.7 mM).In situhybridization on cell monolayers using a cRNA probe encoding the high affinityflgreceptor for FGFs showed an abundant expression of mRNA for the receptor. When chondrocytes were cultured in the presence of exogenous basic FGF both DNA synthetic rate and cell number were increased, the half-maximal effective dose of basic FGF being 0.2 nM. Co-culture with an anti-basic FGF antibody caused a significant decrease of 23% in DNA synthetic rate under basal conditions, and completely blocked the effects of exogenous basic FGF. We conclude that isolated ovine fetal growth plate chondrocytes synthesize and release basic FGF, a proportion of which is in soluble form, which makes a significant contribution to basal DNA synthetic rate, possibly through the expression of theflgreceptor. This suggests an autocrine action of basic FGF in the epiphyseal growth plate during fetal life.

ISSN:0897-7194    

DOI:10.3109/08977199209021540

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractThe expression of IL5 by CD4+CD45RA+, CD4+CD45R0+and CD3+CD8+subsets of human peripheral blood mononuclear cells was assessed. Interleukin 5 expression was detected by RNA extraction, reverse transcription and polymerase chain reaction. Populations of highly purified cells were obtained by a protocol of sequential plastic adherence, magnetic bead separation and flow cytometric cell sorting. IL5 was clearly expressed in the CD4+CD45R0+subset from 3 to 48 hr after activation. The CD4+CD45RA+and CD3+CD8+subsets expressed very much less IL5. By contrast, IL2 expression was readily detected in all sorted populations. Thus, in activated CD4+cells, IL5 was predominantly expressed in the CD4+CD45R0+subset, a pattern of expression corresponding to that reported for a number of other cytokines, and differing from that ofIL2.

ISSN:0897-7194    

DOI:10.3109/08977199209021541

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractThe mouse platelet-derived growth factor (PDGF) A-chain gene has been structurally characterized and compared with its human counterpart. The organization of the two genes is similar. Both consist of 7 exons spaced by 6 introns of corresponding sizes. As in the human gene, exon 6 encodes a sequence which is alternatively spliced. When present, it codes for an alternative C-terminus of the A-chain. In intron 5, conserved stretches of nucleotides, potentially involved in the regulation of the alternative splicing, are identified. The untranslated sequences show a high degree of nucleotide sequence identity and several conserved consensus binding sites for transcription factors are identified within the 5′untranslated as well as in the 5′flanking region.

ISSN:0897-7194    

DOI:10.3109/08977199209021542

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractThe actions of insulin-like growth factors (IGFs) are modulated by several specific binding proteins (IGF BPs). Since the anatomical distribution of IGF BPs is likely to dictate IGF bioavailability we used immunocytochemistry to localize IGF BP-1,-2, and-3 in early second trimester human fetal tissues. Primary antisera were directed against MGF BP-1, bIGF BP-2 and hIGF BP-3 respectively, and showed less than 5% cross-reaction with heterologous IGF BP species. The distribution of immunopositive staining was similar for each IGF BP in many tissues being prominent in the epithelial lining of the gut, kidney and lung; in epidermis, adrenal cortex and pancreatic endocrine tissue; and in association with membranes of skeletal muscle fibres and cardiocytes. Unlike IGF BP-1-2, IGF BP-3 was barely detectable in liver and absent from epiphyseal chondrocytes. Conversely, IGF BP-3 alone was visualized within neurons of the cerebral cortex. The co-distribution of IGF BPs in many human fetal tissues suggests that they may co-ordinately regulate IGF bioavailability in target tissues and modify IGF: receptor interactions.

ISSN:0897-7194    

DOI:10.3109/08977199209021543

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractIt has been shown previously that MCF-7 cells proliferate in response to nanomolar concentrations of IGF-I and IGF-II. It has also been reported that the actions of both peptides are mediated through the IGF-I receptor. To further characterize these observations, we used MCF-7 and Hs578T cell lines in the serum-free/phenol red-free system developed by Ogasawara and Sibarsku, 1988. Cell proliferation was studied in the presence of insulin, IGF-I and-II and a series of growth factor receptor antibodies. No effect was observed on Hs578T cell proliferation with any of the growth factors. However, MCF-7 cells were stimulated 4-5 fold with IGF-I and insulin, while IGF-II was only slightly less potent.αIR3, a monoclonal antibody directed against the IGF-I receptor, was stimulatory when added alone. However,αIR3 blocked approximately 50% of the IGF-I response, only 5% of the insulin response, and did not block the IGF-II effect on cell proliferation. These data suggest thatαIR3 and IGF-I are acting as agonists through the IGF-I receptor, but that insulin and IGF-II are acting through other receptors. Two different IGF-II/M-6-P receptor antibodies and an insulin receptor antibody failed to significantly block IGF-II actions. All three antibodies were stimulatory when added alone.β-gal inhibited 27% of the IGF-II response and had no effect when added alone. Sinceβ-gal decreases the binding affinity of the IGF-II/M-6-P receptor for IGF-II and does not bind to the IGF-I or insulin receptor, these data suggest the possibility that IGF-II mitogenic action is mediated through the IGF-II/M-6-P receptor. In summary, these data indicate that nanomolar concentration of insulin, IGF-I and IGF-II are potent mitogens in MCF-7 cells and can potencially stimulate cell proliferation through all three receptors.

ISSN:0897-7194    

DOI:10.3109/08977199209021544

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractGamma-interferon (γIFN) was found to induce expression of the 150,000 Mrcell surface and the 35,000M, chromatin receptors for nerve growth factor (NGF) in the SW1116 colorectal carcinoma cell line that does not express NGF receptors. In the SW707 colorectal carcinoma cell line that expresses a low level of NGF receptors, ylFN stimulated expression of the cell surface and the nuclear receptors. Induction of NGF receptors in SW1116 cells resulted in internalization and nuclear translocation of125I-NGF. When NGF bound to the chromatin, ribosomal RNA synthesis was inhibited. Two-dimensional gel electrophoresis of [35S]methionine-labeled chromatin proteins indicated significant changes in chromatin protein composition in cells treated and not-treated withγIFN.γIFN effectively stimulated the expression of NGF receptors in two colorectal carcinoma cell lines, but inhibited the expression in melanoma and breast carcinoma cells. It is suggested thatγIFN, by modulating the expression of NGF receptors may affect the NGF-dependent growth of some tumor cell lines.

ISSN:0897-7194    

DOI:10.3109/08977199209021545

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor