摘要:

The SH-SY5Y human neuroblastoma cell line is differentiatedin vitrowith nanomolar concentrations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Untreated cells express insulin receptors, and both type I and type II insulin-like growth factor (IGF) receptors, as has been shown by agonist binding and immunoprecipitation studies. Via interaction with its own receptor and the IGF-I receptor, insulin induced a mitogenic response in these cells. IGF-I and IGF-II are also mitogens for SH-SY5Y cells, as shown by a transient increase of the c-fosmRNA level, ornithin decarboxylase activity, thymidine incorporation, and, finally, cell division. TPA-differentiated cells do not respond mitogenically to any of these factors, although insulin and IGF-I receptors are still present on the cell surface and remain functional, as demonstrated by ligand-stimulated autophosphorylation, actin reorganization, and c-fosinduction. However, other prereplicative responses, i.e., increased ornithin decarboxylase activity and c-mycmRNA levels, cannot be induced. These phenomena, may be part of a receptor uncoupling mechanism(s). The findings are discussed in terms of differentiation stage-dependent signaling of growth factor receptors. We suggest that these receptors switch from controlling cell division in replicative neuronal cells to mediating externally controlled functions related to the differentiated neuronal phenotype.

ISSN:0897-7194    

DOI:10.3109/08977199009167020

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Human insulin-like growth factor II (IGF-II) has been purified to homogeneity from bone which contained 10–15 times more IGF-II than insulin-like growth factor-I (IGF-I). After extraction of IGF-II by demineralization of human bone powder with 10% EDTA containing 4 M guanidine HCl at pH 7.4, IGF-II was separated from IGF binding proteins by hydroxylapatite chromatography in the presence of 4 M guanidine HCl. The hydroxylapatite unbound fraction containing IGF-II was purified by affinity chromatography using Sm 1.2. monoclonal antibodies, which bind both IGF-I and IGF-II. The final purification of IGF-II was achieved by FPLC mono S ion-exchange chromatography in which IGF-II was separated from IGF-I. Human IGF-II thus purified was shown to be pure by (1) HPLC reverse-phase chromatography, (2) SDS-PAGE, and (3) N-terminal amino acid sequence.From 300 g of bone, 0.18 mg IGF-II was obtained with an overall recovery of 42%. These studies demonstrate the usefulness of (1) bone as a source for IGF-II purification and (2) antibodies that cross-react with both IGF-I and IGF-II for affinity purification of IGFs.

ISSN:0897-7194    

DOI:10.3109/08977199009167021

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

A dermal ulcer wound-healing model was established in rabbit ear to examine the effects of recombinant human transforming growth factor-β1 (rhTGF-β1) in wound healing. Histomorphometric examination of the wounds indicate a biphasic healing response 7 days after a single application of rhTGF-β1 at the time of wounding. Statistically significant healing occurred at 5–100 ng but not at higher doses of 500 or 1000 ng rhTGF-β1/wound. Enhanced collagen synthesis as determined by [3H]proline incorporation occurred at 15 and 25 ng and was significantly depressed at 500 ng rhTGF-β1/wound. Multiple doses of 100 ng rhTGF-β1 applied to the wound at the time of wounding and for 3 days after wounding provided results comparable to the single application of growth factor. Delaying treatment 24 hr after wounding did not enhance wound healing compared with vehicle. Our findings suggest that rhTGF-β1 can be a valuable growth factor to improve the healing of ulcer wounds.

ISSN:0897-7194    

DOI:10.3109/08977199009167022

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Anchorage-dependent and -independent MCF-7 cell growth was dose-dependently inhibited by retinoic acid (RA) but was insensitive to TGF-β(from 1 to 100 pm). Growth of MCF-7 monolayer cultures was inhibited (50%) when exposed to 10-6M RA. RA was unable to completely inhibit MCF-7 cell proliferation, as concentrations above 10-6M were rapidly cytotoxic. However, the combination of TGF-βand RA resulted an increase in RA inhibitory effect on MCF-7 monolayer growth and a 80% reduction in colony formation in soft agar. These results demonstrate that although TGF-βdoes not inhibit the growth of MCF-7 cells, it potentiates the antiproliferative effect of RA, suggesting that it may play a part, albeit indirect, in the regulation of MCF-7 cell growth.

ISSN:0897-7194    

DOI:10.3109/08977199009167023

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The human CSF-1 receptor (c-fmsprotooncogene product) was introduced into CSF-1-unresponsive Chinese hamster lung fibroblasts (CCL39 cell line) in order to study its coupling to biochemical signal-transducing systems and to compare the growth-regulating properties of CSF-1 to those of other growth factors. Independent clones expressing different levels of CSF-1 receptors were isolated and characterized. CSF-1 increased [3H]thymidine incorporation in serum-starved cells and potentiated the mitogenic effects of FGF and thrombin. As already observed for other growth factors activating receptor tyrosine kinases (EGF, FGF, IGF-I), CSF-1 alone did not trigger inositol phosphate formation, but slightly enhanced the activity of phospholipase C agonists (thrombin, AIF4- complex). Activation of the CSF-1 receptor by its ligand was evidenced by the rapid activation of the Na+/H+exchanger resulting in amiloride-sensitive cytoplasmic alkalinization (0.1–0.2 pH units) within minutes after stimulation. Whereas pertussis toxin does not affect the action of EGF, FGF, or IGF-I in CCL39 cells, it partially inhibited both DNA synthesis reinitiation and activation of Na+/H+exchange by CSF-1, indicating that the CSF-1 receptor can communicate with a signal-transducing GTP binding protein. A point-mutated form of the c-fmsgene product, in which Tyr 969, a residue negatively modulating signal transduction, had been replaced with Phe [fms(F969)], did not generate responses significantly different from those obtained with the wild-type c-fmsgene product. In the absence of CSF-1, cells expressing either wild-type orfms(F969) showed a considerably higher basal level of thymidine incorporation and decreased anchorage dependence compared with parental CCL39 cells. Monoclonal antibodies that interfere with signal transduction by the human CSF-1 receptor inhibited both basal [3H]thymidine incorporation and soft agar colony formation, indicating that relaxation of growth control was dependent on CSF-1 receptor expression.

ISSN:0897-7194    

DOI:10.3109/08977199009167024

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

A hemopoietic cell line FDC-P1 that requires either IL-3 or GM-CSF to survive and proliferate was infected with retroviruses that expressed either c-fms, which encodes the receptor for M-CSF, or v-fms, which is an oncogenic derivative of c-fms. The expression of c-fmsallowed FDC-P1 to grow in the absence of IL-3 or GM-CSF provided that M-CSF was present. The M-CSF did not, however, induce macrophage differentiation. The expression of v-fmsallowed FDC-P1 to grow in the absence of any added hemopoietic growth factors, including M-CSF, although the addition of M-CSF enhanced v-fmsactivity. V-fmscell lines grew to a higher cell density in suspension and were tumorigenic.

ISSN:0897-7194    

DOI:10.3109/08977199009167025

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. The presence of bFGF in BSMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1.5–1.7 M NaCl, characteristic for bFGF; (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0, 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5–1.7 M NaCl, characteristic for aFGF and bFGF; and second, Northern blot analysis demonstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore, it is shown that aFGF and bFGF are potent mitogens for smooth muscle cellsin vitro.

ISSN:0897-7194    

DOI:10.3109/08977199009167026

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Past work described the partial purification and characterization of a novel serine protein kinase activity designated protein kinase N (PKN) that is activated by nerve growth factor (NGF) in cultured PC12 cells [Rowland et al. (1987)J. Biol. Chem.262; 7504–7513]. We have now devised a rapid, sensitive technique for partially purifying and assaying PKN activity in cell extracts. This methodology was applied to the IARC-EW-1 osteosarcoma and several additional non-neuronal cell lines that possess NGF receptors but that lack both morphological and a variety of additional biochemical responses to NGF. In each case, NGF significantly elevated PKN activity. The assay also revealed activation of PKN activity in IARC-EW-1 cells by additional agents, including epidermal growth factor, fibroblast growth factor, phorbol ester, and a cAMP analog. Also tested were an NGF-receptor-deficient PC12 cell variant and sublines thereof into which human NGF receptors had been introduced [Hempstead et al. (1989)Science243; 373–375]. Acquisition of the NGF receptors resulted in NGF-activatable PKN activity. These findings indicate that detection of PKN activity may serve as a sensitive means to test NGF responsiveness in cells lacking macroscopic responses to the factor and that non-neuronal cells may be useful for studying primary signaling events in the NGF mechanism of action.

ISSN:0897-7194    

DOI:10.3109/08977199009167027

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor