摘要:

AbstractThe mechanism of tumor necrosis factor (TNF)‐induced cytotoxicity has been investigated using two clonal variants of the ME‐180 human cervical carcinoma cell line. The clonal lines were characterized with respect to their expression of TNF receptors, kinetics of cell death, and their ability to communicate intercellularly through gap junctions. The ME‐180.4 and ME‐180.8 clones were identified by their relative sensitivity to TNF induced lysis in a 24‐h assay. The dose of TNF required to kill 50% of the target cells was 60 pM for the sensitive ME‐180.4 and 2.5 nM for the ME‐180.8. However, when assay times were extended, the dose response for both clones was the same, indicating that a difference in the kinetics of cell death and not absolute TNF sensitivity existed between the ME‐180.4 and ME‐180.8 clones. Both clones were gap junction deficient as judged by their inability to transfer Lucifer yellow or 6‐carboxyfluorescein, a characteristic phenotype of cells sensitive to cytotoxicity by TNF. The level of surface receptor expressed on these clones was nearly identical with a Kd = 0.3 nM and 5,000 binding sites per cell. Measurement of the kinetics of cell death revealed that the time between the addition of TNF and the onset of observed cell death (induction phase) was much shorter for the ME‐180.4 (32–55 h) than for the resistant ME‐180.8 (55–80 h). Mitomycin C, a DNA alkylating agent, significantly reduced the length of the induction phase for both clones, although the kinetic difference between the clones remained unchanged. Two epipodophyllotoxins, VP‐16 and VM‐26, which specifically inhibit the rejoining activity of DNA topoisomerase II, showed a 10–100‐fold synergistic effect when combined with TNF as shown by isobologram analysis. VM‐26 when added to the resistant ME‐180.8 clones decreased the length of induction phase and abolished the kinetic difference observed with the ME‐180.4 clone. These results indicate that the variance in the TNF response of these two clones was closely associated with DNA topoisomerase II, and suggest that this enzyme may play an im

ISSN:0730-2312    

DOI:10.1002/jcb.240390202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe effects of transforming growth factor‐β1 (TGF‐β1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor‐α (TNF‐α) and recombinant human interferon‐α (rIFN‐α). Combinations of TNF‐α and TGF‐β1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU‐E) and an additive suppression of granulocyte‐macrophage (CFU‐GM) and multipotential (CFU‐GEMM) progenitor cells. In addition, TGF‐β1 synergized with rIFN‐α to suppress CFU‐GM formation, while the combined suppressive effects of both cytokines on CFU‐GEMM and BFU‐E were additive. When TGF‐β1 was tested with TNF‐α or IFN‐α on granulocyte/macrophage colony‐stimulating factor (GM‐CSF)‐stimulated bone marrow cells in a 5‐day proliferation assay, the antiproliferative effects of TGF‐β1 and TNF‐α were additive, while those with TGF‐β1 and rIFN‐α were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG‐1 where TGF‐β1 in combination with TNF‐α resulted in an additive suppression while inhibition by TGF‐β1 and IFN‐α was synergistic.These results demonstrate for the first time the cooperative effects between TGF‐β and TNF‐α and IFN‐α in the suppression of hematopoietic cell growth, raising the possibility that TGF‐β mig

ISSN:0730-2312    

DOI:10.1002/jcb.240390203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractDuring the last ten years, several groups, including the present authors, have detected growth factor activities in various ocular tissues, and the presence of a ubiquitous Eye‐Derived Growth Factor (EDGF) has been described. More recently, isolation and characterization of this growth factor activity from the retina led to the identification of two molecules. These molecules were shown to be identical to other growth factors isolated from neuronal and non‐neuronal tissues and are now designated as acidic and basic fibroblast growth factor (aFGF, bFGF). The biological function and the reason for the ubiquitous distribution of these factors remain unclear. Understanding may be improved by quantification of this distribution in various tissues during development. In the present study, specific polyclonal antibodies were raised against acidic FGF, aFGF was determined in various ocular tissues by enzyme immunoassay, and the localization of immuno‐reactive aFGF by immunohistological staining with fluorescent antibodies or with enzyme‐ or gold‐labeled antibodies was studied.In almost all tissues tested aFGF was found; but the retina, cornea, and vitreous body contained the highest levels of aFGF per gram of tissue. In the retina, aFGF was associated primarily with the nerve fiber layer and the inner and outer segments of the photoreceptors, whereas corneal aFGF was detected in the cytoplasma of the basal layer of epithel

ISSN:0730-2312    

DOI:10.1002/jcb.240390204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractColony‐stimulating factor 1 (CSF‐1) selectively supports the survival, proliferation, and maturation of hemopoietic cells of the monocyte/macrophage lineage. Although the cellular receptor for CSF‐1, (the c‐fmsprotein) is a protein‐tyrosine kinase activated by the binding of CFS‐1, the role of phosphorylation of cellular proteins in CSF‐1 signal transduction is poorly understood. Therefore, we examined the CSF‐1‐stimulated phosphorylation of cellular proteins in human BeWo choriocarcinoma cell line (known to express the c‐fmsprotein). BeWo cells were metabolically labeled with32Pi, stimulated with recombinant human CSF‐1, and extracted with detergent. Phosphotyrosyl proteins were isolated from detergent extracts by affinity chromatography on a highly specific antibody to phosphotyrosine. Rapid phosphorylation of 170‐kd protein, followed closely by the phosphorylation of a 56‐kd protein, was observed in response to CSF‐1. The 170‐kd phosphotyrosyl protein bound to wheat germ agglutinin and was secondarily immunoprecipitated with a specific anti‐fmsserum, consistent with its identity as the CSF‐1 receptor. Although purified human macrophages that proliferate in culture in response to CSF‐1 are not generally accessible, CSF‐1 did stimulate the phosphorylation of a 56‐kd protein in intact mononuclear leukocytes from human peripheral blood. Thus, the BeWo cell line may represent a good model for the study of CSF‐1‐

ISSN:0730-2312    

DOI:10.1002/jcb.240390205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractAlthough increased free intracellular calcium (Cai) may be one of the main regulators of cell growth and differentiation, studies in cell populations have implied that not all growth factors produce Caiincreases. In order to examine in more detail whether Caiincreases were related to mitogenesis, we used digital image analysis of intracellular Fura‐2 fluorescence to measure Caiin individual BALB/c 3T3 cells stimulated with either platelet‐derived growth factor (PDGF) or fibroblast growth factor (FGF). We found that PDGF induced larger and more prolonged Caiincreases than FGF did, but that both growth factors induced an initial rapid increase in Cai(twofold) correlated with the percentage of cells made competent (subsequent growth in 1% platelet‐poor‐plasma). In contrast, purified bovine basic FGF (200–800 pg/ml) and recombinant human acidic FGF (10–300 ng/ml) produced peak Caiincreases that were not directly correlated with mitogenesis. In addition, concentrations of intracellular Quin 2 that inhibited Caitransients also inhibited PDGF stimulation but not FGF stimulation of mitogenesis. Thus, Caiincreases are necessary for mitogenesis in BALB/c 3T3 cells stimulated by PDGF, but not that stim

ISSN:0730-2312    

DOI:10.1002/jcb.240390206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractTwo retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFRcDNA into two different retroviral vectors. One DNA (pCO11‐EGFR‐neo) also contained a linked selectable marker gene (neoR). The other (pCO12‐EGFR) only expresses hEGFR. When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium. Compared with pCO11‐EGFR‐neo, pCO12‐EGFR induced EGF‐dependent transformation 2–5 times more efficiently and expressed higher numbers of receptors (4 × 105vs. 1 × 105EGF receptors per cell). The results indicate that transforming potential is directly related to the number of EGF receptors. In defined, serum‐free medium that contained only very low concentrations of insulin (0.6 μg/ml) and transferrin (0.6 μg/ml), hEGFR‐virus infected cells were able to grow with EGF as the only growth factor. Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth. NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus. The dose‐dependent growth response to EGF of infected NR6 cells grown in serum‐free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type α (TGFα). This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg–100 ng/ml). Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF sp

ISSN:0730-2312    

DOI:10.1002/jcb.240390207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe c‐erbB‐2 proto‐oncogene is amplified in a high percentage of primary human breast tumors, suggesting that the overexpression of this gene may be involved in the development of human breast cancer. We have investigated five human breast tumor cell lines and have detected amplified c‐erbB‐2 gene copies in two of them. This amplification leads to overexpression of the c‐erbB‐2 protein. In addition, two other cell lines have elevated protein levels without gene amplification, suggesting that other mechanisms can lead to overexpression of the c‐erbB‐2 protein. These results are similar to those that we obtained during a study of primary breast tumors (Berger et al.: Cancer Res 48:1238–1243, 1988). These breast tumor cell lines should be useful for an analysis of c‐erbB‐2 expression and of the mechanisms that in some cases

ISSN:0730-2312    

DOI:10.1002/jcb.240390208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe have recently demonstrated that transforming growth factor (TGF)‐β1 and TGF‐β2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin‐3 (IL‐3) and human bone marrow by IL‐3 or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) was inhibited by TGF‐β1 and TGF‐β2, while the proliferation of murine bone marrow by GM‐CSF or murine and human marrow with G‐CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF‐β1. In particular, CFU‐GM, CFU‐GEMM, BFU‐E, and HPP‐CFC, the most immature colonies, were inhibited by TGF‐β1, whereas the more differentiated unipotent CFU‐G, CFU‐M, and CFU‐E were not affected. TGF‐β1 inhibited IL‐3‐induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF‐β1 results in the resumption of normal growth. TGF‐β1 inhibited the growth of factor‐dependent NFS‐60 cells in a dose‐dependent manner in response to IL‐3, GM‐CSF, G‐CSF, CSF‐1, IL‐4, or IL‐6. TGF‐β1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythorid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF‐β, were also resistant to TGF‐β1 inhibition. These leukemic cells have no detectable TGF‐β1 receptors on their cell surface. Last, TGF‐β1 directly inhibited the growth of isolated Thy‐1‐positive progenitor ce

ISSN:0730-2312    

DOI:10.1002/jcb.240390209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractMutant cells of the HD33 subline of the Ehrlich‐Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high‐molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 × 109) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of β‐type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear

ISSN:0730-2312    

DOI:10.1002/jcb.240390210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractMembranes prepared from rabbit neutrophils exhibit GTPase activity which can be stimulated by the chemotactic factor fMet‐Leu‐Phe. The maximum contribution of the ATPase activities to the basal and the fMet‐Leu‐Phe‐stimulated GTPase activities are less than 20% and 9%, respectively. The basal GTPase activity has a Vmax= 34.2 ± 1.3 (pmol/mg protein, min) and a Km= 0.39 ± 0.03 μM; and the fMet‐Leu‐Phe‐stimulated has a Vmax= 52.3 ± 2.5 (pmol/mg protein, min), and a Km= 0.29 ± 0.02 μM. The GTPase activity can be stimulated by fMet‐Leu‐Phe and leukotriene B4. Unlike these two chemotactic factors, concanavalin A does not stimulate this GTPase activity. In addition, the rise in intracellular concentration of free calcium produced by concanavalin A is not inhibited by pertussis toxin treatment. Both the basal and stimulated GTPase activities are affected by pertussis toxin, cholera t

ISSN:0730-2312    

DOI:10.1002/jcb.240390211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY