ISSN:0897-7194    

DOI:10.3109/08977199209046921

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

ISSN:0897-7194    

DOI:10.3109/08977199209046922

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractGranulocyte-colony stimulating factor (G-CSF) is a haemopoietic growth factor produced by mesenchymal cells but not T lymphocytes after stimulation with specific cytokines or mitogens. A 330 bp promoter fragment of the human G-CSF gene induced reporter gene expression in human embryonic lung fibroblasts in response to tumor necrosis factor-α(TNF-α) or interleukin-1β(IL-1β). The same promoter fragment was not active in Jurkat T cells nor did it respond to phorbol ester in either cell type. At least three distinct elements, the CK-1 sequence, a decanucleotide present in haemopoietic growth factor genes, an NF-IL-6 consensus sequence and a consensus octamer sequence, were essential in the G-CSF promoter for TNF-αand IL-1βresponse. Mutation of any of these sequences abolished promoter function. In contrast, mutation of two other consensus protein binding sequences, i.e. a Pu-1 site and a CK-2-like sequence, did not eliminate promoter function. Both the CK-1 and octamer sequences acted independently as TNF-αand IL-1βresponsive elements upstream of a heterologous promoter. The response of the octamer sequence and the 330 bp promoter but not the CK-1 sequence was greater with IL-1βthan TNF-αreflecting a similar response of the endogenous gene.

ISSN:0897-7194    

DOI:10.3109/08977199209046923

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractWe have previously shown that an autocrine factor (CRDGF) of molecular weight 25,000 is produced by the HT29 human colon cancer cell line. Although CRDGF was shown to inhibit the binding of epidermal growth factor (EGF) to its receptor, several lines of evidence suggested that it was distinct from EGF or transforming growth factor-α(TGF-α). In order to check the possibility that CRDGF represents a new member of the EGF family, a four-step purification protocol involving acid gel filtration, cation-exchange high-performance liquid chromatography (HPLC), C18reversed-phase HPLC and gel permeation HPLC was used to purify this protein to homogeneity. The purified material exhibited a 22 kDa molecular mass on SDS-PAGE. Partial N-terminal amino acid sequence of CRDGF showed identity to amphiregulin (AR), an EGF-related protein. Western blotting experiments using AR-specific antiserum confirmed that CRDGF and AR are identical proteins. In addition, we showed that AR, like EGF or TGF-a stimulated the phosphorylation of the epidermal growth factor receptor (EGF-R) on tyrosine residues. This indicates that the AR intracellular signalling pathway involves the activation of EGF-R kinase.

ISSN:0897-7194    

DOI:10.3109/08977199209046924

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractTransforming growth factorβis a strong growth inhibitor for many types of normal and transformed cells, although little is known on the mechanism of this anti-proliferative effect. The human lung adenocarcinoma cell line A549 is growth arrested by TGF-β1 and serves as a model for studying this effect. We describe that, concurrent with the inhibition of A549 cell growth, TGF-β1 treatment causes a dramatic reduction in the level of expression of the amphiregulin (AR) gene, a recently identified member of the EGF/TGFαfamily. Similar results were also observed with TGF-β2. Peak inhibition occurred at 24 hr of treatment and was reversible upon removal of TGF-β1. The level of AR protein secreted by A549 cells was also decreased by TGF-β1. In contrast, TGF-αmRNA was not detected in these cells regardless of TGF-β1 treatment. Another TGF-βinhibited cell line, PC-3 (human prostatic adenocarcinoma) also exhibited a decrease in AR message levels following exposure to TGF-β1. The growth inhibitory effects of TGF-βmay in part be mediated by modulation of AR expression.

ISSN:0897-7194    

DOI:10.3109/08977199209046925

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractBasic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytesin vitroand is present in developing cartilage invivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58±6% of cells (mean±s.d.,n=3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18±3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiogaphy, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G, with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide. When cells were labelled with [3H] leucine and newly-synthesized basic FGF immunoprecipitated from cytoplasmic or nuclear fractions, peak values of peptide were seen in the cytoplasm 2 hr prior to its transient appearance in the nucleus which was immediately before entry into S phase.The amount of immunoprecipitable, tritiated basic FGF recovered from cytoplasm and nucleus was unaffected by incubation with antibody against basic FGF. Separation of immunoprecipitated, tritiated basic FGF by gel electrophoresis revealed two major forms with molecular sizes 18 kDa and 23 kDa in both the cytoplasm and nucleus. Only the former was present in conditioned culture medium. The results suggest that chondrocytes synthesize and release basic FGF during the second half of G, in response to hormonal signals contained in fetal calf serum. This basic FGF contributes to the subsequent DNA synthetic rate of the cells. During late G1a brief translocation of basic FGF from cytoplasm to nucleus occurs, but this is not directly related to alterations in DNA synthesis.

ISSN:0897-7194    

DOI:10.3109/08977199209046926

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractPolyclonal antibodies capable of reacting with amphibian bone morphogenetic protein (BMP-2 and -4) were raised in rabbits by immunization with a synthetic 21 amino acid peptide which corresponds to a sequence residing in the mature protein ofXenopusBMP-2 (xBMP-2). The antibodies recognized an embryonic BMP as well as mammalian and bacteria expressed recombinant xBMPs. The antibodies detected, under reducing conditions, a 30 kDa protein in the extract of oocytes and embryos during early development. Interestingly, acidification of the extract from each developmental stage yielded a protein band of smaller molecular weight of 18 kDa, which is similar in size to reduced form of mature BMPs purified from mammalian species. Two-dimensional electrophoresis employed to examine the molecular weight of unreduced forms using the antibody, revealed that both molecular forms are monomeric in the embryos. The result suggests that at least BMP-2 mRNA previously detected in early embryos, is translated into peptide but the dimerization may be incomplete or strictly limited in these embryos.

ISSN:0897-7194    

DOI:10.3109/08977199209046927

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

AbstractExpression of the platelet-derived growth factor (PDGF) A-chain gene is known to give rise to multiple transcripts of different sizes. Northern blot analysis, using a set of DNA probes corresponding to various parts of the human PDGF A-chain gene, indicated heterogeneity in both the 5′and 3' end of the transcripts. The 3' heterogeneity was shown to be the result of differential use of three polyadenylation signals. S1-nuclease protection- and chloramphenicol acetyltransferase (CAT)-assays, revealed the 5′heterogeneity to be the consequence of two alternative promoters. Whereas the upstream promoter contained typical TATA- and CAAT-boxes, the downstream promoter lacked a TATA-box but seemed to be composed of several GC-boxes.

ISSN:0897-7194    

DOI:10.3109/08977199209046928

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor