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At least 343 records · Page 19Linked to original sources
PubMed2000

Primary human fibroblasts from a maple syrup urine disease patient undergo apoptosis following exposure to physiological concentrations of branched chain amino acids.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-ketoacid dehydrogenase. We have recently found that MSUD neurodegeneration may result, at least in part, from apoptosis triggered by branched chain amino acids and their alpha-ketoacid derivatives. In the present study, we investigated the sensitivity of MSUD fibroblasts to defined mixtures of MSUD metabolites. Defined combinations of MSUD metabolites, at levels comparable to those in MSUD patients, triggered cell death in skin fibroblasts from a MSUD patient, while control fibroblasts were resistant. The mechanism of cell death was confirmed as apoptosis by in situ end labeling.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2001

TRADD domain of Epstein-Barr virus transforming protein LMP1 is essential for inducing immortalization and suppressing senescence of primary rodent fibroblasts.

Mutation analysis of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-induced B-cell immortalization revealed two transformation effector sites, TES1 and TES2. TES2 mediates the interaction with tumor necrosis factor receptor-associated death domain protein (TRADD) and plays a key role in transactivating NF-kappa B and AP-1. Recombinant EBV containing LMP1 with TES2 deleted induces a limited proliferation of B cells. The present study shows that a mutant with an LMP1 site-specific mutation at TES2, LMP1(TRADD), initially stimulates cell growth and significantly extends the life span of MEF. However, it is not sufficient for the immortalization of MEF, and MEF-LMP1(TRADD) cells eventually enter growth arrest. Further analysis reveals that although LMP1(TRADD) promotes cell growth, it does not prevent the eventual onset of senescence and the expression of tumor suppressor p16(Ink4a).

Animals↗
PubMed2005

Evidence for NF-kappaB- and CBP-independent repression of p53's transcriptional activity by human T-cell leukemia virus type 1 Tax in mouse embryo and primary human fibroblasts.

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein can repress the transcriptional activity of the tumor suppressor protein p53. However, it remains controversial whether Tax requires NF-kappaB factors/activity and/or p300/CBP in order to inactivate p53 function. To address this issue, we have investigated Tax's effect on p53's transcriptional activation in IkappaB-kinase-deficient mouse embryonic fibroblasts (MEFs); some of which are entirely silent for Tax-induced NF-kappaB activity. We found that, in IKKalpha-/-, IKKbeta-/-, and IKKgamma-/- MEFs, p53 activation of a prototypic responsive plasmid (pG13-luciferase) was repressed by wild-type Tax. Curiously, p53's activity in MEFs was also repressed by a p300/CBP-binding deficient Tax protein. Our results highlight the complex nature of Tax-mediated repression of p53- activity, which requires further investigation.

Animals↗
PubMed1992

The mRNA 5' cap-binding protein, eIF-4E, cooperates with v-myc or E1A in the transformation of primary rodent fibroblasts.

We present evidence that eIF-4E, the mRNA 5' cap-binding protein, cooperates with two immortalizing oncogenes, v-myc and E1A, to cause transformation of rat embryo fibroblasts. eIF-4E alone can transform rat embryo fibroblasts when selection is applied. The pattern of transformation by eIF-4E is similar to that of p21 Ras, raising the possibility that eIF-4E shares a common signal transduction pathway with p21 Ras.

Adenovirus Early Proteins↗
PubMed1993

Effect of gamma-interferon or cycloheximide treatment on viral and c-myc transcripts in bovine-papillomavirus-type-1-transformed primary mouse fibroblasts.

We have studied the effect of gamma-interferon (IFN-gamma) treatment on the transcription of viral and c-myc oncogenes in bovine papillomavirus type 1 (BPV-1)-transformed mouse fibroblast cell lines. Upon IFN-gamma treatment, viral transcripts always decreased in cell lines containing episomal BPV-1 DNA, while the effect was variable in cell lines containing integrated BPV-1 DNA. Two series of tumour cell lines established by in vivo passage of B6B71 (episomal) and B6B31-J (integrated) cells via nude mice (NuTu A) into immunocompetent C57BL/6 mice (NuTuA B6Tul) also showed a decrease and an increase, respectively, in viral transcripts upon IFN-gamma treatment. IFN-gamma also reduced c-myc transcription in all cell lines derived from tumours, but increased it in the transformed cell cultures. There was selection of c-myc oncogene transcripts in all the tumour-derived cell lines as compared to their transformed cell cultures. Cycloheximide treatment increased both viral and c-myc gene transcripts 3- to 20-fold in all cell lines. These results imply that IFN-gamma produced locally by immunological mechanisms may influence the expression of Papillomavirus oncogenes, and the response to IFN-gamma treatment may change when the viral DNA is integrated into the host genome.

Animals↗
PubMed1996

Glucocorticoids inhibit keratinocyte growth factor production in primary dermal fibroblasts.

The participation of growth factors in wound healing and tissue repair has been well established. Previous studies demonstrated that the expression of keratinocyte growth factor (KGF) was greatly elevated shortly after injury and that topical application of KGF accelerated healing. Steroidal antiinflammatory agents, specifically glucocorticoids, markedly impair wound healing. The participation of KGF in wound healing led us to examine the effect of glucocorticoids on KGF production. The addition of dexamethasone significantly reduced the level of constitutively produced KGF messenger RNA, protein, and bioactivity in conditioned medium from dermal fibroblasts. This inhibitory effect was observed with a variety of glucocorticoids, whereas nonsteroidal antiinflammatory compounds had little effect on KGF synthesis. The mechanisms by which dexamethasone decreased KGF production include a combination of a diminished transcriptional rate and destabilization of the KGF messenger RNA. Cytokines such as interleukin-1 alpha, platelet-derived growth factor-BB, and transforming growth factor-alpha, typically up-regulated during wound healing, augment KGF expression by dermal fibroblasts. We determined that dexamethasone also blocked this inductive effect. These results suggest that glucocorticoids could inhibit KGF production in the setting of wound repair, which may contribute to the impairment of healing associated with glucocorticoid use.

Animals↗
PubMed1983

Reaction of mineral dusts with primary lung fibroblast cultures.

The complex metabolism of rabbit lung fibroblast cultures maintained over 24 days in vitro and their interaction with the fibrogenic group of asbestos minerals is reviewed. The differential effects of nine dusts given as a single dose to 3-day-old fibroblast cultures which were then analyzed for DNA, protein and cell mat hydroxyproline after 3 weeks are discussed. A comparison is made of the ability of these minerals to promote reticulin deposition in vivo following instillation into rats with the ability of each mineral to alter cell mat hydroxyproline levels in fibroblast cultures. Because this comparison is poor for some of the minerals investigated, it is concluded that with the current methodology the fibroblast cultures do not provide an adequate in vitro test system for assessing the potential fibrogenicity of a mineral in vivo.

Animals↗
PubMed2000

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11.

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Löbrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

Animals↗
PubMed2002

Dose responses for chromosome aberrations produced in noncycling primary human fibroblasts by alpha particles, and by gamma rays delivered at sublimiting low dose rates.

As the total dose of X or gamma rays is delivered at lower and lower rates, the yield of chromosome aberrations progressively diminishes. Simultaneously, the shape of the dose response changes from one exhibiting pronounced upward curvature at high dose rates to one approaching linearity at low dose rates. Although the maximum sparing effect caused by lowering the dose rate can be predicted from classical cytogenetic theory, it has yet to be verified experimentally. Here, noncycling normal human fibroblasts were exposed to graded doses of (137)Cs gamma rays at chronic dose rates of 6.3 and 2.8 cGy h(-1), dose rates that we reasoned should be lower than those required to achieve maximal sparing. This was indeed shown to be the case, after it was determined that the two chronic dose rates produced identical linear dose responses of 0.05 total aberrations per cell Gy(-1). Consistent with cytogenetic theory, this value was statistically indistinguishable from the linear coefficient derived from a fit to aberration frequencies produced by high-dose-rate exposure. Exposure to (238)Pu alpha particles also produced a linear dose response for total aberrations, whose slope-with respect to (137)Cs gamma rays as a reference radiation-implied a maximum RBE of 35 +/- 2.

Alpha Particles↗
PubMed2005

DNA double-strand break misrejoining after exposure of primary human fibroblasts to CK characteristic X rays, 29 kVp X rays and 60Co gamma rays.

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.

Cells, Cultured↗
PubMed2004

Ets-1 regulates TNF-alpha-induced matrix metalloproteinase-9 and tenascin expression in primary bronchial fibroblasts.

Increased subepithelial deposition of extracellular matrix proteins is a key feature in bronchial asthma. Matrix metalloproteinase-9 (MMP-9) is a proteolytic enzyme that degrades the extracellular matrix. Tenascin is an extracellular matrix glycoprotein that is abundant in thickened asthmatic subbasement membrane. The expression of MMP-9 and tenascin reflects disease activity in asthma and airway remodeling. The molecular mechanisms regulating the expression of these proteins remain unknown. Both MMP-9 and tenascin promoters contain an Ets binding site, suggesting control by Ets-1. Thus, we hypothesized that Ets-1 expression is increased in asthma and that it contributed to enhanced MMP-9 and tenascin expression. To test this hypothesis, we determined the expression of Ets-1 in bronchial biopsies obtained from asthmatic subjects and determined the expression of Ets-1, MMP-9, and tenascin by bronchial fibroblasts activated ex vivo. We observed that nuclear extracts from TNF-alpha-activated fibroblasts showed increased Ets-binding activity. In addition, TNF-alpha-activated fibroblasts had increased expression of Ets-1 mRNA and protein, which preceded an increase in MMP-9 and tenascin mRNA. Furthermore, treatment of fibroblasts with Ets-1 antisense oligonucleotides down-regulated TNF-alpha-induced Ets-1, MMP-9, and, to a lesser extent, tenascin protein expression or activity. Taken together, these data demonstrate that TNF-alpha increases MMP-9 and tenascin expression in bronchial fibroblasts via the transcription factor Ets-1, and suggest a role for Ets-1 in airway remodeling in asthma.

Adult↗
PubMed2003

[The "classical" macrophage marker CD68 is strongly expressed in primary human fibroblasts].

AIM: Monoclonal antibodies against the human homologue of mouse macrosialin, CD68, are generally commercialized as markers for human monocytes and macrophages. Indeed, CD68 is considered as a selective marker for human myeloid cells, although several previous immunohistochemical studies indicate that some antibody clones also react with other hematopoietic and non-hematopoietic cell types. The aim of our study was to verify these observations and to evaluate the reliability of CD68 as a macrophage marker. METHODS: We investigated protein and RNA expression of CD68 in various fibroblast types and carcinoma cell lines as compared to monocytes and macrophages using immunohistochemistry, flow cytometry, and specific RT-PCR. Different monoclonal antibody clones against CD68 were applied including KP-1 and EBM11. RESULTS: As expected, the intensity of immunohistochemical and flow cytometric CD68 staining was dependent on both the antibody clone and the fixation procedure. However, fibroblasts isolated from normal skin, normal breast, breast tumor tissue, and osteoarthritis synovia clearly expressed CD68 protein at levels comparable to macrophages. The specificity of CD68 expression in fibroblasts was verified by RT-PCR which also showed some tumor cell types to express CD68 mRNA. CONCLUSION: Our findings clearly demonstrate that the expression of CD68 is not restricted to the macrophage lineage. This is highly relevant for experimental and diagnostic purposes, since anti CD68 antibodies cannot be accepted without reservations for the discrimination of myeloid cells and fibroblasts even in paraffin sections after formalin fixation.

Antigens, CD↗
PubMedDate not supplied

Characterization of an antiviral agent from primary murine fibroblast cultures: murine tissue culture CVI.

We have previously described a class of virus inhibitors which are produced spontaneously by many types of cells in culture and present in a number of physiological fluids. These inhibitors are differentiated from all other known naturally occurring antiviral substances in regard to their (i) lack of species specificity, (ii) broad antiviral activity (iii) absence of high affinity binding by the inhibitor to the virus, (iv) mechanism of the action of the inhibitor is through inhibition of viral attachment, and (v) extreme thermal stability. In this report, we show that this class of inhibitors can be divided into two distinct subclasses. The first category includes the inhibitor spontaneously produced by cells in culture, originally described as contact-blocking viral inhibitor (CVI), and has a polypeptide component associated with its antiviral activity. The second category includes the inhibitors detected in body fluids and tissue extracts and has no essential peptide structure. Further characterization of CVI with respect to molecular size and stability to heat and a number of chemical reagents and enzymes indicate that the antiviral activity of CVI is associated with a large molecule (90s or approximately 4 million daltons), is stable at 100(8)C, and is resistant to the action of RNase, DNase, sulfhydral reagents, protein denaturants, and extraction by organic solvents.

Animals↗