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At least 469 records · Page 26Linked to original sources
PubMed2002

Cell type-specific responses of human cells to inhibition of replication licensing.

Replication origins are 'licensed' for a single initiation event by loading Mcm2-7 complexes during late mitosis and G1. Licensing is blocked at other cell cycle stages by the activity of cyclin-dependent kinases and a small protein called geminin. Here, we describe the effects of over-expressing a non-degradable form of geminin in various cell lines. Geminin expression reduced the quantity of Mcm2 bound to chromatin and blocked cell proliferation. U2OS (p53+/Rb+) cells showed an early S phase arrest with high cyclin E and undetectable cyclin A levels, consistent with the activation of an intra-S checkpoint. Saos2 (p53-/Rb-) cells showed an accumulation of cells in late S and G2/M with approximately normal levels of cyclin A, consistent with loss of this intra-S phase checkpoint. Geminin also induced apoptosis in both these cell lines. In contrast, IMR90 primary fibroblasts over-expressing geminin arrested in G1 with reduced cyclin E levels and no detectable apoptosis. A 'licensing checkpoint' may therefore act in primary cells to prevent passage into S phase in the absence of sufficient origin licensing. These results suggest that inhibition of the licensing system may cause cancer-specific cell killing and therefore represent a novel anti-cancer target.

Adenoviridae↗
PubMed1997

Abnormalities of copper accumulation in cell lines established from nine different alleles of mottled are the same as those found in Menkes disease.

Menkes disease (MD) is caused by a defect in copper homeostasis and has a recognised mouse model, mottled (Atp7aMo). Copper uptake and retention assays performed on fibroblast cultures have been used successfully for pre- and postnatal diagnosis of Menkes disease. We report here the results of these assays applied to primary fibroblast cultures established from nine independent mottled alleles associated with phenotypes of varying severity maintained on identical genetic backgrounds. No significant differences were found between the different alleles, or between the mottled cultures and fibroblasts established from MD patients. Thus, in the mouse, the data obtained for copper retention/uptake at the cellular level do not correlate with the severity of the phenotype.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Cysteine proteinases at various stages of neoplastic transformation of rat fibroblasts].

Cysteine-dependent proteinases were studied in a model system of rat embryonal fibroblasts which enabled to evaluate two discrete steps of malignant transformation in vitro: immortalization and tumoral transformation. Dynamics of intracellular and secreted activities of cysteine proteinases was estimated in relation to the stage of transformation and duration of cells cultivation. Activation of cysteine proteinases cathepsins L and B correlated with developed transformation of fibroblasts; each step of the process was characterized. Intracellular and secreted activities of the proteinases were distinctly increased in immortalized cells as compared with primary fibroblasts. The highest activity of the enzymes was detected both in intra- and extracellular fractions at the stage of complete fibroblast transformation. Cysteine-dependent cathepsin L-like proteinase was mainly responsible for intracellular and secreted activities of the fibroblast cultures studied. This proteinase was found to be produced in the form of precursor in all the cell strains studied and/or main part of the enzyme developed complexes with an inhibitor.

Adenoviruses, Simian↗
PubMed1980

Lysosomal acridine orange uptake in fibroblasts transformed by SV40 or human cytomegalovirus.

Lysosomes of living human fibroblasts, SV40-transformed rat fibroblasts and human CMV-transformed hamster fibroblasts were examined by fluorescence microscopy after pretreatment with acridine orange at a supravital concentrations (5 x 10(6) M). Dye uptake by human primary fibroblast lysosomes was considerable and independent of the age of the cultures. In the transformed cultures, cytoplasmic granular red fluorescence indicating lysosomal acridine orange uptake could not be observed in part of the cells; cells showing no cytoplasmic granular fluorescence appeared as early as after 48 hr incubation and were growing in dependence on the age of the culture. Staining of living cells by acridine orange solutions at supravital concentration is a practicable method for the examination of functional changes of lysosomes.

Acridine Orange↗
PubMed2006

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts.

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimer's disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Abeta 1-40 and Abeta 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

Adult↗
PubMed2006

Adaptive stress response in segmental progeria resembles long-lived dwarfism and calorie restriction in mice.

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD(G602D/R722W)/XPA(-/-)) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80(-/-) mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage.

Aging↗
PubMed2003

Molecular mechanisms of interleukin-4-induced up-regulation of type I collagen gene expression in murine fibroblasts.

OBJECTIVE: There is evidence that interleukin-4 (IL-4) plays a major role in the induction of extracellular matrix protein synthesis in fibrotic disease. We therefore examined the effect of IL-4 on collagen synthesis in primary fibroblasts isolated from normal and TSK/+ mice, which spontaneously develop a scleroderma-like syndrome characterized by diffuse cutaneous hyperplasia. METHODS: Expression of the IL-4 receptor was determined by flow cytometry and Western blotting. The IL-4 signal transduction cascade was analyzed by Western blotting. We assessed the role of signal transducer and activator of transcription 6 (STAT-6) in IL-4 induction of alpha2(I) collagen promoter activity and message levels via luciferase reporter assay and real-time polymerase chain reaction. The activation status of the transcription factors activator protein 1 (AP-1) and Sp-1 upon stimulation with IL-4 in normal and TSK/+ fibroblasts was examined by electrophoretic mobility shift assay. RESULTS: Flow cytometry and Western blotting showed that IL-4 receptor alpha expression was elevated in TSK/+ fibroblasts compared with normal fibroblasts. After IL-4 stimulation, janus-activated kinase 1 (JAK-1) and JAK-2 were phosphorylated to a greater degree in TSK/+ fibroblasts than in C57BL/6 fibroblasts. TSK/+ fibroblasts appeared to be hyperresponsive to IL-4, displaying increased synthesis of alpha1(I) collagen messenger RNA (mRNA), collagen protein, and activity of a luciferase reporter construct containing the -300 to +54 murine alpha2(I) collagen promoter. Overexpression of STAT-6 enhanced this effect, whereas expression of a dominant-negative STAT-6 abrogated the ability of IL-4 to induce alpha1(I) collagen mRNA in TSK/+ fibroblasts. Moreover, IL-4 induced increased DNA binding activity of transcription factors that are important for collagen synthesis. CONCLUSION: Our observations indicate that IL-4 has a profound effect on several factors that have been identified as playing major roles in the regulation of collagen synthesis and suggest that IL-4 increases the expression of type I collagen through a mechanism involving the activation of transcription factors that bind to and activate collagen promoter.

Animals↗
PubMed2005

[Expression of thymosin beta 4 mRNA expression in keloid tissues and fibroblasts cultured from keloid and its significance].

OBJECTIVE: To investigate the relations between thymosin beta 4 and pathologic scars by comparing the mRNA levels of thymosin beta 4 in keloid, hypertrophic scar and normal skin. METHODS: The primary fibroblasts from the patients of keloid (KFB), hypertrophic scar (HFB) and normal skin (NFB, n=7) were cultured in vitro with tissue culture system. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was used to assess the thymosin beta 4 mRNA levels in the tissues and fibroblasts obtained from the patients of three groups. RESULTS: The mRNA levels of thymosin beta 4 in the keloid tissues were lower than those in the tissues of hypertrophic scar and normal skin(both, P<0.01). The thymosin beta 4 mRNA level in keloid group was lower by 66.98% than hypertrophic scar, and 62.48% than normal skin. In addition, the same significant change was found in the cultured KFB compared with HFB group(the mean value was lower by 27.13%), but no difference was found between KFB and NFB. CONCLUSION: Expression of thymosin beta 4 is closely related with keloid. The inadequate expression of thymosin beta 4 may be one of the key factors for keloid formation.

Cells, Cultured↗
PubMed2004

Telomere dynamics in Fancg-deficient mouse and human cells.

A number of DNA repair proteins also play roles in telomere metabolism. To investigate whether the accelerated telomere shortening reported in Fanconi anemia (FA) hematopoietic cells relates to a direct role of the FA pathway in telomere maintenance, we have analyzed telomere dynamics in Fancg-deficient mouse and human cells. We show here that both hematopoietic (stem and differentiated bone marrow cells, B and T lymphocytes) and nonhematopoietic (germ cells, mouse embryonic fibroblasts [MEFs]) Fancg(-/-) mouse cells display normal telomere length, normal telomerase activity, and normal chromosome end-capping, even in the presence of extensive clastogen-induced cytogenetic instability (mitomycin C [MMC], gamma-radiation). In addition, telomerase-deficient MEFs with humanlike telomere length and decreased Fancg expression (G5 Terc(-/-)/Fancg shRNA3 MEFs) display normal telomere maintenance. Finally, early-passage primary fibroblasts from patients with FA of complementation group G as well as primary human cells with reduced FANCG expression (FANCG shRNA IMR90 cells) show no signs of telomere dysfunction. Our observations indicate that accelerated telomere shortening in patients with FA is not due to a role of FANCG at telomeres but instead may be secondary to the disease. These findings suggest that telomerase-based therapies could be useful prophylactic agents in FA aplastic anemia by preserving their telomere reserve in the context of the disease.

Animals↗
PubMed2006

Collagen expression in fibroblasts with a novel LMNA mutation.

Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

Adolescent↗
PubMed2003

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice.

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 +/- 0.1 per 106 cell divisions. Interestingly, the rate is approximately 10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximately 15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.

Animals↗
PubMed1988

Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.

Animals↗
PubMed2002

Instability of extracellular matrix gene expression in primary cell culture of fibroblasts from human vocal fold lamina propria and tracheal scar.

Primary fibroblast cell cultures were established from lamina propria of human vocal fold and tracheal scar. There exists a crucial need to provide new tools for studying voice biology, and one of the first steps is the development of a human primary laryngeal cell culture bank. Because cell lines can lose their differentiated phenotype in culture across passages, documentation of gene expression must be determined for passage populations, for us to have knowledge of cell behavior in vitro. Comparison of messenger RNA gene expression of extracellular matrix proteins (procollagen I, collagenase, elastin, hyaluronic acid synthase 2, hyaluronidase, fibronectin, cd44, fibromodulin, and decorin) across cell passages (3, 4, 5, 6, 10, and 12 fornormal laminapropria and 3, 4, 5, 6, and 10 for tracheal scar) revealed varied growth patterns. Cytogenetic analysis demonstrated relative stability of the karyotypes across passages for the tracheal scar cell cultures, whereas the karyotypes of the normal lamina propria fibroblasts showed instability in in vitro cultures. Recommendations for use of primary cell cultures for further studies of gene expression are made.

Adolescent↗
PubMed2000

Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts.

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.

Amino Acid Sequence↗
PubMed2002

Werner syndrome diploid fibroblasts are sensitive to 4-nitroquinoline-N-oxide and 8-methoxypsoralen: implications for the disease phenotype.

The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

4-Nitroquinoline-1-oxide↗
PubMed1998

Effect of roxithromycin on IL-8 synthesis and proliferation of nasal polyp fibroblasts.

Although several studies have demonstrated that low-dose, long-term 14-member macrolides (erythromycin (EM), roxithromycin (RXM), clarithromycin (CAM)) are effective in the treatment of chronic airway diseases like chronic sinusitis and diffuse panbronchiolitis (DPB), the mechanism of action of these drugs is not yet clear. Both these airway diseases are associated with an increase in the proliferation of fibroblasts. Moreover, fibroblasts are also an important source of proinflammatory cytokines such as interleukin-8 (IL-8), that play an important role in the pathogenesis of nasal polyps. Therefore, using primary fibroblast lines derived from nasal polyps, we investigated the effect of RXM on the synthesis of IL-8 and proliferation of nasal polyp fibroblasts (NPF). These fibroblasts were either treated with lipopolysaccharide (LPS) and RXM for 24 h, or pre-incubated with RXM for 24 h and then treated with LPS and RXM for 24 h. The level of IL-8 mRNA in NPF was analysed by reverse transcriptase-polymerase chain (RT-PCR) and the level of IL-8 in culture supernatants was measured by ELISA. Next, the proliferative capacity of NPF after treatment with RXM was analysed by cell counting and 3H-thymidine uptake. RXM had no effect on LPS-induced IL-8 synthesis by NPF. On the other hand, RXM suppressed the proliferation of NPF in a dose-dependent manner. These findings suggest that, although RXM cannot directly inhibit the synthesis of IL-8, it probably reduces IL-8 production by inhibiting the proliferation of NPF.

Anti-Bacterial Agents↗
PubMed2001

Loss of poly(ADP-ribose) polymerase-1 causes increased tumour latency in p53-deficient mice.

PARP-1-deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage-induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour-free survival of parp-1(-/-)p53(-/-) mice increased by 50% compared with that of parp- 1(+/+)p53(-/-) mice. Tumour formation in nude mice injected with oncogenic parp-1(-/-)p53(-/-) fibroblasts was significantly delayed compared with parp-1(+/+)p53(-/-) cells. Upon gamma-irradiation, a partial restoration of S-phase radiosensitivity was found in parp-1(-/-)p53(-/-) primary fibroblasts compared with parp-1(+/+)p53(-/-) cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp-1(-/-)p53(-/-) mice compared with parp-1(+/+)p53(-/-) mice. The abrogation of the oxydated status of p53(-/-) cells, due to the absence of parp-1, may be the cause of the delay in the onset of tumorigenesis in parp-1(-/-)p53(-/-) mice.

Animals↗
PubMed2001

N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro.

N-t-butyl hydroxylamine (NtBHA) delays senescence-dependent changes in human lung fibroblasts (IMR90) (Atamna et al., J. Biol. Chem. 275, 6741-6748). The current study examines the effect of NtBHA on mitochondria in old and young rats and human primary fibroblasts (IMR90). In NtBHA-treated rats, the age-dependent decline in food consumption and ambulatory activity was reversed without affecting body weight. The respiratory control ratio of mitochondria from liver of old rats improved after feeding NtBHA. These findings suggest that NtBHA improved mitochondrial function in vivo. The age-dependent increase in proteins with thiol-mixed disulfides was significantly lower in old rats treated with NtBHA. NtBHA was effective only in old rats; no significant effect was observed in young rats. In IMR90 cells, NtBHA delayed senescence-associated changes in mitochondria and cellular senescence induced by maintaining the cells under suboptimal levels of growth factors. Proteasomal activity was also higher in cells treated with NtBHA than in untreated cells. NtBHA accumulates in cells 10- to 15-fold the extracellular concentration and is maintained by mitochondrial NADH. NtBHA is an antioxidant that is recycled by mitochondrial electron transport chain and prevents radical-induced toxicity to mitochondria.

Aging↗