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PubMed2002

The role of fibroblasts in thymocyte-positive selection.

Mice with fibroblast-specific expression of TAP-1 were generated by expressing the TAP-1 transgene under the control of the fibroblast-specific protein (FSP) 1 promoter/enhancer on TAP-1-deficient background. MHC class I expression in primary fibroblast cultures isolated from the resulting strain mimicked that of wild-type counterparts. MHC class I was detected in both types of fibroblasts following treatment with IFN-alphabeta. Positive selection of CD4(-)CD8(+) thymocytes was observed in neither adult nor fetal/neonatal thymus of transgenic mice. IFN-alphabeta-induced expression of MHC class I rescued positive selection of CD4(-)CD8(+) T cells in fetal thymic organ cultures, but not in adult mice. Contrary to previous suggestions, our results indicate a limited role of fibroblasts in promoting positive selection. In addition, the results suggest that positive selection may occur by a different mechanism in fetal vs adult thymus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Regulation of proliferation-survival decisions during tumor cell hypoxia.

Hypoxia may influence tumor biology in paradoxically opposing ways: it is lethal as a direct stress trigger, yet hypoxic zones in solid tumors harbor viable cells which are particularly resistant to treatment and contribute importantly to disease relapse. To examine mechanisms underlying growth-survival decisions during hypoxia, we have compared genetically related transformed and untransformed fibroblast cells in vitro for proliferation, survival, clonogenicity, cell cycle, and p53 expression. Hypoxia induces G0/G1 arrest in primary fibroblasts but triggers apoptosis in oncogene-transformed derivatives. Unexpectedly, the mechanism of apoptosis is seen to require accumulated acidosis and is rescued by enhanced buffering. The direct effect of hypoxia under nonacidotic conditions is unique to transformed cells in that they override the hypoxic G0/G1 arrest of primary cells. Moreover, when uncoupled from acidosis, hypoxia enhances tumor cell viability and clonogenicity relative to normoxia. p53 is correspondingly upregulated in response to hypoxia-induced acidosis but downregulated during hypoxia without acidosis. Hypoxia may thus produce both treatment resistance and a growth advantage. Given strong evidence that hypoxic regions in solid tumors are often nonacidotic (G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, Nat. Med. 3:177-182, 1997), this behavior may influence relapse and implicates such cells as potentially important therapeutic targets.

Animals↗
PubMed2003

An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes.

One of the critical challenges for cellular genetic studies in primary human skin cells is lack of a gene delivery system that provides efficient transduction and sustained expression of the transgenes. Due to the limited time of survival in culture, the processes of drug selection and clonal expansion for establishing gene stably expressing cell lines are not a realistic option for primary skin cells. We have examined various gene transduction techniques in primary dermal fibroblasts and epidermal keratinocytes of human skin. We report here that vectors based on the human immunodeficiency virus (HIV, lentivirus) offer more than 90% gene transduction efficiency and sustained expression of transgenes in both human skin cell types. In contrast, most of the commonly used techniques have at best 30% transduction efficiency in these cells. Using two previously reported migration control genes, protein kinase Cdelta and p38alpha-MAPK, as examples, we provide evidence that the unprecedented efficiency of the lentiviral system enables a clear detection of the genes' dominant negative effects, which are otherwise greatly compromised by ordinary transfection techniques. We believe that a wide application of this gene transduction system will greatly benefit studies of gene function in human skin cells.

Fibroblasts↗
PubMed1999

alpha-thrombin inhibits interleukin-6-induced Stat3 signaling and gp130 gene expression in primary cultures of human lung fibroblasts.

Exposure of primary human lung fibroblasts (HLF) to interleukin-6 (IL-6) rapidly induced Stat3 (signal transducers and activators of transcription 3) tyrosine phosphorylation. In these cells, alpha-thrombin did not induce tyrosine phosphorylation of Stat3; however, it potently induced its serine phosphorylation. Interestingly, a short pretreatment of cells with alpha-thrombin significantly inhibited IL-6-induced tyrosine phosphorylation of Stat3. The inhibition by alpha-thrombin was attenuated if cells were pretreated with U0126, a specific inhibitor of the mitogen-activated protein (MAP) kinase kinase 1 (MAPKK1). Exposure of HLF cells to IL-6 induced a twofold increase in gp130 mRNA levels; however, alpha-thrombin inhibited this IL-6-induced response almost to control levels. These results demonstrate, for the first time, that in HLF cells alpha-thrombin inhibits IL-6-induced Stat3 signaling via activation of MAPKK1 and that this cross-talk regulates IL-6-induced gp130 gene expression.

Antigens, CD↗
PubMed2000

JunD protects cells from p53-dependent senescence and apoptosis.

JunD is the most broadly expressed member of the Jun family and the AP-1 transcription factor complex. Primary fibroblasts lacking JunD displayed p53-dependent growth arrest, upregulated p19(Arf) expression, and premature senescence. In contrast, immortalized cell lines lacking JunD showed increased proliferation and higher cyclinD1 levels. These properties are reminiscent of the effects of oncogenic Ras expression on primary and established cell cultures. Furthermore, JunD(-/-) fibroblasts exhibited increased p53-dependent apoptosis upon ultraviolet irradiation and were sensitive to the cytotoxic effects of TNF-alpha. The antiapoptotic role of JunD was confirmed using an in vivo model of TNF-mediated hepatitis. We propose that JunD protects cells from senescence, or apoptotic responses to stress stimuli, by acting as a modulator of the signaling pathways that link Ras to p53.

3T3 Cells↗
PubMed2006

Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection.

Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.

Biomarkers↗
PubMed1998

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.

Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

Actins↗
PubMed2005

Dose-dependent misrejoining of radiation-induced DNA double-strand breaks in human fibroblasts: experimental and theoretical study for high- and low-LET radiation.

Misrejoining of DNA double-strand breaks (DSBs) was measured in human primary fibroblasts after exposure to X rays and high-LET particles (helium, nitrogen and iron) in the dose range 10-80 Gy. To measure joining of wrong DNA ends, the integrity of a 3.2-Mbp restriction fragment was analyzed directly after exposure and after 16 h of repair incubation. It was found that the misrejoining frequency for X rays was nonlinearly related to dose, with less probability of misrejoining at low doses than at high doses. The dose dependence for the high-LET particles, on the other hand, was closer to being linear, with misrejoining frequencies higher than for X rays, particularly at the lower doses. These experimental results were simulated with a Monte Carlo approach that includes a cell nucleus model with all 46 chromosomes present, combined with realistic track structure simulations to calculate the geometrical positions of all DSBs induced for each dose. The model assumes that the main determinant for misrejoining probability is the distance between two simultaneously present DSBs. With a Gaussian interaction probability function with distance, it was found that the data for both low- and high-LET radiation could be fitted with an interaction distance (sigma of the Gaussian curve) of 0.25 microm. This is half the distance previously found to best fit chromosomal aberration data in human lymphocytes using the same methods (Holley et al., Radiat. Res. 158, 568-580, 2002). The discrepancy may indicate inadequacies in the chromosome model, for example insufficient chromosomal overlap, but may also be partly due to differences between fibroblasts and lymphocytes.

Cells, Cultured↗
PubMed1999

Muscle-specific transcription factors in fibroblasts expressing the alpha-striated tropomyosin 3' untranslated region.

The alpha-striated tropomyosin 3' untranslated region (TM UTR) promotes differentiation of fibroblasts into cells resembling skeletal muscle. To investigate the mechanism of this observation, RNA harvested from transfected primary fibroblasts was used for semiquantitative RT-PCR with primers specific for muscle transcription factors, showing that myoD and myogenin transcripts are detected in these cells, but that differentiation after TM UTR expression is independent of a detectable increase in these transcripts. Double immunofluorescent staining with antibodies to myoD family members and to titin confirms that muscle differentiation in TM UTR-transfected fibroblasts is independent of production of any transcription factor in this family. In contrast, the muscle transcription factor myocyte enhancer factor 2 (mef-2) is strongly expressed after transfection of fibroblasts with the TM UTR. The increase in mef-2 protein is due to an increase in the steady-state level of its mRNA, as shown by Northern analysis. The expression of p21 ordinarily observed in skeletal myogenesis before the expression of muscle-specific proteins is not seen in fibroblasts induced to differentiate by the TM UTR. These results demonstrate that post-transcriptional regulation of myoD family members is seen in fibroblasts, and that the TM UTR induces muscle differentiation independent of the myoD transcription factors and without expressing proteins characteristic of terminal withdrawal from the cell cycle. Finally, an increase in the steady-state level of mef-2 transcripts appears in the proximal pathway of myogenic activation in response to expression of the TM UTR. These results imply that fibroblasts can utilize an additional differentiation route upon TM UTR expression resulting in mature muscle other than that requiring myoD family members.

3' Untranslated Regions↗
PubMedDate not supplied

Functional inactivation of pRB results in aneuploid mammalian cells after release from a mitotic block.

The widespread chromosome instability observed in tumors and in early stage carcinomas suggests that aneuploidy could be a prerequisite for cellular transformation and tumor initiation. Defects in tumor suppressors and genes that are part of mitotic checkpoints are likely candidates for the aneuploid phenotype. By using flow cytometric, cytogenetic, and immunocytochemistry techniques we investigated whether pRB deficiency could drive perpetual aneuploidy in normal human and mouse fibroblasts after mitotic checkpoint challenge by microtubule-destabilizing drugs. Both mouse and human pRB-deficient primary fibroblasts resulted, upon release from a mitotic block, in proliferating aneuploid cells possessing supernumerary centrosomes. Aneuploid pRB-deficient cells show an elevated variation in chromosome numbers among cells of the same clone. In addition, these cells acquired the capability to grow in an anchorage-independent way at the same extent as tumor cells did suggesting aneuploidy as an initial mutational step in cell transformation. Normal Mouse Embryonic Fibroblasts (MEFs) harboring LoxP sites flanking exon 19 of the Rb gene arrested in G2/M with duplicated centrosomes after colcemid treatment. However, these cells escaped the arrest and became aneuploid upon pRB ablation by CRE recombinase, suggesting pRB as a major component of a checkpoint that controls cellular ploidy.

Aneuploidy↗
PubMed1995

Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease.

Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.

Animals↗
PubMed1995

Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice.

Continuous delivery of factor VIII (FVIII) protein in hemophiliacs by gene therapy will represent a major clinical advance over the current practice of infrequent administration of purified FVIII. Conceptually, retroviral vectors that can permanently insert the FVIII gene into the DNA of the host cell appear the most suitable vehicles for this specific purpose. However, most retroviral vector systems have shown a poor performance in the production of FVIII from primary cells in vitro and in vivo. Here we report the retroviral-mediated gene delivery of a B-domain-deleted human FVIII by using the MFG vector system. This vector permitted efficient transduction of the majority of the primary cells in culture without the use of a selectable marker. High levels of FVIII were produced by various transduced primary cells in vitro. Upon transplantation of primary fibroblasts into mice, therapeutic levels of FVIII in the circulation were obtained for > 1 week. The capacity of primary cells to deliver the FVIII into the circulation was strongly dependent on the site of implantation. These results represent a major step forward in development of gene therapy for treating hemophilia A.

3T3 Cells↗
PubMed1999

Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras.

The cell-cycle inhibitor p21 is upregulated during senescence and upon induction of senescence-like arrest by oncogenic Ras. We have used primary fibroblasts derived from p21-null mice to evaluate the role of p21 in these processes. We find that primary p21-/- cells enter senescence and have a lifespan similar to wild-type cells. Upon immortalization, most wild-type and p21-/- cultures acquire alterations in either p53 or p16INK4a, further indicating that p21-deficiency is not sufficient by itself to allow immortalization. Primary p21-/- cells, like wild-type cells, respond to oncogenic Ras by accumulating p53 and p16INK4a, and by decreasing their proliferation rate. In agreement with this, p21-/- cells are refractory to neoplasic transformation by oncogenic Ras when compared to p53-/- cells. We conclude that, in murine fibroblasts, p21 is not essential neither for senescence nor for preventing neoplasic transformation by oncogenic Ras.

3T3 Cells↗
PubMed1999

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts.

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

Animals↗
PubMed2004

Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.

Amyloid↗
PubMed1999

Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3.

Increased collagen and extracellular matrix (ECM) deposition within the lung is a characteristic feature of lung fibrosis. Transforming growth factor (TGF)-beta isoforms play a pivotal role in the production of collagen and ECM. In this study, we investigated the effects of TGF-beta1 and TGF-beta3 on the main processes controlling ECM deposition using primary human lung fibroblasts. We analyzed 1) collagen metabolism by [3H]proline incorporation, 2) matrix metalloproteinase (MMP) expression by substrate gel zymography, and 3) tissue inhibitor of metalloproteinases (TIMP) expression by Western blot analysis. TGF-beta1 and TGF-beta3 increased the percentage of secreted collagens in supernatants of primary fibroblasts from 8.0 +/- 1.2 (control) to 23.6 +/- 4.6 and 22.3 +/- 1.3%, respectively. The collagen percentage in deposited ECM was increased from 5.8 +/- 0.3 (control) to 9.0 +/- 0.5 and 8.8 +/- 0.5% by TGF-beta1 and TGF-beta3, respectively. Secretion of MMP-1 (interstitial collagenase) by fibroblasts was reduced by both TGF-beta isoforms, whereas secretion of MMP-2 (gelatinase A) was unaffected by either of the two isoforms. Both TGF-beta isoforms increased TIMP-1 protein expression, whereas TIMP-2 protein was decreased. We thus conclude that TGF-beta1 and TGF-beta3 are equally potent in increasing ECM deposition. Their fibrotic effect in lung fibroblasts results from 1) an increase in the secretion and deposition of total ECM and collagens, 2) a decrease in MMP-1 secretion, and 3) an increase of TIMP-1 expression.

Blotting, Western↗
PubMed2001

Gelatinase A (MMP-2) activation by skin fibroblasts: dependence on MT1-MMP expression and fibrillar collagen form.

The respective requirements of collagen and MT1-MMP in the activation of MMP-2 by primary fibroblast cultures were explored further. Three-dimensional gels enriched in human collagen types I and III or composed of recombinant human type II or III collagen, caused increased MT1-MMP production (mRNA and protein) and induced MMP-2 activation. Only marginal induction was seen with dried monomeric collagen confirming the need for collagen fibrillar organisation for activation. To our surprise, relatively low amounts (as low as 25 microg/ml) of acid soluble type I collagen added to fibroblast cultures also induced potent MMP-2 activation. However, the requirement for collagen fibril formation by the added collagen was indicated by the inhibition seen when the collagen was pre-incubated with a fibril-blocking peptide, and the reduced activation seen with alkali-treated collagen preparations known to have impaired fibrilisation. Pre-treatment of the collagen with sodium periodate also abrogated MMP-2 activation induction. Further evidence of the requirement for collagen fibril formation was provided by the lack of activation when type IV collagen, which does not form collagen fibrils, was added in the cultures. Fibroblasts derived from MT1-MMP-deficient mice were unable to activate MMP-2 in response to either three-dimensional collagen gel or added collagen solutions, compared to their littermate controls. Collectively, these data indicate that the fibrillar structure of collagen and MT1-MMP are essential for the MMP-2 activational response in fibroblasts.

Animals↗
PubMed2004

Tissue plasminogen activator is a potent activator of PDGF-CC.

Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.

Animals↗