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PubMed1975

[Electron microscopic study of the interaction between neoplastic fibroblasts and the substrate in tissue culture].

Electron microscope study of neoplastic L fibroblasts was carried out in 15, 30, 60, and 90 min after their attachment to solid substratum. Comparative analysis of neoplastic and normal fibroblasts at the same stages was carried out. Spreading rate of L fibroblasts proved slower than that of normal fibroblasts. Primary reaction of neoplastic cells was disturbed on the contact with substratum: it was morphologically manifested in changing of structure of the cell lower surface. Bundles of microfilaments were absent from the neoplastic fibroblasts' cytoplasm. The above changes, apparently, may entail the lesser degree of spreading and the weaker attachement of neoplastic fibroblasts to the substratum.

Animals↗
PubMed1993

Choroid tissue supports the survival of ciliary ganglion neurons in vitro.

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.

3T3 Cells↗
PubMed1980

[Glucosamine: its value for the metabolism of articular cartilage. 1. Biochemistry of proteoglycans, studies on in-vitro cultures of embryonal mouse fibroblasts and bone germs].

Mucopolysaccharides (glycosaminoglycanes) are essential constituents of the cartilage (chondroitin sulfate) and the synovial fluid (hyaluronic acid). They are formed within the chondrocyte preferably from glucosamine or from glucose and bound to protein constituting proteoglycanes. Non steroidal anti-inflammatory drugs and corticosteroids inhibit this synthesis. Primary fibroblast cultures and in vitro cultured mouse embryo tibiae show a dose-dependent increased synthesis of mucopolysaccharides and an enhanced growth of the bones after addition of glucosamine to the culture medium. This finding underlines the importance of a sufficient supply of the joints with glucosamine.

Adrenal Cortex Hormones↗
PubMed2003

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription.

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription.

Alternative Splicing↗
PubMed2003

Transformation of rodent fibroblasts by the jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain.

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). Expression of the JSRV envelope protein (Env) is sufficient to transform immortalized and primary fibroblasts, but the precise mechanisms of this process are not known. The cellular receptor for JSRV is hyaluronidase 2 (Hyal-2), the product of a putative tumor suppressor gene that in humans maps to a chromosomal region frequently deleted in the development of lung and breast cancers. Here we report studies to determine whether the Hyal-2-JSRV Env interaction plays a role in virus-induced transformation of rodent fibroblasts. Chimeric Env proteins between JSRV and the unrelated murine retroviruses Moloney murine leukemia virus (MMuLV) and mouse mammary tumor virus (MMTV) showed cell surface expression comparable to that of wild-type MMuLV Env and rescued infection of MMuLV particle pseudotypes. Interestingly, an MMuLV-JSRV chimera in which the putative receptor binding domain (RBD) and proline-rich region (PRR) of JSRV Env were replaced by the RBD and PRR of MMuLV induced transformation of 208F, a rodent fibroblast line. Cell lines derived from foci of MMuLV-JSRV chimera-transformed 208F cells grew in soft agar and showed Akt activation, a hallmark of JSRV-transformed rodent fibroblasts. Transformation assays performed using proteins with amino-terminal deletion mutations showed that the carboxy-terminal 141 amino acids of the transmembrane subunit (TM) were sufficient to induce cell transformation when targeted to the membrane with a myristoylation signal. Thus, the JSRV TM is necessary and sufficient to transform rodent fibroblasts. Taken together these results indicate that the interaction with Hyal-2 at least is not an essential determinant of JSRV-induced transformation of fibroblasts and that the viral TM functions essentially as an oncoprotein.

3T3 Cells↗
PubMed2003

Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B.

Recent data have implicated the serine/threonine protein kinase Akt/protein kinase B (PKB) in a diverse array of physiological pathways, raising the question of how biological specificity is maintained. Partial clarification derived from the observation that mice deficient in either of the two isoforms, Akt1/PKBalpha or Akt2/PKBbeta, demonstrate distinct abnormalities, i.e. reduced organismal size or insulin resistance, respectively. However, the question still persists as to whether these divergent phenotypes are due exclusively to tissue-specific differences in isoform expression or distinct capacities for signaling intrinsic to the two proteins. Here we show that Akt2/PKBbeta-/- adipocytes derived from immortalized mouse embryo fibroblasts display significantly reduced insulin-stimulated hexose uptake, clearly establishing that the partial defect in glucose disposal in these mice derives from lack of a cell autonomous function of Akt2/PKBbeta. Moreover, in adipocytes differentiated from primary fibroblasts or immortalized mouse embryo fibroblasts, and brown preadipocytes the absence of Akt2/PKBbeta resulted in reduction of insulin-induced hexose uptake and glucose transporter 4 (GLUT4) translocation, whereas Akt1/PKBalpha was dispensable for this effect. Most importantly, hexose uptake and GLUT4 translocation were completely restored after re-expression of Akt2/PKBbeta in Akt2/PKBbeta-/- adipocytes, but overexpression of Akt1/PKBalpha at comparable levels was ineffective at rescuing insulin action to normal. These results show that the Akt1/PKBalpha and Akt2/PKBbeta isoforms are uniquely adapted to preferentially transmit distinct biological signals, and this property is likely to contribute significantly to the ability of Akt/PKB to play a role in diverse processes.

Adipose Tissue, Brown↗
PubMed2005

Long-term therapeutic effects on parkinsonian rats of intrastriatal co-grafts with genetically engineered fibroblasts expressing tyrosine hydroxylase and glial cell line-derived neurotrophic factor.

The long-term improvement of intrastriatal co-grafts with genetically engineered fibroblasts expressing tyrosine hydroxylase (TH) and glial cell line-derived neurotrophic factor (GDNF) was investigated in the present study. Two recombinant vectors, pCMV-TH and pCI-neo-GDNF, were transfected respectively into the primary fibroblasts, and their expression was further identified by in situ hybridization and immunocytochemistry. The engineered fibroblasts expressing TH, GDNF, or both were transplanted into the striatum of parkinsonian rats, and the therapeutic effects were observed for 20 weeks. Data revealed that only animals with fibroblasts expressing both TH and GDNF exhibited a stable and significant behavioral and biochemical recovery. Moreover, persistence of both TH and GDNF expression in grafts was demonstrated 20 weeks after transplantation. These results suggest that combined transplantation of fibroblasts expressing TH and GDNF can lead to long-term and remarkable therapeutic effects on parkinsonian rat model.

Animals↗
PubMed2005

Development and characterization of a spheroidal coculture model of endothelial cells and fibroblasts for improving angiogenesis in tissue engineering.

Neovascularization is a critical step in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of central tissues. We have developed a three-dimensional spheroidal coculture system consisting of human umbilical vein endothelial cells (HUVECs) and human primary fibroblasts (hFBs) to improve angiogenesis in tissue engineering applications. Morphological analysis of cryosections from HUVEC/hFB cospheroids revealed a characteristic temporal and spatial organization with HUVECs located in the center of the cospheroid and a peripheral localization of fibroblasts. In coculture spheroids, the level of apoptosis of endothelial cells was strongly decreased upon cocultivation with fibroblasts. Collagen-embedded HUVEC spheroids develop numerous lumenized capillary-like sprouts. This was also apparent for HUVEC/hFB cospheroids, albeit to a lesser extent. Quantification of cumulative sprout length revealed an approximately 35% reduction in endothelial cell sprouting upon cocultivation with fibroblasts in cospheroids. The slight reduction in endothelial cell sprouting was not mediated by a paracrine mechanism but is most likely due to the formation of heterogenic cell contacts between HUVECs and hFBs within the cospheroid. The model system introduced in this study is suitable for the development of a preformed lumenized capillary-like network ex vivo and may therefore be useful for improving angiogenesis in in vivo tissue engineering applications.

Apoptosis↗
PubMed2004

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells.

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.

Analysis of Variance↗
PubMed1996

Tenascin-X expression in tumor cells and fibroblasts: glucocorticoids as negative regulators in fibroblasts.

Tenascin-X has recently been shown to be a novel member of the tenascin family and its distribution is often reciprocal to that of tenascin-C in the developing mouse embryo. We have investigated the expression of tenascin-X in fibroblasts and carcinoma cells in culture. Tenascin-X protein was secreted in vitro in the conditioned media at an apparent molecular mass of approximately 450 kDa. In addition fibroblasts contained a major tenascin-X isoform of 220 kDa. On northern blots, a single major transcript with a size of approximately 13 kb was detected. No overexpression of tenascin-X protein was found in primary fibroblasts of the tenascin-C-gene knockout mice. Steroid hormone glucocorticoids, were found to downregulate tenascin-X mRNA levels and protein synthesis in fibroblasts but not carcinoma cells at physiological concentrations. None of the growth factors or cytokines examined affected the expression level of tenascin-X. As in vivo study, carcinoma cells were transplanted into nude mice. In contrast to the ubiquitous presence of tenascin-X in adult skin, expression of tenascin-X protein during tumorigenesis was found to be down-regulated considerably not only in tumor cells themselves but also in tumor stroma. These findings provide evidence that the expression of tenascin-X can be influenced by stromal-epithelial interactions. We have identified glucocorticoids as physiological inhibitors of tenascin-X and suggest that glucocorticoids may in part participate in the downregulation of tenascin-X in fibroblasts in vivo.

Animals↗
PubMed2004

Porcine collagen crosslinking, degradation and its capability for fibroblast adhesion and proliferation.

Porcine dermal collagen permanently crosslinked with hexamethylene diisocyanate was investigated for its suitability as a dermal tissue engineering matrix. It was found that the chemically crosslinked collagen had far fewer free lysine groups per collagen molecule than did the uncrosslinked matrix. The ability of the matrix to support human primary fibroblast outgrowth from explants was compared for matrices that had been presoaked in various solutions, including fibroblast media, cysteine and phosphate buffered saline (PBS). It was found that superior cell outgrowth was obtained after soaking with fibroblast media and PBS. The fibroblast attachment properties of the matrix were compared against tissue culture plastic and PET. The collagen matrix showed the least amount of cell retention compared to the other to matrices, however, the general trends were similar for all three scaffolds. Longer term cultures on the collagen showed fibroblasts covering the matrix stacking up on each other and bridging natural hair follicles. However, it was also observed that the fibroblasts were not able to penetrate into the matrix structure. This was believed to result from the chemical crosslinking, as shown by the resistance of the matrix to degradation by collagenases.

Absorbable Implants↗
PubMed2006

Human cytomegalovirus infection modulates DNA base excision repair in fibroblast cells.

Regulation of DNA repair mechanisms during the viral replication cycle may have consequences for the virus with regards to genomic variability, adaptation, and replication of viral DNA. We have studied the activities and expression patterns of key enzymes involved in the first two steps of base excision repair (BER) of DNA in primary fibroblasts infected by human cytomegalovirus (HCMV). Infected cells were very proficient for removal of uracil and 5' hydrolysis of AP sites (AP endonuclease activity) as compared to the mock-infected cells, suggesting a direct role in generating free ends at uracil lesions in DNA for initiation of viral replication. Furthermore, the capacity to initiate repair of alkylated and oxidized base lesions were reduced in HCMV-infected cells, indicating increased mutation frequencies that could promote genetic variability. We hypothesize that modulation of BER activities may play an important role in HCMV pathogenesis to ensure efficient replication and genomic variation of viral DNA.

Base Sequence↗
PubMed2020

Human NK cell deficiency as a result of biallelic mutations in MCM10.

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Alleles↗
PubMed1999

Instability of the CAG repeat in immortalized fibroblast cell cultures from Huntington's disease transgenic mice.

Huntington's Disease transgenic mice were used for an exploration into the stability of a trinucleotide repeat. The brain shows heterogeneous somatic instability that increases quantitatively with age. To test somatic CAG-repeat alterations during long-term culture, DNA was extracted from transgenic tissue, primary fibroblasts, and SV40-immortalized fibroblasts at intervals of approximately 100 cell doublings. In fibroblasts derived from an adult mouse, there was an initial short truncation of the repeat, followed by an emerging population of cells showing continuous slow expansion. After 15 months in continuous culture (approximately 600 cell doublings following transformation) the major CAG peak has increased from 155 to approximately 170 triplets. This in vitro system can now be used to assay factors that affect instability.

Animals↗
PubMed2002

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence.

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.

Adult↗
PubMedDate not supplied

Liposome-mediated gene transfer into established CNS cell lines, primary glial cells, and in vivo.

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a beta-galactosidase (beta-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 microg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A "boost-lipofection" markedly increased (twice) lipofection efficiency in C6 cells. Expression of beta-GAL reached a maximum after 3-5 days. When the liposome-DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.

3T3 Cells↗
PubMed1999

Tyrosine phosphorylation of caldesmon is required for binding to the Shc.Grb2 complex.

S3-v-erbB is a retroviral oncogene that encodes a ligand-independent, transforming mutant of the epidermal growth factor receptor. This oncogene has been shown to be sarcomagenic in vivo and to transform fibroblasts in vitro. Our previous studies (McManus, M. J., Lingle, W. L., Salisbury, J. L., and Maihle, N. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11351-11356) showed that expression of S3-v-erbB in primary fibroblasts results in the tyrosine phosphorylation of caldesmon (CaD), an actin- and calmodulin-binding protein. This phosphorylation is transformation-associated, and the phosphorylated form of CaD is associated with a signaling complex consisting of Shc, Grb2, and Sos in transformed fibroblasts. To identify the tyrosine phosphorylation site(s) in the CaD molecule and to further elucidate the functional role of CaD tyrosine phosphorylation in S3-v-ErbB oncogenic signaling, we have generated a series of mutant CaDs in which one or more tyrosine residues have been replaced with phenylalanine. Using a CaD null cell line, DF1 cells (an immortalized chicken embryo fibroblast cell line), and transient transfection assays, we demonstrated that Tyr-27 and Tyr-393 are the major sites of tyrosine phosphorylation on CaD. Interestingly, Tyr-27 is located within the myosin binding domain of CaD, and Tyr-393 is adjacent to one of the major actin binding and actomyosin ATPase inhibitory domains. Our studies also show that the tyrosine phosphorylation of CaD enhances its binding to the Shc.Grb2 complex. Specifically, replacement of Tyr-27, but not of Tyr-165 or Tyr-393, significantly reduces the ability of CaD to interact with the Shc. Grb2 complex. Together, these studies demonstrate that the major sites of tyrosine phosphorylation on CaD are located in the myosin and actin binding domains of CaD and that Tyr-27 is the major tyrosine phosphorylation site through which CaD interacts with the Shc.Grb2 complex.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Aberrant dysferlin trafficking in cells lacking caveolin or expressing dystrophy mutants of caveolin-3.

Mutations in the dysferlin (DYSF) and caveolin-3 (CAV3) genes are associated with muscle disease. Dysferlin is mislocalized, by an unknown mechanism, in muscle from patients with mutations in caveolin-3 (Cav-3). To examine the link between Cav-3 mutations and dysferlin mistargeting, we studied their localization at high resolution in muscle fibers, in a model muscle cell line, and upon heterologous expression of dysferlin in muscle cell lines and in wild-type or caveolin-null fibroblasts. Dysferlin shows only partial overlap with Cav-3 on the surface of isolated muscle fibers but co-localizes with Cav-3 in developing transverse (T)-tubules in muscle cell lines. Heterologously expressed dystrophy-associated mutant Cav3R26Q accumulates in the Golgi complex of muscle cell lines or fibroblasts. Cav3R26Q and other Golgi-associated mutants of both Cav-3 (Cav3P104L) and Cav-1 (Cav1P132L) caused a dramatic redistribution of dysferlin to the Golgi complex. Heterologously expressed epitope-tagged dysferlin associates with the plasma membrane in primary fibroblasts and muscle cells. Transport to the cell surface is impaired in the absence of Cav-1 or Cav-3 showing that caveolins are essential for dysferlin association with the PM. These results suggest a functional role for caveolins in a novel post-Golgi trafficking pathway followed by dysferlin.

Animals↗