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PubMed2000

Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase.

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilić et al. 1998. J. Cell Biol. 143:547-560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.

Animals↗
PubMed2002

[Hypotensive effect of encapsulated genetically engineered fibroblasts expressing mutant atrial natriuretic peptide in hypertensive rats].

OBJECTIVE: To study the inhibitive effect of subcutaneous implantation of capsule filled with fibroblasts engineered to secrete the mutant human atrial natriuretic peptide (mhANP) on blood pressure in young spontaneously hypertensive rats (SHR) and to explore the feasibility of gene therapy for the treatment of hypertension. METHODS: A recombinant retroviral vector pLHY24 bearing mhANP cDNA was constructed. Primary fibroblasts derived from the skin of new born SHR were cultured and transfected with the vector pLHY24 to establish a genetically modified fibroblast line or transfected with the blank vector pLNCX. Then the two kinds of cell culture were put into specially made capsules with microholes. The capsules filled with the genetically modified allogenic fibroblasts and those with blank vector were implanted into the dorsal subcutaneous tissues of two groups of 10 young SHR respectively. The plasma ANP, blood pressure, urine volume, potassium and sodium concentrations in urine, and body weight were determined every week for 7 weeks. RESULTS: After delivery of retroviral vector bearing mhANP gene into the packaging cell PA317 and the primary fibroblasts, immunoreactive mhANP were detected in the cell culture medium at the concentration of (5.84 +/- 0.07) and (13.37 +/- 2.36) ng.10(-6) cells.24 h(-1) respectively. One week after implantation of the genetically modified allogenic fibroblasts the plasma level of mhANP was 131 pg/ml +/- 8 pg/ml, significantly higher than that in control group (104 pg/ml +/- 7 pg/ml, t = 8.62, P < 0.001). Although the blood pressure increased along with aging after the gene transfer, an obvious delay of blood pressure increase was seen significantly lower in test group [from (129 +/- 9) to (169 +/- 9) mm Hg] than that in the control group [from (145 +/- 10) to (181 +/- 9) mm Hg, P < 0.05 or 0.01]. A maximal blood pressure reduction of 28 mm Hg in young SHR was observed 7 days after transplantation as compared with controls. In addition, there was an obvious increase in urine volume of test group 2 weeks after transplantation and the effect lasted for more than 2 weeks. However, there were no statistical differences in body weight and the concentrations of K(+) and Na(+) in urine. CONCLUSION: Subcutaneous implantation of the encapsulated genetically modified fibroblasts engineered to secrete mutant ANP causes a lowering effect of blood pressure in young SHR.

Animals↗
PubMed1999

A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency.

Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCDelta97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, the UL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.

Animals↗
PubMedDate not supplied

Differential CDK-inhibitor gene expression in aging human diploid fibroblasts.

Cellular aging is accompanied by a reduction in proliferative activity and changes in gene expression. To further elucidate the mRNA phenotype of aging fibroblasts we have monitored the expression of an array of genes implicated in regulating cell-cycle progression. Fourteen genes, including 3 cyclin-dependent kinase (CDK) inhibitors (p16INK4, p21SDI/CIP/WAF and p27KIP), 5 cyclins, 4 CDKs, Cdi-1, and PCNA were tested in four primary fibroblast strains. Relative mRNA expression levels were assessed using a rapid and sensitive Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay called the "Primer-dropping" method. p16INK4, a specific inhibitor of the cyclin D-associated kinases CDK4 and CDK6, was, in addition to p21 and cyclin D1, overexpressed in higher passage cells, while the abundance of the D-type kinase mRNAs remained relatively constant. Levels of cyclin H, a component of the CDK-activating kinase (CAK) were markedly reduced in all strains examined, suggesting that the activity of target cyclin/CDK complexes may not be activated in aging cells. These results corroborate and extend previous observations demonstrating elevated expression of specific cell cycle genes in higher passage cells and suggest that overexpression of the CDK-inhibitors p16INK4 and p21SDI/CIP/WAF, but not p27KIP, may contribute to lower proliferative activity of senescing primary fibroblasts.

Base Sequence↗
PubMed1977

Diagnosis of Pompe's disease in cultured skin fibroblasts and primary amniotic fluid cells using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate.

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.

Amniotic Fluid↗
PubMed1975

Separation of human epidermal cells from fibroblasts in primary skin culture.

To obtain pure culture of epidermal cells from small human biopsies, two different techniques were tested and compared, i.e. separation of epidermis from corium before cultivation by trypsin and suction, and after cultivation by trypsin and collagenase. The most active growth of epidermal cells was obtained by the third technique, since short-term trypsin treatment released only fibroblasts from the culture. Crude collagenase (type I) was less effective than trypsin. Collagenase type II, III and IV had no effect on fibroblast release. Neither trypsin nor collagenases dispersed epidermal cells.

Biopsy↗
PubMed2006

Induction of TIMP-1 and HSP47 synthesis in primary keloid fibroblasts by exogenous nitric oxide.

BACKGROUND: The excessive accumulation of extracellular matrix is a hallmark of many fibrotic diseases, including the hypertrophic scar and keloid. Recent reports from this research team had shown that exogenous nitric oxide (NO) participates in the keloid formation; however, its role on the synthesis of fibrotic factor (TGF-beta1, TIMP-1 and HSP47) in the keloid fibroblasts (KF) remained unclear. OBJECTIVE: In this study, to better define the potential effect of exogenous NO on the expression of fibrotic factors in KF, the enhancing effect of exogenous NO, released from a NO donor, on the synthesis of fibrotic factors in KF was investigated. METHODS: The seven primary KF cultures were set up to measure the effect of exogenous NO on enhancing the expression of fibrotic factor. RESULTS: Elevation of cellular cGMP levels was observed to be induced by NO or blocked by the hydrolysis activity of phosphodiesterase (PDE) by the PDE inhibitor. The elevated levels of cellular cGMP were noted to enhance the expression of TIMP-1 and HSP47 in KF. Exogenous NO was found to significantly accelerate the production of TIMP-1 and HSP47 in the seven primary KFs with a corresponding increase in the production of TGF-beta1. CONCLUSION: The results have led to a conclusion, that is: the excess collagen formations in the keloid lesion may be attributed to the NO/cGMP signal pathway by initiating a rapid increase in the expression of TGF-beta1, TIMP-1 and HSP47 in the KF cells.

Adult↗
PubMed2002

Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome).

AIMS: Hunter syndrome is a rare X-linked lysosomal storage disorder caused by the deficiency of the housekeeping enzyme iduronate-2-sulphatase (IDS). Deficiency of IDS causes accumulation of undegraded dermatan and heparan-sulphate in various tissues and organs. Approaches have been proposed for the symptomatic therapy of the disease, including bone marrow transplantation and, very recently, enzyme replacement. To date, gene therapy strategies have considered mainly retroviral and adenoviral transduction of the correct cDNA. In this paper, two non-viral somatic gene therapy approaches are proposed: encapsulated heterologous cells and muscle electro-gene transfer (EGT). METHODS: Hunter primary fibroblasts were co-cultured with either cell clones over-expressing the lacking enzyme or with the same incorporated in alginate microcapsules. For EGT, plasmid vector was injected into mouse quadriceps muscle, which was then immediately electro-stimulated. RESULTS: Co-culturing Hunter primary fibroblasts with cells over-expressing IDS resulted in a three- to fourfold increase in fibroblast enzyme activity with respect to control cells. Fibroblast IDS activity was also increased after co-culture with encapsulated cells. EGT was able to transduce genes in mouse muscle, resulting in at least a tenfold increase in IDS activity 1-5 weeks after treatment. CONCLUSION: Although preliminary, results from encapsulated heterologous cell clones and muscle EGT encourage further evaluations for possible application to gene therapy for Hunter syndrome.

Animals↗
PubMed1997

Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells.

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae-induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.

Bacterial Proteins↗
PubMed2000

Ras-independent oncogenic transformation by an EGF-receptor mutant.

Mutations in the ligand-binding domain of the epidermal growth factor receptor have been identified in several types of human cancers, including malignant gliomas. These mutations render signaling by this receptor to be constitutively ligand-independent. In fibroblasts transformed with ligand-independent epidermal growth factor receptor mutants, there is a correlation between the formation of a unique phosphotyrosine protein complex and oncogenic transformation. This phosphoprotein complex includes Grb2, Shc, Sos, tyrosine-phosphorylated form of caldesmon, and two, as yet, unidentified proteins. The presence of Grb2, Shc, and Sos in this complex implicates Ras in ligand-independent signaling by these oncogenic epidermal growth factor receptor mutants. We, therefore, have used retroviral co-infections of cultured primary fibroblasts to determine if Ras activation is required for phosphoprotein complex formation, stress fiber loss, or transformation. As predicted, expression of a dominant-negative Ras mutant (N17Ras) completely abrogates ligand-stimulated soft agar colony growth of primary fibroblasts. In contrast, N17Ras expression has no effect on v-ErbB mediated stress fiber disassembly, soft agar colony growth, or phosphoprotein complex assembly. In addition, our data suggest that ligand-dependent Ras activation may be suppressed by oncogenic v-ErbB expression. Together these observations suggest that oncogenic signaling by v-ErbB does not require Ras activation, and implicate an alternative signal transduction pathway in ligand-independent epidermal growth factor receptor oncogenic signaling.

Animals↗
PubMed2004

Interventional magnetic resonance imaging for guiding gene and cell transfer in the heart.

BACKGROUND: Interventional magnetic resonance imaging (iMRI) has the potential for guiding interventional cardiac procedures in real time. OBJECTIVES: To test the feasibility of iMRI guided gene and cell transfer to the heart and to monitor myocardial remodelling after myocardial infarction in a rat model. METHODS: The MRI contrast agent GdDTPA, together with either Evans blue dye, or a recombinant adenovirus encoding the LacZ gene, or primary fibroblasts tagged by BrdU, were injected into the myocardium of rats under iMRI guidance. Rats were killed seven days after the injection and the hearts sectioned to identify the blue dye, LacZ expression, or fibroblast presence, respectively. In a parallel study, left ventricular area was measured before and after myocardial infarction and in sham operated rats by T1 weighted MRI and by echocardiography. RESULTS: Location of GdDTPA enhancement observed with iMRI at the time of injection was correlated with Evans blue stain, beta-gal expression, and the primary fibroblast location in histological studies. iMRI and echocardiography measured a comparable increase in left ventricular area at seven and 30 days after myocardial infarction. A good correlation was found between the iMRI and echocardiographic assessment of left ventricular area (r = 0.70; p < 0.0001) and change in left ventricular area with time (r = 0.75; p < 0.0001). CONCLUSIONS: The results show the feasibility and efficiency of iMRI guided intramyocardial injections, and the ability to monitor heart remodelling using iMRI. Genes, proteins, or cells for tissue engineering could be injected accurately into the myocardial scar under iMRI guidance.

Adenoviridae↗
PubMed2007

UV-induced apoptosis in XPG-deficient fibroblasts involves activation of CD95 and caspases but not p53.

Mildly affected individuals from xeroderma pigmentosum complementation group G (XP-G) possess single amino acid substitutions in the XPG protein that adversely affects its 3' endonuclease function in nucleotide excision repair. More serious mutations in the XPG gene generate truncated or unstable XPG proteins and result in a particularly early and severe form of the combined XP/CS complex. Following UV irradiation, cells from such XP-G/CS patients enter apoptosis more readily than other DNA repair-deficient cells. Here, we explore the mechanisms by which UV triggers the apoptotic cell death program in XP-G and XP-G/CS primary fibroblasts. Activation of the CD95 signalling pathway occurs within minutes and it is the earliest detectable post-UV event in such cells. This is rapidly followed by activation of caspase-8 then caspase-3. Several hours later caspase-9 becomes activated and the mitochondrial membrane potential drops, but without any obvious prior release of cytochrome c. Although p53 accumulates in XPG-deficient cells after UV irradiation, use of RNA interference demonstrates that p53 is not required for their UV-induced apoptotic response. p53 ablation of wild-type fibroblasts reduces MDM2 mRNA levels, inhibits accumulation of the 90kDa/92kDa Mdm2 isoforms, and prevents the nuclear relocalisation of Mdm2 after UV treatment. The same post-UV effects occur in XPG-deficient cells that express normal p53 levels. These results emphasise the importance of the extrinsic apoptotic pathway and aberrant Mdm2 events for the severe UV-induced apoptosis of XPG-deficient primary fibroblasts. XP-G/CS cells constitutively overexpress the pro-apoptotic Bax protein and a long isoform of the E2F1 transcription factor that controls S phase entry, which may prime them to enter apoptosis very readily after UV irradiation.

Apoptosis↗
PubMed2001

p38 mitogen-activated kinase is a bidirectional regulator of human fibroblast collagenase-1 induction by three-dimensional collagen lattices.

When fibroblasts are cultured in contracting collagen matrices, matrix metalloproteinase-1 (MMP-1, collagenase-1) is induced. In the present study we demonstrate that p38alpha mitogen-activated protein kinase (p38alpha MAPK) plays a bi-directional role in the MMP-1 response to contracting floating collagen lattices (fl-coll). fl-coll, but not attached collagen lattices (att-coll), co-ordinately increased expression of MMP-1 and activities of p38alpha and MKK3/6 (MAPK kinase 3/6). However, treatment of primary fibroblasts cultured in fl-coll with increasing doses of SB203580, an inhibitor of p38alpha and p38beta, caused a bipolar pattern of MMP-1 expression. Partial inhibition of p38 MAPK activity resulted in the lowest level of MMP-1 expression, whereas total inhibition of p38 activity led to MMP-1 levels as high as in the absence of inhibitor. The activation/inhibition of p38alpha was apparently responsible for the observed phenomena, as supported by three lines of evidence. (1) p38alpha was the predominant isoform sensitive to SB203580 in primary fibroblasts. (2) Fibroblasts transfected with increasing dose of a dominant negative p38alpha (p38DN) similarly demonstrated the bipolar pattern of MMP-1 expression induced by fl-coll. (3) The bipolar MMP-1 expression occurred during the gradual, linear inhibition of p38alpha kinase activity by both inhibitors, SB203580 and p38DN. Nuclear factor-kappaB (NF-kappaB), a previously identified positive regulator of MMP-1 expression induced by fl-coll [Xu, Zutter, Santoro and Clark (1998) J. Cell Biol. 140, 709-719] was mediated by fl-coll-activated p38alpha. However, the fl-coll-induced expression of MMP-1 facilitated by p38alpha suppression was maintained independent of NF-kappaB activity, suggesting the existence of a p38alpha-dependent antagonistic pathway. We conclude that fl-coll-induced MMP-1 expression is the net outcome of opposing effects mediated by p38alpha. Therefore, the level of p38alpha kinase activity may provide a fine-tuned control of MMP-1 gene expression in response to biomechanical signals.

Adult↗
PubMed2001

NF-kappaB-inducing kinase is dispensable for activation of NF-kappaB in inflammatory settings but essential for lymphotoxin beta receptor activation of NF-kappaB in primary human fibroblasts.

The transcription factor NF-kappaB is of major importance in the biology of pro-inflammatory cytokines, such as TNF-alpha and IL-1alpha, and thereby is intimately involved in the process of inflammation. Understanding the mechanisms by which NF-kappaB is activated in response to inflammatory stimuli has become a major goal of inflammation research. The discovery of NF-kappaB-inducing kinase (NIK) as a TNFR-associated factor-interacting enzyme and a potential activator of the IkappaBalpha-kinase complex appeared to have identified an important element of the NF-kappaB activation pathway, a view that was supported by several subsequent studies. However, recent experiments in the alymphoplasia (aly/aly) mouse, which has missense point mutation (G885R) in NIK, has challenged that view. The reasons for the discrepancy between the different studies is unclear and could be due to multiple factors, such as cell type, species of cell, or primary vs transformed cell lines. One system that has not been investigated is primary human cells. Using an adenoviral vector encoding kinase-deficient NIK, we have investigated the role of NIK in LPS, IL-1, TNF-alpha, and lymphotoxin (LT) betaR signaling in primary human cells and TNF-alpha expression from rheumatoid tissue. These data show that, in the primary systems tested, NIK has a restricted role in LTbetaR signaling and is not required by the other stimuli tested. Also, there is no apparent role for NIK in the process of TNF-alpha production in human rheumatoid arthritis. These data also highlight the potential problems in extrapolating the function of signaling pathways between primary and transfected cell lines.

Adenoviridae↗
PubMed2002

Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells.

Defects in chromosome segregation play a critical role in producing genomic instability and aneuploidy, which are associated with congenital diseases and carcinogenesis. We recently provided evidence from immunofluorescence and electron microscopy studies that merotelic kinetochore orientation is a major mechanism for lagging chromosomes during mitosis in PtK1 cells. Here we investigate whether human primary fibroblasts exhibit similar errors in chromosome segregation and if at least part of lagging chromosomes may arise in cells entering anaphase in the presence of mono-oriented chromosomes. By using in situ hybridization with alphoid probes to chromosome 7 and 11 we showed that loss of a single sister is much more frequent than loss of both sisters from the same chromosome in anatelophases from human primary fibroblasts released from a nocodazole-induced mitotic arrest, as predicted from merotelic orientation of single kinetochores. Furthermore, the lagging of pairs of separated sisters was higher than expected from random chance indicating that merotelic orientation of one sister may promote merotelic orientation of the other. Kinetochores of lagging chromosomes in anaphase human cells were found to be devoid of the mitotic checkpoint phosphoepitopes recognized by the 3F3/2 antibody, suggesting that they attached kinetochore microtubules prior to anaphase onset. Live cell imaging of H2B histone-GFP-transfected cells showed that cells with mono-oriented chromosomes never enter anaphase and that lagging chromosomes appear during anaphase after chromosome alignment occurs during metaphase. Thus, our results demonstrate that the mitotic checkpoint efficiently prevents the possible aneuploid burden due to mono-oriented chromosomes and that merotelic kinetochore orientation is a major limitation for accurate chromosome segregation and a potentially important mechanism of aneuploidy in human cells.

Cells, Cultured↗
PubMed1990

Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation.

The human insulinlike growth factor I receptor was overexpressed in NIH 3T3 cells as well as human and rat primary fibroblast strains. The NIH 3T3 cells displayed a ligand-dependent, highly transformed phenotype. When exposed to insulinlike growth factor I or supraphysiologic levels of insulin, NIH 3T3 cells that expressed high levels of receptors formed aggregates in tissue culture dishes, colonies in soft agar, and tumors in nude mice. Expression of 1 million receptors per cell, a 40-fold increase above the base-line level, was required for anchorage-independent growth. Primary fibroblasts that expressed high levels of receptors displayed a ligand-dependent change in morphology and an increase in saturation density but did not acquire a fully transformed phenotype. The results demonstrate that when amplified, this ubiquitous growth factor receptor behaves like an oncogenic protein and is capable of promoting neoplastic growth in vivo.

Animals↗
PubMed1997

Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein.

The tumour suppressor protein RB restricts cellular growth. This may involve inhibiting the synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III. We have shown previously that RB can repress pol III transcription when overexpressed either in vitro or in vivo. We also demonstrated that pol III activity is elevated substantially in primary fibroblasts from RB-deficient mice. Here we address the molecular mechanism of this regulation. RB is shown to repress all types of pol III promoter. It can do this even if added after transcription complex assembly. Functional assays demonstrate that RB targets specifically the general pol III factor TFIIIB. A physical interaction between TFIIIB and RB is indicated by fractionation, pull-down and immunoprecipitation data. We show that TFIIIB activity is elevated in primary fibroblasts from RB-deficient mice. TFIIIB is a multisubunit complex that includes the TATA-binding protein (TBP) and a TFIIB-related factor called BRF. We show that RB itself contains regions of homology to both TBP and BRF and propose a model in which RB disrupts TFIIIB by mimicking these two components.

Amino Acid Sequence↗