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Biomedical subjects

C A Lambert

Publications and source records attributed to C A Lambert.

At least 19 recordsLinked to original sources
PubMed2006

Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases.

Small GTP-binding proteins of the Rho family (RhoA, Cdc42, Rac1) regulate the organisation and the turnover of the cell's cytoskeleton and adhesion structures. A significant function of these cellular structures is to translate and counterbalance forces applied to, or generated by, cells in order to maintain homeostasis and control cell movement. We therefore hypothesised that Rho-GTPases are directly involved in cellular gravity perception and may participate in the alterations induced in microgravity. To define an adequate cellular model allowing to investigate this issue, we have established stable cell lines constitutively expressing active forms of either RhoA, Cdc42, or Rac1. The three cell lines differ by morphology and by their ability to form filopodia, lamellipodia, and bundles of actin stress fibers. Overexpression of the active form of either RhoA, Cdc42, or Rac1 is compatible with cell viability and does not affect cell population doubling time. Thus, our series of mutant cells appear well suited to gain further knowledge on the molecular mechanisms of cellular gravity perception.

Actin Cytoskeleton↗
PubMed2006

Effects of constitutively active GTPases on fibroblast behavior.

The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing.

Actins↗
PubMed2006

The in vitro influences of neurotensin on the motility characteristics of human U373 glioblastoma cells.

Astrocytic tumours are associated with dismal prognoses due to their pronounced ability to diffusely invade the brain parenchyma. Various neuropeptides, including gastrin, are able to modulate tumour astrocyte migration. While neurotensin has been shown to influence the proliferation of glioma cells and the migratory ability of a large set of other cell types, its role in glioma cell migration has never been investigated. Neurotensin-induced modifications to the motility features of human U373 glioblastoma cells therefore constitute the topic of the present study. We evidenced that three subtypes of neurotensin receptors (NTR1, NTR2 and NTR3) are expressed in U373 glioblastoma cells, at least as far as their mRNAs are concerned. Treating U373 tumour cells with 10 nM neurotensin markedly modified the morphological patterns of these cells and also profoundly altered the organization of their actin cytoskeletons. Pull-down assays revealed that neurotensin induced the activation in U373 cells of both Rac1 and Cdc42 but not RhoA. Scratch wound assays evidenced that neurotensin (0.1 and 10 nM) very significantly inhibited wound colonization by U373 cells cultured in the absence of serum. In addition, quantitative phase-contrast videomicroscopy analyses showed that neurotensin decreases the motility levels of U373 glioblastoma cells when these cells are cultured on plastic. In sharp contrast, neurotensin stimulates the motility of U373 cells when they are cultured on laminin, which is a pro-adhesive extracellular matrix component ubiquitously secreted by glioma cells. Our data thus strongly suggest that, in addition to gastrin, neurotensin is a neuropeptide capable of modulating tumour astrocyte migration into the brain parenchyma.

Actins↗
PubMed2001

Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.

The aim of the work was to analyze, on a comparative basis, the signaling pathways operating in the regulation of a panel of matrix metalloproteinases (MMP) expressed by human dermal fibroblasts submitted to mechanical stress relaxation by cytochalasin D (CD) and in a retracting collagen gel (RCG). The mRNA steady-state level of MMPs was measured by a quantitative RT-PCR procedure using a synthetic RNA as internal standard. In monolayer, most MMPs were barely detected, except MMP-2. Disruption of the actin stress fibers by CD induced a moderate increase of MMP-2 mRNA and a much larger stimulation of MMP-3, -9, -13 and -14 mRNAs. In RCG, a significant up-regulation of these MMPs was also observed although to a lower extent than in CD-treated monolayers. Among the investigated MMPs, the MMP-8 and -11 were not reproducibly detected. MMP-2 was processed to its active form both by CD and in RCG. The CD-induced up-regulation of gene expression was largely repressed by blocking protein synthesis by cycloheximide for all the MMPs, by inhibiting the tyrosine-kinases of the src family by herbimycin A for all MMPs, except MMP-2, and by inhibiting the TPA-inducible PKC isoforms by bisindoyl maleimide for all MMPs, except MMP-14. The up-regulation induced by stress relaxation in RCG was protein synthesis-dependent for MMP-2 and MMP-13, tyrosine kinases-dependent for MMP-3 and MMP-13, as previously described for MMP-1. Inhibiting TPA-inducible PKC did not affect any MMP in RCG except MMP-13, which was strongly induced. The processing of MMP-2 was tyrosine kinases-dependent but PKC-independent. Inhibitors of the ERK1,2 and p38 MAP kinases pathways diversely affected the MMPs expression. Inhibiting the Rho-kinase activity by Y-27632 was inactive. These results point to the potent regulation operated by the status of the cytoskeleton on the cell phenotype, and to distinct regulatory pathways involved in the control of different MMPs expression.

Animals↗
PubMed2001

Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxy- and amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.

Administration, Topical↗
PubMed2001

Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts.

Mechanical tension governs fibroblast proliferation and survival and the homeostasis of the extracellular matrix to adapt its resistance to the mechanical requirements of the organs. To consolidate this view, we analysed the effect of tension release on the expression of molecules involved in the architecture and stabilisation of the collagen fibres, namely the procollagens type I and III, the amino- and carboxy-procollagen peptidases (N-pCP and C-pCP) and lysyl oxidase. Cells were cultured in conditions of high mechanical stress in monolayer on a collagen coat and under reduced tension by disruption of the cytoskeleton upon treatment with cytochalasin D in monolayer on a collagen coat or by integrin-mediated stress relaxation in a freely retracting collagen gel. The mRNAs were measured by quantitative RT-PCR monitored by simultaneous reverse-transcription and amplification of an original internal standard. Tension relaxation resulted in a decreased expression of the procollagens type I and III, of the two expressed forms of C-pCP, of the two forms of N-pCP and of lysyl oxidase. Type III collagen, known to control diameter of the fibres, was less down-regulated than type I collagen. Interestingly, the expression of the two alternatively spliced forms of the N-pCP was dissimilarly regulated. These data suggest that mechanical tension may modulate the stiffness of the extracellular matrix by controlling not only the level of expression of its fibrillar constituents but also that of the enzymes participating in their extracellular processing and mechanical stabilisation.

Adolescent↗
PubMed1998

Decreased protein levels of the c-Cbl protooncogene in murine AIDS.

Murine acquired immunodeficiency syndrome (MAIDS) can be viewed as a lymphoproliferative disease which involves B cells as well as T cells from spleen and lymph nodes while thymus and Peyer's patches do not participate in the process. The 120-kDa protooncogene product c-Cbl was initially cloned from the murine Cas NS-1 B cell lymphoma. It is a main target of immunoreceptor (TCR and BCR)-mediated protein tyrosine kinase activity. Moreover, recent data suggest that c-Cbl might play a crucial role in the regulation of cell proliferation through regulation of GTP-binding proteins. Therefore, the involvement of c-Cbl was evaluated in the lymphoproliferative disease induced by the MAIDS virus. The expression of the c-Cbl protein was dramatically reduced in the lymph node of infected mice while it remained normal in the thymus. In contrast, the expression of actin, TCR-zeta chain, ZAP-70, and p59(fyn) remained similar in controls and infected mice. Identical results were obtained with sorted B cells and T cells. Surprisingly, a B cell lymphoma line derived from late stage MAIDS mice displayed a normal level of c-Cbl.

Animals↗
PubMed1998

An interleukin-1 loop is induced in human skin fibroblasts upon stress relaxation in a three-dimensional collagen gel but is not involved in the up-regulation of matrix metalloproteinase 1.

The integrin-mediated stress relaxation as it occurs in a retracting three-dimensional collagen gel (RCG) is accompanied by a large up-regulation of the interstitial collagenase, matrix metalloproteinase 1 ((MMP-1), EC 3.4.24.7), regulated notably by interleukin-1 (IL-1), phorbol esters, and cytoskeleton-disrupting drugs as cytochalasin D (CD). The repression of MMP-1 up-regulation in RCG by cycloheximide suggested the participation in the regulation process of a de novo synthesized intermediary component. We demonstrate here that culture of human skin fibroblasts in RCG or in CD- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated monolayers resulted in the activation of an IL-1 autocrine feedback loop that was switched off by the naturally occurring IL-1 receptor antagonist (IL-1RA), a blocker of the common IL-1 receptor. The IL-1RA did not suppress the MMP-1 up-regulation induced in RCG nor in CD-treated cells, indicating that the up-regulation of MMP-1 and the IL-1 autocrine loop occurred in an independent way, while the TPA-induced MMP-1 expression was suppressed by the receptor antagonist. The RCG- as well as the TPA-, IL-1-, and CD-induced up-regulation of both MMP-1 and IL-1 was totally suppressed by protein tyrosine kinases inhibitors. In contrast bisindoylmaleimide, at a concentration (5 microM) that inhibits the TPA-induced protein kinase C activity, suppressed the CD-induced MMP-1 expression but did not or barely altered that induced in RCG or by IL-1. None of the other tested inhibitors of a variety of signaling pathways including those used by integrins was able to suppress the RCG or CD-induced MMP-1. These results point to a potent regulation of MMP-1 by mechanical stress relaxation, a process depending on de novo protein synthesis and occurring independently of the activation of an IL-1 autocrine feedback loop.

Calmodulin↗
PubMed1998

Endothelial cell intracellular Ca2+ concentration is increased upon breast tumor cell contact and mediates tumor cell transendothelial migration.

Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca2+ concentration ([Ca2+]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca2+]i. This [Ca2+]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca2+ stores by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca2+]i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca2+ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca2+]i may govern multiple steps of tumor cell extravasation.

Adenocarcinoma↗
PubMed1998

Reciprocal regulation of protein tyrosine kinases p56lck and p59fyn, and altered tyrosine phosphorylation in murine AIDS.

Murine AIDS (MAIDS), caused by a defective murine leukemia virus, is a severe lymphoproliferative disease associated with profound immunodeficiency and increased susceptibility to opportunistic infections. Most subsets of lymphocytes, including CD4+ and CD8+ T cells, are refractory to mitogen stimulation. As a first step to examine proximal signal transduction in the infected mice, Western and Northern blot analyses were performed, and showed that p56lck is dramatically decreased at the protein as well as the mRNA level in the lymph nodes (LN). In contrast, p59(fyn) and its mRNA were slightly increased in the LN of the same mice. Similar results were obtained with purified T cells. Interestingly, the thymus of the infected animals did not show any abnormality regarding p56(lck) or p59(fyn). Tyrosine phosphorylation was constitutively increased in the infected mice and was barely amplified by anti-CD3 mAb stimulation. A similar pattern was observed when tyrosine phosphorylation was selectively examined at the level of ZAP-70. Our results suggest that a reciprocal regulation of p56(lck) and p59(fyn) protein tyrosine kinases, previously described in various models of anergy, could also be involved in the pathogenesis of MAIDS.

Animals↗
PubMed1996

[Low intensity electromagnetic fields produce a wave of calcium in the fibroblasts].

Human fibroblasts display a Ca2+ wave after irradiation with an electromagnetic field (EMF) of low intensity (100 to 900 microT) as seen by LASER confocal microscopy and excitation of Fluo 3. The number of excited cells is proportional to the intensity of EMF between 100 and 900 microT. Cellular activation by a dialysable serum factor is required to induce the Ca2+ wave. It also depends on extracellular Ca2+ and active tyrosine kinases and phospholipase C gamma.

Calcium↗
PubMed1993

Modulation of expression of endogenous collagenase and collagen genes by electroporation: possible involvement of Ca2+ and protein kinase C.

We have investigated the effect of electroporation on the expression of collagen alpha 1(I), collagenase, c-fos and c-jun genes in human dermal fibroblasts (HDF), human smooth muscle cells (HSMC) and HeLa cells. Collagenase and collagen mRNA levels were respectively increased and decreased in a voltage-dependent manner in HDF harvested 2 days after a sham electroporation. These effects were still observed 10 days after electroporation. Similar effects occurred in electroporated HSMC. Neither collagen nor collagenase mRNAs were detected in control or electroporated HeLa cells. c-fos and c-jun mRNA levels were also increased in electroporated HDF, HSMC and HeLa cells harvested 1 h after plating. This suggests that factor AP1 (fos/jun) could mediate the up-regulation of collagenase expression in electroporated HDF and HSMC. When electroporation of HDF was performed in the presence of H7, an inhibitor of protein kinase C, no increase in collagenase mRNA level was observed, suggesting that protein kinase C might be involved in the transduction of the effect. All the effects reported were also suppressed when cells were electroporated in a medium containing EGTA, suggesting that Ca2+ might mediate the transduction of this effect.

Aluminum↗
PubMed1992

Effect of cell-cell and cell-matrix interactions on the response of fibroblasts to epidermal growth factor in vitro. Expression of collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases.

Investigations of the effect of epidermal growth factor (EGF) on the expression of four genes involved in the turnover of the extracellular matrix, collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases (TIMP) were performed on four strains of skin fibroblasts in vitro. Addition of EGF to subconfluent cultures for increasing periods of time up to 5 days induced an inhibition of procollagen alpha 1(I) mRNA and a strong stimulation of collagenase (100-fold) and stromelysin (1000-fold) mRNAs, whereas the mRNA of TIMP was increased to a lesser extent (5-fold). After a 40 h pulse with EGF, these effects persisted for 24-48 h after withdrawal of the growth factor and slowly diminished thereafter to attain control values after several days. By culturing fibroblasts for increasing periods of time, different levels of confluence were obtained allowing for the deposition of an extracellular biomatrix. The steady-state level of collagenase and stromelysin mRNAs were profoundly depressed in confluent as against non-confluent cultures, whereas no major change for TIMP and procollagen alpha 1(I) mRNAs was observed. Upon treatment of these cultures with EGF for 48h, the steady-state level of collagenase, stromelysin and TIMP increased, whereas procollagen alpha 1(I) mRNA was slightly reduced. These modifications were, at least in part, dependent upon a regulation of the transcription rate, as suggested from run-off experiments. Similar states of confluence were obtained by seeding cells at increasing densities in short-term cultures in which cell-cell contact predominated. In such culture conditions, the collagenase and stromelysin mRNAs were enhanced in high as compared to low density cultures. The response to EGF was progressively decreased for collagenase, stromelysin and, to a lesser extent, TIMP mRNAs at most densities and a complete lack of response to EGF at the highest cell density was observed. Under all culture conditions the modulation of collagenase mRNA was paralleled by similar modifications of enzyme activity. These results emphasize the importance of the cell-cell contacts and cell-matrix interactions in the expression of the genes coding for metalloproteinases or their inhibitor and their modulation by growth factors.

Cell Communication↗
PubMed1992

Modulation of collagen and fibronectin synthesis in fibroblasts by normal and malignant cells.

The influence of various normal and malignant human cells on the level of collagen synthesis by human fibroblasts was tested in coculture. As revealed by immunoperoxidase staining, in cocultures with breast adenocarcinoma cells (MCF7, SA52, T47D) fibroblasts synthesized collagen while tumor cells did not. Fibroblasts displayed increased collagen production without change in the overall protein synthesis. Several other types of cells derived from normal human tissues (keratinocytes, normal mammary cells) or from fibrosarcoma, melanoma, cervical carcinoma, choriocarcinoma, or other breast adenocarcinoma (SW613, MDA, BT20) did not affect collagen synthesis of fibroblasts. Although to a lesser extent, this stimulating effect was reproduced by using the conditioned medium (CM) of the active cells but not with CM of the other cell types. A slight stimulation was also obtained when tumoral MCF7 cells and fibroblasts shared the same medium but were physically separated, suggesting that close contact was required for optimal stimulation of collagen synthesis. The collagen synthesis stimulating activity was not related to a modification of fibroblast proliferation rate. The production of collagen types I, III, and VI and fibronectin were increased in cocultures of fibroblasts with MCF7 cells. The increased synthesis of collagen types I and III and fibronectin was paralleled by similar changes in the steady-state level of their mRNAs. On the contrary, the increased production of collagen type VI appeared regulated at a post-transcriptional level.

Adenocarcinoma↗
PubMed1992

Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces.

In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.

Actins↗