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M Serres

Publications and source records attributed to M Serres.

16 recordsLinked to original sources
PubMed2003

Withdrawal of TNF-alpha after the fifth day of differentiation of CD34+ cord blood progenitors generates a homogeneous population of Langerhans cells and delays their maturation.

Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) generate a heterogeneous population of dendritic cells (DC), including Langerhans cells (LC). This combination of cytokines has been shown to be crucial for differentiation into LC. After day 5 of culture, TNF-alpha has been maintained in the medium in most studies despite the observation of spontaneous maturation of LC after day 12. Five-day samples of in vitro differentiated LC were cultured in parallel with or without TNF-alpha. The absence of TNF-alpha was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogate the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-alpha abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-alpha-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-alpha. These data indicate that the suppression of TNF-alpha after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.

Antigens, CD34↗
PubMed2000

The disruption of adherens junctions is associated with a decrease of E-cadherin phosphorylation by protein kinase CK2.

The down-regulation of E-cadherin is a common event in carcinogenesis. Phosphorylation/dephosphorylation is one posttranscriptional process which may regulate intercellular junctions. Here we show that in okadaic acid-treated keratinocytes, E-cadherin expression is shifted from the membrane to the cytoplasm, preventing cells from forming aggregates. These changes of E-cadherin localization and function are associated with a decrease in its phosphorylation state. The decrease in E-cadherin phosphorylation was essentially detected in okadaic acid-treated cell lysates isolated from 0.5% Triton-soluble fraction and not in the Triton-insoluble fraction linked to the cytoskeleton, suggesting a role of E-cadherin phosphorylation in cell-cell interactions. E-cadherin was markedly phosphorylated by CK2, either the purified recombinant enzyme or the endogenous enzyme. Using specific CK2 inhibitors such as heparin and 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, endogenous CK2 was confirmed as the main enzyme phosphorylating E-cadherin. The decrease in E-cadherin phosphorylation by endogenous CK2 was not restored by the addition of purified CK2, confirming that it is not due to a defect in CK2 expression or to its reduced activity, but rather to the incapacity of CK2 to phosphorylate E-cadherin. The co-immunoprecipitation and colocalization of E-cadherin and CK2 suggests that CK2 may play a critical role in the maintenance of epidermis cohesion.

Cadherins↗
PubMed2000

Expression of E-, P-, n-cadherins and catenins in human bladder carcinoma cell lines.

PURPOSE: Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classical cadherins, E-, P- and N-cadherins, are known to self-associate from their extracellular domain, while their cytoplasmic domain interacts with either beta-catenin or plakoglobin (gamma-catenin), which in turn is bound to alpha-catenin that links the complex to the actin cytoskeleton. The aim of the present study was to analyze the expression of E-, P- and N-cadherins and catenins in human bladder carcinoma cells. MATERIALS AND METHODS: Five human bladder carcinoma cell lines, representing a variety of differentiation states, were grown in cell culture. We performed a cell aggregation assay, specific for biological cadherin activity. The expression of cadherins and catenins was analyzed by immunocytochemistry, Western blotting and RT-PCR. The interactions between cadherins and catenins were assessed by immunoprecipitation. RESULTS: We observed a reduced E-cadherin expression in the poorly differentiated and invasive-tumor derived cells. Interestingly, immunofluorescence study reveals the persistent localization of catenins at intercellular contacts in two E-cadherin deficient cell lines (T24 and TCCSUP) which yet exhibit an epithelial-like morphology and a calcium-dependent adhesive capacity. This suggests that other cadherin(s) are expressed in these both cell lines. P-cadherin, another epithelial cadherin, is expressed only in E-cadherin positive cells. On the other hand, N-cadherin is present at cell-cell borders in the very anaplastic cell lines, T24 and TCCSUP, and is able to link beta-catenin or plakoglobin. CONCLUSION: These results indicate that N-cadherin may participate in intercellular adhesion, while facilitating bladder tumorigenesis.

Actins↗
PubMed1999

Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat.

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.

Cell Adhesion↗
PubMed1998

Cell migration and MMP-9 secretion are increased by epidermal growth factor in HaCaT-ras transfected cells.

Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H-ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT-ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT-ras. However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT-ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT-ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT-ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H-ras mutation, they could be implicated in the local invasion by the HaCaT-ras clones.

Cell Line↗
PubMed1997

Hyperphosphorylation of beta-catenin on serine-threonine residues and loss of cell-cell contacts induced by calyculin A and okadaic acid in human epidermal cells.

Phosphorylation and dephosphorylation events may critically control junction assembly and stability, as well as regulate the formation of the cadherin-cytoskeleton complex, thus influencing the adhesive function of cells. In the present study, we have used specific activators and inhibitors of protein kinases and phosphatases to analyze the role of protein phosphorylation in the maintenance of epithelial architecture. Okadaic acid and calyculin A cell treatments induced two major effects: a dramatic alteration of the keratin network of epidermal cells and a complete disruption of cell-cell contacts. This loss in cell-cell contacts was not tissue and species restricted and the interactions of keratinocytes with the matrix were not involved. The observed changes were highly specific for these drugs and were obtained in the range of concentrations corresponding to the inhibition of protein phosphatase 1 (PP1). They were time- and dose-dependent, and reversible, excluding a cytotoxic effect of the drugs. A decrease in electrophoretic mobility of beta-catenin, a major protein involved in the regulation of intercellular adherens junctions, was observed in keratinocytes and fibroblasts treated with okadaic acid and calyculin A, suggesting a change in the protein phosphorylation level and/or protein conformation. Data from beta-catenin immunocomplex autoradiography performed after 32P in vivo incorporation in untreated and okadaic acid or calyculin A-treated HaCaT cells, demonstrated a higher level of phosphorylation of beta-catenin in treated cells compared to untreated ones. Analysis of 32P-labeled phosphoaminoacids demonstrated that beta-catenin was exclusively phosphorylated on serine-threonine residues but not on tyrosine residues. Immunoprecipitations and Western blotting using anti-phosphoserine and anti-phosphotyrosine antibodies confirmed these data. The change in beta-catenin phosphorylation on serine-threonine residues may play a role in the control of the cohesion between epithelial cells and may be involved in the regulation of the transduction signal.

Apoptosis↗
PubMed1996

Glucocorticoid receptor localization in human epidermal cells.

Glucocorticoids, which are widely used in therapy, exert their immunosuppressive actions through specific receptors. These receptors have been characterized in cultured human skin fibroblasts and keratinocytes, but their localization in vitro and in vivo has not been established. To determine the tissue and cellular distribution of glucocorticoid receptors (GR), two specific polyclonal rabbit anti-human GR antibodies were used to detect these receptors in skin biopsy specimens, in freshly isolated and cultured human epidermal cells and in keratinocyte cell lines. Immunoreactive GR were only faintly detected in normal and abnormal differentiated cells and as well as those in the stratum granulosum and corneocytes. These immunolocalization studies were confirmed by fluorescence cell sorter analysis of isolated basal and suprabasal keratinocytes. Immunoreactive GR were highly expressed in normal cultured human keratinocytes, Langerhans cells and several cell lines whereas they were less expressed in melanocytes. Based upon these results the main targets of glucocorticoids in the epidermis appear to be basal and Langerhans cells.

Animals↗
PubMed1994

Expression of annexin I in freshly isolated human epidermal cells and in cultured keratinocytes.

Annexin I belongs to a newly characterized family of intracellular proteins involved in the regulation of the production of inflammatory lipid mediators such as prostaglandins and leucotrienes. Annexin I (named p35, lipocortin I or calpactin II) was initially described as a protein inducible by glucocorticoids. In the skin, the role of annexins has still not been elucidated. In the study reported here we investigated the expression of annexin I both in freshly isolated epidermal cells and in cultured keratinocytes using immunofluorescence, FACS analysis and immunoblotting techniques. Using epidermal cells freshly isolated from normal skin, annexin I was detected by double immunostaining mainly in basal and suprabasal keratinocytes. Langerhans cells isolated from Ficoll gradient were faintly stained compared with keratinocytes. Annexin I was also highly expressed in keratinocytes maintained in culture in a serum-free medium without hydrocortisone. By confocal microscopy, annexin I was shown to be mainly localized in the cytoplasm of the cells. The protein was characterized by Western blot and immunoprecipitation as a 35-kDa protein in freshly isolated epidermal cells and cultured keratinocytes. Results from in vivo studies confirmed the presence of annexin I in the basal and suprabasal layers of normal human skin with modified reactivity patterns in hyperproliferative lesions.

Annexin A1↗
PubMed1994

Annexin 1 regulation in human epidermal cells.

Annexin 1 (named p35, lipocortin I or calpactin II), initially described as a glucocorticoid induced protein, belongs to a new characterized family of intracellular proteins. In the skin, the role of annexins has still not been elucidated. In a previous study, we reported the localization of annexin 1 in both freshly isolated human epidermal cells and in cultured keratinocytes using immunofluorescence, FACS analysis and immunoblotting techniques. The protein was characterized by Western blot and immunoprecipitation as a 35 kDa protein. Results from in vivo studies confirmed the presence of annexin 1 in basal and suprabasal layers of normal human skin with modified reactivity patterns in hyperproliferative lesions. In the present study, the role of glucocorticoids in annexin 1 regulation was investigated in epidermal cells by Western blot and immunoprecipation assays. In contrast to other studies, we found that glucocorticoid treatment of epidermal cells led to a decrease in annexin 1 content in the cytoplasm and the membranes of cells. As annexin 1 was not detected in the nucleus of cells, we conclude that there was a down regulation of annexin 1 after glucocorticoid treatments rather than a translocation of the protein to the nucleus. Despite the absence of the signal peptide sequence necessary for protein secretion, annexin 1 was released in the keratinocyte culture medium. We found that the protein was secreted only in low Ca2+ medium (0.15 mM), this process required an active metabolism.

Annexin A1↗
PubMed1990

Modulation of O6-methylguanine-DNA methyltransferase in rat and hamster liver after treatment with dimethylnitrosamine.

Distinct species differences exist between BDIV rats and Syrian Golden hamsters in the repair of methylated DNA lesions, after single exposures to dimethylnitrosamine (DMN). The promutagenic lesions O6-methylguanine (O6-MeG) and O4-methylthymidine were actively repaired in rat liver; in contrast, in hamster liver the levels of O6-MeG remained relatively stable while O4-methylthymidine levels were reduced. Species differences in the levels of two enzymes involved in the repair of DNA alkylation damage were also noted. An increase in the methylpurine-DNA glycosylase levels was seen in both species following DMN exposure; however, significant species differences in the inactivation and subsequent time course of recovery of the "suicide protein" O6-MeG-DNA methyltransferase were observed. In the rat a rapid recovery of activity began within 24 h of DMN exposure (20 mg/kg) and an approximately 3-fold induction in enzyme levels was observed at 96 h. In hamster liver, in which the constitutive level of expression of this enzyme is similar, no activity was detectable up to 96 h after treatment (25 mg/kg DMN). Only in animals in the lowest treatment group (2.5 mg/kg DMN) was a significant recovery seen, 264 h after treatment. The data presented suggest that the schedule of DMN treatment, in particular the time between doses of the carcinogen and the regeneration of the O6-MeG-DNA methyltransferase, would evoke different carcinogenic responses in hamster and rat liver following chronic exposure to alkylating agents.

Animals↗
PubMed1988

Detection in human cells of alkylated macromolecules attributable to exposure to nitrosamines.

Various methods for detecting DNA alkylation adducts are described briefly, with emphasis on immunoassays using antibodies against O6-methyldeoxyguanosine (O6-medGua), O4-methylthymidine (O4-meThy) and 7-methyldeoxyguanosine (7-medGua). The application of these methods to epidemiological studies is discussed, and results obtained so far on the presence of DNA alkylation adducts in human tissues are presented.

Alkylation↗
PubMed1983

Macromolecular, histological, ultrastructural and immunocytochemical characteristics of the neointima developed within PTFE vascular grafts. Experimental study in dogs.

The nature and characteristics of the tissue that develops on the inner surfaces of vascular arterial PTFE prostheses have been investigated by histological, morphometrical, ultrastructural, and immunocytochemical criteria. The ability of this tissue to synthetize glycoproteins and glycosamino-glycuronoglycans (previously called mucopolysaccharides) has been compared to that of the normal arterial wall. PTFE prostheses were used for carotid replacement in the dog and studied until the 90th postoperative day. These prostheses were mainly characterized by (1) a limited or even absent neointimal tissue proliferation; (2) the absence of an endothelial-like structure on the prosthesis using scanning electron microscopy and immunocytochemical staining with rabbit antidog factor VIII-related antigen sera; (3) limited activities of both microsomial enzymes (sialyl transferase and N-acetyl-glucosaminyl-transferase) but marked xylosyl transferase activity; and (4) inverse qualitative distribution of the glycosaminoglycans, i.e., decrease of heparan sulfate and chondroitin-6-sulfate and increase of hyaluronic acid and dermatan sulfate. The absence of morphological evidence of an endothelial structure at the blood--prosthesis interface even 3 months after implantation and the marked functional impairment in the biosynthesis of macromolecular components responsible for the normal blood--vessel interface suggest that this newly developed tissue cannot be considered a true vascular endothelium.

Animals↗
PubMed1980

Macromolecular and histological studies of new tissue formation in velour vascular prostheses.

The nature and characteristics of the tissue which develops on the inner surfaces of vascular arterial prostheses have been investigated by means of histological and biochemical criteria. Velour prosthetic tubes were implanted as aortic segmental replacements in the dog. On the 60th postoperative day, a neointimal layer of collagen covered with flattened endothelial-like cells was noted. However, despite this morphological evidence the microsomic and cytosolic enzymes implicated in the biosynthesis of glycosamino-glycuronoglycans were significantly modified in comparison with the normal aortic wall. The marked decrease in enzymes corresponding to the initiation of the glucidic linkages suggests that the biosynthesis of the macromolecular components responsible for the normal blood vaessel interfact was impaired in these newly developed tissues. Therefore, it is suggested that this newly developed tissue cannot be considered as a true vascular endothelium.

Acetylglucosamine↗
PubMedDate not supplied

The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor.

The induction of tumor angiogenesis is mediated in particular by an increased production of VEGF. As ras oncogene is implicated in tumorigenesis, the inhibition of farnesyl transferase activity has recently been developed. The purpose of this study was to evaluate whether expression of mutated Ha-ras oncogene is associated with an altered expression of VEGF in an in vitro model of human skin carcinogenesis and to appreciate the effect of a new farnesyl transferase inhibitor on this VEGF expression. The amounts of VEGF secreted by an HaCaT cell line and two cell clones (metastatic or not) obtained after mutated c-Ha-ras transfection were compared. Our findings showed that the release of VEGF is greater for HaCaT-ras than for HaCaT cells and could be down-regulated using a protein farnesyl transferase inhibitor, in a reversible and dose-dependent manner. These results confirm that the Ha-ras oncogene can contribute to tumor development and progression of epidermal tumors through neoangiogenesis and that farnesyl transferase inhibitors as anticancer drugs may be efficient for the reduction of skin tumor growth.

Alkyl and Aryl Transferases↗