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M Kedinger

Publications and source records attributed to M Kedinger.

At least 55 records · Page 3Linked to original sources

Experimental photodynamic therapy with a copper metal vapor laser in colorectal cancer.

In an attempt to define the best conditions for an adjunctive treatment of residual colonic microtumors by photodynamic therapy (PDT), an experimental model has been defined. S.c. HT29 colonic-cancer-cell tumors grown in nude mice were used and, 48 hr after i.p. administration of 30 mg/kg Photofrin (PH), laser illumination was performed with 75 or 150 Joules/cm2. The efficiencies of 2 lasers, the classically used rhodamine laser (RL) and a copper metal vapor laser (CMVL), were compared. The effects of PDT were assessed by histological and immunocytochemical (detection of a digestive enzyme, dipeptidyl-peptidase IV, as a marker of cell viability) follow-up and by the growth curve of the tumors after illumination. We conclude that, although the depth of necrosis resulting from PDT was nearly 3 mm at 75 J/cm2 and nearly 4-5 mm at 150 J/cm2 with both lasers, complete necrosis was obtained only with the CMVL at 150 J/cm2 (in 50% of the tumors). Under the other conditions, a layer of unaffected cells persisted at the pole opposite to laser illumination, resulting in growth curves lower than but parallel to those of the controls. Analysis of drug concentrations in the tumors and various organs, 48 hr after injection, i.e., at the time of laser illumination, revealed the presence of 21 micrograms/g dry weight PH in the tumors. The tumor vs. host-organ ratios were equal to or higher than 1 for the small bowel, colon, stomach, lung, skin and muscle. In contrast, the ratios were below 1 for the spleen, pancreas, kidney and liver.

Animals↗

Dual and asynchronous deposition of laminin chains at the epithelial-mesenchymal interface in the gut.

The production of laminin by 14-day fetal rat intestinal endoderm and mesenchyme was investigated. The amount of neosynthesized laminin was measured after purification using affinity chromatography. Chain composition of laminin was analyzed by immunoblotting and immunofluorescence staining. The data show that both embryonic intestinal tissue components synthesize laminin and that A and B1/B2 chains were detected in both endodermal and mesenchymal cells. The cellular source of laminin found at the epithelial basement membrane has been studied by immunocytochemistry in rat/chick or mouse/chick interspecies hybrid intestines taken at various stages of development. Immunodetection of the whole laminin molecule and of the individual A and B1/B2 chains by rodent-specific polyclonal and monoclonal antibodies at the basement membrane level in these hybrid intestines revealed (a) laminin molecules, which originate from both mesenchymal and endodermal cells; (b) deposition of A and B1/B2 chains by endodermal cells, regardless of the stage of growth of the hybrid intestines; and (c) asynchronous deposition of the various chains of laminin into the basement membrane by the mesenchyme. B1/B2 chains are deposited concomitant with contact with the epithelium, whereas A chains appear only later (13 days after grafting). These data reinforce the suggestion from previous studies that cooperation between epithelium and mesenchyme is necessary for the formation of a complete basement membrane in the developing intestine.

Animals↗

Mesenchyme-mediated effect of dexamethasone on laminin in cocultures of embryonic gut epithelial cells and mesenchyme-derived cells.

Previous studies have shown that glucocorticoids accelerate intestinal maturation and that this process is mediated by the mesenchymal cells. The possible involvement of laminin (LN), a basement membrane component, in this mesenchymal mediation has been analyzed. For this purpose, the influence of dexamethasone (DX) on the synthesis of LN, its chain composition and its cellular distribution has been examined biochemically and immunocytochemically in two different mesenchyme-derived cell populations, fetal intestinal mesenchymal cells and fetal skin fibroblasts, as well as in cocultures of intestinal endodermal cells seeded on top of confluent fetal skin fibroblasts. Neither the amount of metabolically labeled LN purified by affinity chromatography (expressed per mg cell proteins), nor the A versus B chain ratio monitored after separation on gel electrophoresis and immunoblotting, showed significant differences after 5 days of DX treatment. However, glucocorticoids induced a shift from secreted to cell-associated LN molecules paralleling a striking difference in the immunostaining pattern of intracellular and surface LN in the mesenchyme-derived cell monocultures; the granular intracytoplasmic LN staining in the control cultures was replaced by a fibrillar organization of LN molecules concomitantly with an increased accumulation at the cell surface. In 2-day DX-treated cocultures, there was an acceleration of LN deposition at the epithelial-fibroblastic interface, which accompanied the enhanced expression of epithelial cell differentiation markers (brush border digestive enzymes). These DX-induced changes can be blocked by the addition of anti-LN antibodies in the culture medium. These findings further support the concept that glucocorticoid action on intestinal epithelial cells involves alterations in the extracellular microenvironment, assessed here for LN molecules, occurring at the level of the mesenchymal cell compartment. These changes may contribute to an accelerated organization of LN at the epithelial-mesenchymal interface and subsequently to epithelial differentiation.

Animals↗

Altered deposition of basement-membrane molecules in co-cultures of colonic cancer cells and fibroblasts.

Two human colon carcinoma cell lines, HT29 and Caco-2 were co-cultured with fetal rat or human skin fibroblasts. Their morphological features, ultra-structural characteristics at the heterologous cell interface, and the deposition of basement-membrane molecules [laminin, type-IV collagen, heparan sulfate proteoglycan (HSPG)] at the epithelial-stromal junction were analyzed. The 2 cell lines behaved differently. HT29 cells did not spread on the fibroblasts and grew as clusters, while Caco-2 cells formed a monolayer over the fibroblastic feeder layer. Only the latter carcinoma cells exhibited cytoplasmic processes towards the fibroblasts and, after 5 days in co-cultures, a structured basement membrane (BM). The immunocytochemical analysis of the BM constituents revealed the absence of the molecules studied at the sites of heterologous contacts in the case of HT29 cells. In contrast, in the co-cultures comprising Caco-2 cells, laminin and type-IV collagen were progressively deposited in a polar fashion at the epithelial-fibroblastic interface which, however, remained devoid of HSPG molecules. Together with earlier data indicating a dual origin of the BM molecules located at the epithelial-fibroblastic interface in normal intestine, the present study shows that the cancer cells as well as the fibroblastic ones under the influence of carcinoma cells display an altered capacity to synthesize and/or secrete BM molecules. The extent of such abnormalities correlates with the differentiation of the cells. Finally, these modifications occur concomitantly with alterations in cell interactions which vary among cell lines.

Adenocarcinoma↗

Changes in glycosaminoglycan expression in the rat developing intestine.

Synthesis of glycosaminoglycan (GAG) chains was studied in the developing rat intestine. Intestinal segments, taken at various developmental stages, were exposed to 3H-glucosamine and 35S-sulfate for 6 hours. The amounts of 3H-GAGs (total GAGs) and of 35S-GAGs (sulfated GAGs) showed a clear age-dependence, with a broad maximum in the fetal period when dramatic growth and morphogenesis occur. Characterization of individual GAG species indicated that hyaluronic acid (HA), heparan and chondroitin sulfate (HS and CS) synthesis was modified quantitatively or qualitatively during development: decrease of HA with age; production of undersulfated HS molecules during embryonic life; shift towards a lower hydrodynamic form of HA and HS molecules after birth. We postulate that these alterations are crucial in the elaboration of an age-related specific extracellular microenvironment allowing intestinal growth and differentiation.

Aging↗

Changes in the expression of laminin during intestinal development.

The expression of laminin, a major glycoprotein constituent of basement membranes, was investigated in the rat developing intestine. The biosynthesis of laminin was studied after metabolic labeling of intestinal segments taken at various stages of development; the neosynthesized laminin was purified by affinity chromatography on heparin-Sepharose. Immunoblotting and immunoprecipitation experiments allowed us to analyze its constitutive chains. The data show that laminin is synthesized in very large amounts at 16-18 days of gestation concomitant with the onset of intestinal morphogenetic movements, i.e. villus emergence. Evaluation of the relative proportion of individual laminin polypeptides shows that laminin B1/B2 chains are produced in excess of A chains whatever the developmental stage considered. Interestingly at 17 days of gestation, levels of laminin A subunits are maximal. A second rise in the A/B chain ratio starts around birth and continues until adulthood. These quantitative data are corroborated by the immunocytochemical detection of laminin A and B chains, which revealed a specific spatiotemporal pattern. The finding that laminin A chains are located in the basement membrane of growing villi and of adult crypts raises the possibility that they may be involved in the process of cell growth and/or in the establishment of cell polarity by creating a specialized extracellular microenvironment.

Animals↗

Changes in glycosaminoglycan synthesis and in heparan sulfate deposition in human colorectal adenocarcinomas.

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

Adenocarcinoma↗

Type IV collagen mRNA accumulates in the mesenchymal compartment at early stages of murine developing intestine.

The expression of type IV collagen mRNA during mouse intestinal morphogenesis was examined by in situ hybridization using a cDNA probe corresponding to mRNA for alpha 1 (IV) chain. Type IV collagen mRNA is detected in the embryonic mesenchymal cells at early stages of development (12 d of gestation). A segregation of mesenchymal cells expressing high levels of type IV collagen mRNA in close vicinity of the epithelium occurs just before villus formation. During villus outgrowth, type IV collagen mRNA, still confined to mesenchyme-derived tissues, is progressively restricted to the mucosal connective tissue (the lamina propria) and to a lesser extent to the muscular layers. In the adult, the amount of messenger is quite low as compared to the level found in the developing intestine and the in situ hybridization signal, indistinguishable from the background, is uniform throughout the whole intestinal wall. At all developmental stages no detectable specific hybridization signal is virtually observed over the epithelium cell layer. These results show that high amounts of the type IV collagen messenger are detected during phases of intensive morphogenetic events. Furthermore, they reinforce the notion already gained previously (Simon-Assmann et al. 1988) that the mesenchymal compartment is the principal endogenous source of type IV collagen. They also indicate that the continuous migration of epithelial cells along the basement membrane of intestinal villi in the mature organ is not accompanied by a significant remodeling of the collagen IV network.

Aging↗

Smooth muscle actin expression during rat gut development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium.

Cytodifferentiation of smooth muscle cells has been analyzed immunocytochemically during rat intestinal development and in chimaeric intestines by using monoclonal antibodies reacting specifically with smooth muscle actin species (CGA7 [10] and anti-alpha SM-1 [40]). As development proceeds, the various intestinal muscle layers differentiate in the following order: (1) cells expressing smooth muscle actin appear within the mesenchyme of the 15-day fetal rat intestine, in the circular muscle-forming area, the differentiation of cells in the presumptive longitudinal muscle layer starting with a 48-h delay; (2) smooth muscle fibers appear within the connective tissue core of the villi shortly after birth, in parallel with a progressive formation of the muscularis mucosae, which becomes clear-cut only in the course of the 2nd week after birth; (3) a distinct cell layer in the innermost part of the circular muscle layer arises during the perinatal period. Thereafter, the fluorescence pattern remains unchanged until the adult stage. Chimaeric intestines were constructed by the association of 14-day fetal intestinal epithelium and cultured fetal rat or human skin fibroblasts. These fibroblastic cells did not express actin at the time at which they were associated. The immunocytochemical analysis of smooth muscle actin in the hybrid intestines, which had developed as intracoelomic grafts for 12 days, revealed that the skin fibroblastic cells had been induced by the intestinal epithelial cells to differentiate into smooth muscle cells. Such a result was also obtained with allantoic endoderm. It was not obvious in cocultures of intestinal epithelium with skin fibroblastic cells. However, when intestinal epithelial cells were cocultured with intestinal mesenchymal cells, actin expression was stimulated in the latter cell population.

Actins↗

Synthesis of basement membrane proteins in the small intestine.

Are the basement membrane (BM) molecules involved in epithelial-mesenchymal cell interactions known to be instrumental in intestinal development and differentiation? Several findings argue in favor of this assumption. First, quantitative and/or qualitative changes in type IV collagen, laminin-nidogen and heparan sulfate proteoglycan (HSPG) are obvious at the phases of intensive morphogenesis. Second, BM molecules deposited at the epithelial-mesenchymal interface are of dual origin: HSPG being produced by the epithelial cell population, while others like type IV collagen and laminin are mainly produced by the mesenchymal compartment. Third, the formation of the BM requires an actual contact between the epithelial and mesenchymal cells and always precedes the expression of differentiation markers in the epithelial cells. These data suggest that BM molecules display an instructive role in intestinal cell interactions.

Animals↗

Development of the vertebrate small intestine and mechanisms of cell differentiation.

The intestinal epithelium represents an attractive biological model of differentiation from stem cells to highly differentiated epithelial cells, not only during particular developmental events depending upon the vertebrate species considered but also throughout adult life. The ontogenic maturation of the intestinal epithelium arises from both a programmed expression of specific genes and epigenetic influences mainly due to epithelial and mesenchymal interactions and hormonal participation. In the present paper we review the structural and functional changes that occur in the amphibian, avian and mammalian intestine during embryonic and/or post-embryonic development. Furthermore, we review the data concerning the mechanisms which control the cytodifferentiation of the intestinal epithelium.

Animals↗

Sucrase-isomaltase expression and enterocytic ultrastructure of human colorectal tumors.

We report the relative frequency of sucrase-isomaltase (SI) antigen expression in human colonic adenocarcinoma (22/57), in peritumoral mucosa taken next to the tumor (31/41) or distant from it (29/42) as well as in 21/23 polyps. Our results are based on indirect immunofluorescence with a monoclonal antibody (MAb) specific for human intestinal SI. A regular and intense expression of SI occurred only in 6 tumor specimens. In the remaining 16 SI-positive tumor samples, labelling was heterogeneous, i.e., scattered over more or less extensive areas. A similar irregular staining pattern was also found in polyps and in peritumoral mucosa, irrespective of its distance from the tumor. Electron microscopic examination of 19 carcinomas mostly revealed altered brush-border membrane features, irrespective of histological SI staining pattern. Brush-border enzyme activities of sucrase, alkaline phosphatase and maltase showed no difference between tumor specimens and peritumoral mucosa, but aminopeptidase was depressed in the former. Sucrase activity was extremely low (mean values 1.1 to 1.8 mU/mg protein) and rose only exceptionally to 17.5 mU/mg prot.

Adenocarcinoma↗

Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine.

The deposition of intestinal heparan sulfate proteoglycan (HSPG) at the epithelial-mesenchymal interface and its cellular source have been studied by immunocytochemistry at various developmental stages and in rat/chick interspecies hybrid intestines. Polyclonal heparan sulfate antibodies were produced by immunizing rabbits with HSPG purified from the Engelbreth-Holm-Swarm mouse tumor; these antibodies stained rat intestinal basement membranes. A monoclonal antibody (mAb 4C1) produced against lens capsule of 11-d-old chick embryo reacted with embryonic or adult chick basement membranes, but did not stain that of rat tissues. Immunoprecipitation experiments indicated that mAb 4C1 recognized the chicken basement membrane HSPG. Immunofluorescent staining with these antibodies allowed us to demonstrate that distribution of HSPG at the epithelial-mesenchymal interface varied with the stages of intestinal development, suggesting that remodeling of this proteoglycan is essential for regulating cell behavior during morphogenesis. The immunofluorescence pattern obtained with the two species-specific HSPG antibodies in rat/chick epithelial/mesenchymal hybrid intestines developed as grafts (into the coelomic cavity of chick embryos or under the kidney capsule of adult mice) led to the conclusion that HSPG molecules located in the basement membrane of the developing intestine were produced exclusively by the epithelial cells. These data emphasize the notion already gained from previous studies, in which type IV collagen has been shown to be produced by mesenchymal cells (Simon-Assmann, P., F. Bouziges, C. Arnold, K. Haffen, and M. Kedinger. 1988. Development (Camb.). 102:339-347), that epithelial-mesenchymal interactions play an important role in the formation of a complete basement membrane.

Animals↗

Perinatal expression of brush-border hydrolases in rat colon: hormonal and tissue regulations.

The evolution pattern of brush-border digestive hydrolases and their hormonal regulation were studied in the proximal colon of newborn rats. The potentiality of the colon to express a small intestinal enzymatic pattern was also examined in associations made up of colonic endoderm and small intestinal mesenchyme, developed as either intracelomic grafts in 3-day-old chick embryos or as intrarenal grafts in adult rats. A transient increase of lactase- and aminopeptidase-specific activities occurred in the colon from the 19th day of gestation to 14 days after birth, but sucrase activity could never be detected. Immunocytochemical studies with antibodies specific for rat lactase, aminopeptidase, and sucrase confirmed these results. However, the levels of hydrolase activities were lower in the colon than in the jejunum at the same age. Thyroxine or hydrocortisone treatment during the first 4 days postpartum decreased lactase activity by 70 and 30%, respectively, but did not affect aminopeptidase activity. A slight but significant induction of sucrase activity was obtained with both hormones. In contrast, in the jejunum, only thyroxine decreased lactase activity with a lesser effect (30%), but both hormones increased aminopeptidase activity and induced the marked well-known appearance of sucrase activity. The fetal small intestinal mesenchyme was not able to induce the colonic endoderm to achieve a small intestinal-like differentiation. But the exposure of the developed hybrid intestines to glucocorticoids in organ culture allowed expression of sucrase in one-third of the cases. These results demonstrate the presence of brush-border hydrolases in the proximal colon of newborn rats, normally expressed in the small intestine, but never in the adult colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures. Fibroblastic cell-mediated effects of glucocorticoids.

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.

Animals↗

Modulation of HT-29 human colonic cancer cell differentiation with calmidazolium and 12-O-tetradecanoylphorbol-13-acetate.

The effects of a protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and of a calmodulin antagonist calmidazolium (CZ), on a human colonic cancer cell line HT-29 were analyzed. HT-29 cells are undifferentiated in standard culture conditions (HT-29 G+) and display an enterocytic differentiation when cultured in glucose-deprived medium (HT-29 G-). Early effects of TPA and CZ on the localization of cytoskeletal proteins (caldesmon, alpha-actinin and vinculin) and on cell proliferation were examined. Differentiation of the cells was assessed after 4 weeks on the basis of ultrastructural and functional characteristics of enterocytic polarity, presence of apical brush borders, expression of brush border membrane antigens (Caco 5/50 and sucrase-isomaltase), and segregation of calmodulin to the brush border cytoskeleton. TPA treatment of HT-29 G+ or G- cells induced early morphological and cytoskeletal alterations: the cells rounded up and lost their stress fibers with the associated caldesmon, alpha-actinin, and vinculin. TPA did not modify the differentiation of G- cells, but induced in G+ cells the expression, although limited, of enterocytic differentiation characteristics. Addition of CZ to HT-29 G- cells enhanced their differentiation state but did not provoke any early morphological or cytoskeletal alterations. No effects of CZ on HT-29 G+ cells were obvious. The results suggest that protein kinase C, the TPA receptor, is involved in the triggering of HT-29 G+ cell differentiation whereas calmodulin-dependent functions would be implicated in HT-29 G- cell maturation.

Antigens, Neoplasm↗

Expression of brush border calmodulin-binding proteins during human small and large bowel differentiation.

The expression and immunocytochemical localization of three brush border cytoskeletal calmodulin-binding proteins, caldesmon, fodrin, and the 110 kDa subunit of the 110 kDa calmodulin complex, have been studied in human intestinal epithelial cells as a function of their ontogenic differentiation. At immature stages (fetal week 8), caldesmon and fodrin were present in undifferentiated intestinal epithelial cells. However, no 110 kDa protein was detectable except a 135 kDa immunoreactive species. The 110 kDa form appeared at week 12, when microvilli differentiate, and became prominent at week 14 simultaneously with the disappearance of the 135 kDa species. Finally at week 14, the calmodulin-binding protein pattern was identical to that found in adults. Immunocytochemical experiments revealed that at week 8, antibodies to caldesmon and fodrin gave a fluorescence lining at the periphery of the cells, whereas the 110 kDa immunoreactive species was hardly detectable. Then, as early as week 12 of gestation, with the three antisera, a bright fluorescence lined the apex of the cells, as in adults. In the colon, the events were delayed. This study demonstrates that the developmental pattern of the three calmodulin-binding proteins investigated, caldesmon, fodrin and the 110 kDa subunit, parallels the temporal differentiation of human intestinal brush borders and the proximo-distal morphological intestinal maturation.

Calmodulin-Binding Proteins↗