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S Robine

Publications and source records attributed to S Robine.

At least 19 recordsLinked to original sources
PubMed2007

Conditional mouse models of cancer.

The development of inducible and conditional technologies allowed us to generate transgenic mouse models that faithfully recapitulate human tumorigenesis. It is possible to control, in time and space, the development of tumors in almost every mouse tissue. The result is that now we have available mouse models for all major human cancers. Novel noninvasive approaches to tumor imaging will enable us to follow tumor development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumor models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumor development. In this review, we focus on the different mouse models for colon cancer.

Adenomatous Polyposis Coli↗
PubMed2001

Immunocytochemical detection of bone marrow micrometastases in colorectal carcinoma patients, using a monoclonal antibody to villin.

The search continues to find methods to more effectively distinguish colorectal carcinoma patients who could be separated into high-risk and low-risk categories. Investigators have reported on the detection of occult micrometastases in bone marrow using antibodies to cytokeratin, which is a marker of epithelial cells but which has no tissue specificity, as opposed to villin, a cytoskeletal protein that is specifically involved in the formation of brush-border microvilli in the small intestine and colon epithelium. Specificity and sensitivity of antibody to villin (ID2C3) and antibody to cytokeratin (A45-B/B3) were first studied in normal bone marrow and in a test system in which cancer cell lines were mixed in normal bone marrow. In a preliminary study including 16 colorectal carcinoma patients, we compared the number of villin-positive cells with cytokeratin-presenting cells. As A45-B/B3, ID2C3 was determined to be sensitive enough to detect one cancer cell in 10(6) hematopoietic cells. Staining of hematopoietic cells with irrelevant antibody and a light staining of megakaryocytes with ID2C3 limited the specificity of the method. In colorectal carcinoma patients, correlation between ID2C3 and A45-B/B3 was 94%. Sensitivity and specificity of ID2C3 antibody to villin were satisfactory. Its clinical relevance must be investigated in further studies.

Aged↗
PubMed2000

Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.

Cell Line↗
PubMed1999

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

Actin Cytoskeleton↗
PubMed1999

Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines.

Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent beta-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted beta-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa.

Animals↗
PubMed1999

Immunodetection of the microvillous cytoskeleton molecules villin and ezrin in the parasitophorous vacuole wall of Cryptosporidium parvum (Protozoa: Apicomplexa).

Microvilli - actin - villin - ezrin - Cryptosporidium parvum The sporozoites and merozoites of the Apicomplexan protozoan Cryptosporidium parvum (C. parvum) invade the apical side of enterocytes and induce the formation of a parasitophorous vacuole which stays in the brush border area and disturbs the distribution of microvilli. The vacuole is separated from the apical cytoplasm of the cell by an electron-dense layer of undetermined composition. In order to characterize the enterocyte cytoskeleton changes that occur during C. parvum invasion and development, we used both confocal immunofluorescence and immunoelectron microscopy to examine at the C.parvum-enterocyte interface the distribution of three components of the microvillous skeleton, actin, villin and ezrin. In infected cells, rhodamine-phalloidin and anti-villin and anti-ezrin antibodies recognized ring-like structures surrounding the developing parasites. By immunoelectron microscopy, both villin and ezrin were detected in the parasitophorous vacuole wall surrounding the luminal and lateral sides of the intracellular parasite. In contrast, anti-beta and anti-gamma actin antibodies showed no significant labelling of the vacuolar wall. These observations indicate that the parasitophorous vacuole wall contains at least two microvillus-derived components, villin and ezrin, as well as a low amount of F-actin. These data suggest that C.parvum infection induces a rearrangement of cytoskeleton molecules at the apical pole of the host cell that are used to build the parasitophorous vacuole.

Actins↗
PubMed1998

I-SceI-induced gene replacement at a natural locus in embryonic stem cells.

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 x 10[-6]) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

Animals↗
PubMed1997

Epithelial cell growth and differentiation. IV. Controlled spatiotemporal expression of transgenes: new tools to study normal and pathological states.

The gut epithelium represents a dynamic, well-organized developmental system for examining self-renewal, differentiation, repair, and tumorigenesis. The apical pole of the enterocytes, the brush border, is composed of an array of well-organized actin microfilaments that support the plasma membrane. Villin, one actin-binding protein that contributes to the assembly and dynamics of the microvillus bundle, exhibits special features such as restricted tissue specificity and early expression in the immature crypt cells. The regulatory elements of the villin gene are suitable to control the expression of transgenes in intestinal cells. Engineering genetically modified animals by classic transgenesis using the villin promoter or by gene targeting in the villin locus will allow the establishment of animal models that may recapitulate human intestinal disorders.

Animals↗
PubMed1995

Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells.

We have used an antisense RNA strategy to investigate the role of the actin-associated protein, villin, in the brush-border morphogenesis of human intestinal CaCO2 cells. Stable expression of a cDNA encoding antisense villin RNA resulted in the permanent down-regulation of the endogenous villin message and dramatically affected brush-border assembly. Ultrastructural and immunolocalization studies revealed that epithelial cell polarity was largely maintained. However, in contrast to brush-border markers such as dipeptidyl-peptidase IV, the apical localization of sucrase-isomaltase was specifically impaired. Retransfection of the villin antisense-expressing cell line with a cDNA encoding a partial sense villin RNA restored both brush-border assembly and sucrase-isomaltase apical expression. The suggestion that brush-border morphogenesis may be important for the trafficking of certain proteins is discussed.

Actins↗
PubMed1993

Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line.

To develop a molecular tool for tissue-specific targeting of gene expression in immature and differentiated epithelial cells of the small and large intestinal mucosa, we have isolated the 2-kb 5'-flanking region of the human villin gene. This region contains numerous short sequences that are conserved among other tissue-specific promoters of genes expressed in differentiated enterocytes. This DNA fragment promotes the transcription and expression of the luciferase reporter gene in villin-positive intestinal, renal, and hepatoma cell lines but not in a villin-negative keratinocyte cell line. The pattern of expression corresponds that of the endogenous gene, indicating that this sequence can direct intestine-specific transcription. In the differentiating HT29 intestinal cell line, expression of the reporter gene is already detectable in undifferentiated cells, and dramatically increases when terminal differentiation is induced. Thus, as previously reported for the endogenous gene the isolated 5'-flanking region of the villin gene responds positively to conditions known to stimulate terminal differentiation of these cultured epithelial intestinal cells. The reported results indicate that this genomic fragment contains sufficient regulatory elements to recapitulate the expression pattern of the villin promoter during intestinal differentiation.

Adenocarcinoma↗
PubMed1993

Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

Aminopeptidases↗
PubMed1993

Functional properties of proximal tubule cell lines derived from transgenic mice harboring L-pyruvate kinase-SV40 (T) antigen hybrid gene.

This study describes the functional characterization of two cell lines derived from the proximal convoluted (PKSV-PCT cells) and proximal straight (PKSV-PR) tubules microdissected out from kidneys of transgenic mice harboring the simian virus 40 (SV40) large T and small t antigens placed under the control of the rat L-type pyruvate kinase (L-PK) 5' regulatory sequence. Both cell lines exhibited cellular cyclic AMP stimulated by parathormone (PTH) and calcitonin (CT) and a sodium-dependent glucose transporter. Uptake of the fluid-phase marker [3H]inulin showed that both cell lines grown on filters exhibited biphasic apical and basolateral endocytic rates. Results from Northern blot analysis indicate that the expression of the T antigen gene (Tag) is dependent on the concentration of D-glucose in the medium and show that the L-PK construct has maintained its capacity for up- or down-regulation by carbohydrates. Replacement of D-glucose by neoglucogenic substrates (lactate, oxaloacetate) blunted the expression of Tag transcripts and induced arrest of cell growth. Compared to cell grown in D-glucose-enriched medium, the hormonal sensitivities to PTH and CT and the sodium-dependent glucose uptake were unchanged whereas quiescent cells exhibited increased hydrolase content. Thus the proximal function has been preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice. As the expression of Tag transcripts is controlled by D-glucose, the structural and physiological characteristics of these cell lines can be studied in either quiescent or active growth conditions.

Animals↗
PubMed1992

Developmental regulation of villin gene expression in the epithelial cell lineages of mouse digestive and urogenital tracts.

The expression of villin, an actin-binding protein and major structural component of the brush border of specialized absorptive cells, was studied during mouse embryogenesis. We show that the ontogeny of villin expression is limited to the epithelial cell lineages of the digestive and uro-genital tracts and accounts for the tissue-specific expression observed in adult mice. This spatiotemporal pattern of villin expression is distinctive in sequence, intensity, regional distribution and polarization. During the development of the primitive gut, villin is faintly and discontinuously expressed in the invaginating foregut but it is expressed in every cell bordering the hindgut pocket. Later, villin expression increases along the developing intestine and concentrates in the brush border of the epithelium bordering the villi. In gut derivatives, villin is present in liver and pancreas primordia but only biliary and pancreatic cells maintain a faint villin expression as observed in adults. In the urogenital tract, mesonephric tubules are the first mesodermal derived structures to express villin. This expression is maintained in the ductuli efferents, paradidymis and epoöphoron. Villin then appears in the proximal metanephric tubules and later increases and concentrates in the brush border of the renal proximal tubular epithelial cells. Thus villin expression can be considered as an early marker of the endodermal cell lineage during the development of the digestive system. Conversely, during the development of the excretory and genital system, villin is only expressed after the mesenchyme/epithelium conversion following the appearance of tubular structures. These observations emphasize the multiple levels of regulation of villin gene activity that occur during mouse embryogenesis and account for the strict pattern of tissue-specific expression observed in adults. In the future, regulatory elements of the villin gene may be used to target the early expression of oncogenes to the digestive and urogenital tracts of transgenic mice.

Animals↗
PubMed1992

Amphibian intestinal villin: isolation and expression during embryonic and larval development.

An actin-binding protein of M(r) 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian M(r) 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal M(r) 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal M(r) 105,000 protein is immunologically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.

Actins↗
PubMed1991

Structure of the human villin gene.

We have isolated and characterized the complete human villin gene. The villin gene is located on chromosome 2q35-36 in humans and on chromosome 1 in mice. Villin belongs to a family of calcium-regulated actin-binding proteins that share structural and functional homologies. The villin gene is expressed mainly in cells that develop a brush border, such as mucosal cells of the small and large intestine and epithelial cells of the kidney proximal tubules. Villin gene expression is strictly regulated during adult life and embryonic development in the digestive and urogenital tracts and, thus, may be used as a marker of the digestive and renal cell lineages. The human villin gene has one copy per haploid genome, encompasses about 25 kilobases, and contains 19 exons. Analysis of the structural organization of this gene shows that the two mRNAs that encode villin in humans arise by alternative choice of one of the two polyadenylylation signals located within the last exon. The overall organization of the exons reflects the gene duplication event from which this family of actin-binding proteins originated.

Actins↗
PubMed1990

Presence of villin, a tissue-specific cytoskeletal protein, in sera of patients and an initial clinical evaluation of its value for the diagnosis and follow-up of colorectal cancers.

Villin is an actin-binding protein found in a few normal adult epithelia, namely epithelial cells in the digestive and urogenital tracts. Moreover, villin production is maintained in malignant cells. We assumed that cell lysis and necrosis of solid tumors producing villin might result in villin release into blood. We analyzed the villin content of sera from 788 patients and controls using an enzyme-linked immunosorbent assay. Patients and controls were classified into healthy donors, patients with benign diseases of the gastrointestinal tract, patients with colorectal cancers, and patients with malignant nondigestive diseases. In the panel of sera analyzed, the sensitivity of the assay for colorectal cancers was 50.5%, and its overall specificity for malignant digestive tumors was 94.5%. Results were statistically analyzed comparing each group of sera with each other. We conclude that the presence of villin is indicative of a pathological state in the gastrointestinal tract (P less than 0.001). Finally, we followed villin levels after tumor resections (60 patients). We found that the villin level in sera remains low in remissions but is raised in recurrences. We suggest that the villin assay may have clinical utility as a diagnostic adjunct for adenocarcinoma of the gastrointestinal tract. It may also have some value in monitoring patients with advancing colorectal carcinomas after resection of these tumors.

Adult↗