Isolation and chromosomal location of mE4, a novel murine gene of the immunoglobulin superfamily.
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Amino Acid Sequence↗
Biomedical subjects
M G Denis
Publications and source records attributed to M G Denis.
Glycosylation of a novel member of the immunoglobulin gene superfamily expressed in rat carcinoma cell lines.
MAb E4 recognizes a 66-kDa glycoprotein, pE4, which is a member of the immunoglobulin gene superfamily. This protein is expressed at the cell surface in rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. Since expression of aberrant carbohydrate structures is often associated with malignant transformation, glycosylation of pE4 was analyzed. Reactivity of lectins with pE4 suggested the absence of N-acetylneuraminic acid, terminal galactose and O-linked glycan, and the presence of N-linked glycans. Tunicamycin treatment reduced the binding of MAb E4 to cancer cells suggesting that the E4 epitope is at least partially glycosylated. Digestions with neuraminidases, O-glycosidase and peptide-N-glycosidase F confirmed these results. Pronase treatment abolished the binding of MAb E4, indicating that E4 epitope involves not only a carbohydrate determinant but also a peptide moiety. Mild periodate oxidation abolished the binding of MAb E4, indicating that non-reducing terminus carbohydrates are part of the E4 epitope. Neutral sugar analysis revealed the absence of galactose and the presence of fucose. Since fucose is sensitive to periodate oxidation, this sugar could be the carbohydrate part of the determinant recognized by MAb E4. Reactivity of lectins specific for fucose indicated the presence of alpha(1-6)-fucose on pE4.
Adenocarcinoma↗
Qualitative low-level internal control for nested RT-PCR.
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Base Sequence↗
Evidence for two distinct alpha(1,2)-fucosyltransferase genes differentially expressed throughout the rat colon.
Blood-group-ABH antigens are carbohydrate structures widely distributed in numerous tissues. These structures are fucosylated by an alpha(1,2)-fucosyltransferase. The occurrence of at least two alpha(1,2)-fucosyltransferase genes in the human genome has been strongly suggested by genetic studies, but only one of them has been cloned so far. Specific primers deduced from this human cDNA were used to amplify a fragment of rat genomic DNA (FTA). Screening of a rat colon cDNA library with this probe allowed us to isolate a clearly distinct, but related, cDNA clone (FTB). Both sequences showed considerable sequence similarity to the human alpha(1,2)-fucosyltransferase cDNA previously cloned. Furthermore, cells transfected with these DNA fragments in antisense orientation displayed a decreased alpha(1,2)-fucosyltransferase activity, indicating that they both correspond to fragments of alpha(1,2)-fucosyltransferase genes. Finally, differential expression of these genes was demonstrated in two rat colon-cancer cell lines and throughout the rat colon.
Animals↗
A novel member of the immunoglobulin gene superfamily expressed in rat carcinoma cell lines.
Defined by monoclonal antibody E4, the pE4 antigen is a 66,000-Da glycoprotein which is expressed at the cell surface of rat colon and mammary carcinomas, but only in trace amounts in normal adult rat tissues. To determine the structure of this tumor-associated antigen and to identify its functional domains, we have cloned a cDNA coding for this protein. It encodes a 416-amino acid protein with an expected molecular weight for the core protein of approximately 42,000. The predicted amino acid sequence reveals that pE4 contains the conserved amino acids and domain structures characteristic of members of the immunoglobulin gene superfamily. Comparison of this sequence with data banks revealed a significant homology with the human and mouse receptors for polio-virus. However, pE4 is not the rat receptor for poliovirus, as different patterns were obtained by hybridization of rat genomic DNA with both probes. A major approximately 2.2-kilobase transcript of the pE4 gene was detected in all the rat tumor cell lines tested. In contrast, barely detectable levels of pE4 mRNA were found in normal adult rat tissues.
Amino Acid Sequence↗
Over-expression of the S13 ribosomal protein in actively growing cells.
In order to define biological markers of aggressiveness, 2 rat colon-carcinoma cell lines differing by their tumorigenicity were used to clone genes over-expressed in colon carcinoma as compared with normal epithelial cells. A progressive rat colon-carcinoma clone (PROb) cDNA library was hybridized with 32P-cDNA synthesized from mRNA prepared from these PROb cells, or from regressive cells (REGb) derived from the same tumor. Several clones were isolated after the initial screening. The specificity of each clone was confirmed by RNA blotting. One of these (B9) was found to hybridize to an mRNA 30-fold more abundant in PROb cells than in normal adult rat colon, and was therefore selected for further study. No gene amplification was detected by Southern blot analysis, indicating that the difference in mRNA content was most likely due to an increased transcription of this gene. Sequencing of the cDNA revealed approximately 98% homology with the rat S13 ribosomal protein. The expression level of this gene was determined in a series of rat cell lines with different growth rates. A good correlation was found between these 2 parameters. Our data suggest that the S13 ribosomal-protein gene can be used to evaluate the growth rate of tumor cells, which might be correlated with their aggressiveness. In an initial trial experiment, S13 ribosomal-protein mRNA was detected in a series of human colorectal tumors by in situ hybridization. A strong signal was seen in the 4 tumors analyzed.
Adenocarcinoma↗
Cloning and analysis of the human S13 ribosomal protein cDNA.
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Amino Acid Sequence↗
Analysis of the relationship between stage of differentiation and NK/LAK susceptibility of colon carcinoma cells.
NK and LAK cells which are able to lyse tumor target cells in an MHC-unrestricted manner are not equally effective against targets of the same nature. In the case of colorectal tumors, some cells are highly sensitive, whereas others are resistant to NK and can even be quite resistant to LAK-mediated lysis. In the present paper, we tried to correlate the stage of differentiation of 17 human colorectal tumor cell lines with their NK- or LAK-cell susceptibility. It was observed that NK cells killed colorectal target cells independently from their stage of differentiation defined according to histopathological criteria from xenografting in nude mice. NK susceptibility was not correlated either with in vitro-defined criteria of differentiation, such as cell polarity and morphology, brush-border enzyme expression and CEA production. A LAK-resistant HT-29 sub-line (HT-29 LAK) was selected which could not be distinguished from HT-29 in terms of features of differentiation. It was further observed that HT-29 Glc-/+ cell line, a highly differentiated enterocytic-like variant of HT-29, obtained after glucose starvation, was killed by LAK cells as efficiently as the moderately differentiated parental HT-29, and that Caco-2 cells, which differentiate spontaneously after confluence in standard culture conditions, were equally sensitive to NK-mediated lysis whatever their stage of differentiation. In contrast, HT29 MTX10(-5), a highly differentiated mucus-secreting variant of HT29 obtained by methotrexate selection, was much more resistant to LAK cells than parental HT29 cells.
Animals↗
Isolation of cDNA clones corresponding to genes differentially expressed in two colon-carcinoma cell lines differing by their tumorigenicity.
In an effort to isolate genes involved in the progression of colonic cells leading to a carcinoma, we used as a model 2 rat colon-carcinoma cell lines selected from the same tumor, differing by their tumorigenicity. When soluble, Triton-X-100 extracted, or cytoskeletal proteins from the progressive PROb cells and the regressive REGb cells were analyzed by SDS-PAGE, minor differences were seen. Furthermore, mRNA-cDNA hybridization analyses showed extensive homology between the 2 mRNA populations. Thus, the homology between the 2 clones is high at both the protein and the mRNA levels. A PROb cDNA library was hybridized with 32P-cDNA synthesized from PROb or REGb mRNA. The clones giving a stronger signal when hybridized with the homologous PROb probe were isolated. The specificity of each clone was confirmed by RNA blotting. Most of the positive clones showed a 2- to 3-fold higher expression in PROb cells when compared with REGb cells. One clone (J 13) corresponded to an mRNA 7- to 10-fold more abundant in PROb cells, and was further studied. No gene amplification was detected by Southern blot analysis, indicating that the difference in mRNA content was most likely due to an increased transcription of this gene in PROb cells. Sequencing of the cDNA showed high homology with the rat ferritin light sub-unit. Over-expression of ferritin in PROb cells as compared with REGb cells was confirmed at the protein level using specific antibodies.
Adenocarcinoma↗
Biological effects of glucocorticoid hormones on two rat colon adenocarcinoma cell lines.
Glucocorticoid hormones are thought to play a role in carcinogenesis as they regulate cell differentiation and proliferation. We have investigated the effect of dexamethasone on two cell lines derived from a colon carcinoma, which differ by their tumorigenicity. Dexamethasone was found to inhibit growth of both the progressive (PROb) and the regressive clone (REGb). Upon glucocorticoid treatment, PROb cells were found to secrete an additional Mr approximately 40,000 protein. The synthesis and the release in the culture medium of this protein is stimulated specifically by glucocorticoid agonists, and not by other steroid hormones. The anti-glucocorticoid RU 38486 is inefficient and suppresses the induction of this protein by dexamethasone. Induction is sensitive to actinomycin D, suggesting that regulation may be related to an alteration of the rate of mRNA synthesis. The cellular effect of glucocorticoid hormones being mediated through a specific soluble receptor, we have characterized this protein. The PROb cells contained more specific glucocorticoid-binding sites (approximately 170,000 sites per cell) than the regressive ones (REGb cells; approximately 100,000 sites per cell). In both clones, the receptor was associated with the Mr approximately 90,000 heat shock protein to yield large complexes (Stokes radius Rs approximately 7.5 nm), which were dissociated to the same extent upon heat- and salt-treatment. The steroid- and DNA-binding unit of the receptor, characterized under denaturing conditions using an anti-receptor monoclonal antibody, was found to be more degraded in the PROb cell line.
Adenocarcinoma↗
Possible involvement of TGF beta 1 in the distinct tumorigenic properties of two rat colon carcinoma clones.
The presence in tumors of numerous cytokines suggests that they potentially modulate tumor cell activities and host tissue remodelling. To investigate the possible involvement of transforming growth factor type beta (TGF beta) in the metastatic process of cancer development, we have studied the effect of this factor on two rat colon carcinoma cell lines. These cell clones had been previously tested and selected for their ability to develop metastases in syngenic animals or lack of it. The two cell lines were characterized for their production of TGF beta. Production of active and latent forms of TGF beta 1 in the medium conditioned by the rat colon cancer cells were quantified using a bioassay. The presence of active TGF beta 1 was demonstrated in conditioned medium from the progressive tumor (PROb) cells and significant expression of latent forms of TGF beta 1 were found in the conditioned media from both cell clones. TGF beta 1 slightly inhibited proliferation of PROb cells which had been previously described as moderately differentiated, and significantly stimulated proliferation of the regressive (REGb) cells, described as poorly differentiated. On the basis of our observations, we suggest that this endogenous factor could be involved in autocrine regulation of tumor cell activities and in paracrine regulation of stroma cell and immune responses. Active and/or latent expression of TGF beta 1 by the two rat colon carcinoma cell lines, and their variable responses to the growth factor, strongly suggest that this polypeptide is involved in the regulation of tumorigenic expression of adenocarcinoma cells.
Adenocarcinoma↗
A secreted Mr approximately 40,000 glycoprotein specifically induced by glucocorticoids in a rat colon carcinoma cell line.
Glucocorticoid hormones are thought to play a role in carcinogenesis, as they regulate cell differentiation and proliferation. We have previously shown that dexamethasone inhibits the growth of a rat colon carcinoma cell line, and induces the secretion of an Mr approximately 40,000 protein. We now report that the synthesis and the release in the culture medium of this protein is stimulated specifically by glucocorticoid agonists, and not by other steroid hormones. The anti-glucocorticoid RU 38486 is inefficient and suppresses the induction of this protein by dexamethasone. Induction is sensitive to actinomycin D, suggesting that regulation may be exerted by altering the rate of mRNA synthesis. Characterization of culture medium from dexamethasone-treated cells revealed that the Mr approximately 40,000 protein is glycosylated, and can be further separated from other secreted proteins by high-performance anion-exchange chromatography.
Adenocarcinoma↗
Characterization, isolation and amino terminal sequencing of a rat colon carcinoma-associated antigen.
Monoclonal antibodies have been raised against a cell line derived from a dimethylhydrazine-induced rat colon carcinoma. One of these antibodies (MAb E4) has previously been shown to react slightly with normal small intestine and colon, and not with other normal tissues as determined by immunohistochemistry. Using Western immunoblotting we confirmed this tumor specificity. Therefore, the Mr of approx. 66,000 glycosylated antigen (pE4) recognized by MAb E4 appeared to be a potential marker of colon carcinoma. Fifteen human tumor cell lines were tested by flow cytometry for the expression of pE4. This antigen was not detected on these cells. In the rat colon carcinoma cell, pE4 was exclusively found on the cell membrane. pE4 was purified to near homogeneity by immunoaffinity chromatography. The first 20 N-terminal amino acids were identified. Comparison with the NBRF data bank did not reveal a complete homology with known sequenced proteins but similarities were found with the mouse L3T4 precursor, the env polyprotein of human immunodeficiency virus type I, flagellin from Halobacterium halobium and the gp30 from hepatitis B surface antigen. Homology was always found in transmembranous or hydrophobic domains of these proteins. By indirect immunofluorescence analysis of adherent cells and size exclusion chromatography under native conditions, pE4 was found to interact with other molecules and perhaps to be involved in intercellular contact.
Amino Acid Sequence↗
Glucocorticoid effects and receptors in two rat colon carcinoma cell lines differing by their tumorigenicity.
Steroid hormones, regulators of cell differentiation and proliferation, are believed to play a role in carcinogenesis. Glucocorticoid hormones in particular modulate the expression of a number of proteins implicated in this process. We have investigated the effect of dexamethasone on two cell lines derived from a colon carcinoma, which differ by their tumorigenicity. Dexamethasone was found to inhibit growth of both the progressive (PROb) and the regressive clone (REGb). Upon hormonal treatment, glucocorticoid hormones induced fibronectin secretion by the two clones, whereas PROb cells were found to secrete an additional Mr approximately 43,000 protein. The cellular effect of glucocorticoid hormones being mediated through a specific soluble receptor, we have characterized this protein. The progressive cells (PROb) contained more specific glucocorticoid-binding sites (approximately 170,000 sites per cell) than the regressive ones (REGb cells; approximately 100,000 sites per cell). In both clones, the receptor was associated with the Mr approximately 90,000 heat shock protein to yield large complexes (Stokes radius Rs approximately 7.5 nm), which were dissociated to the same extent upon heat- and salt-treatment. The steroid- and DNA-binding unit of the receptor, characterized under denaturing conditions using an anti-receptor monoclonal antibody was found to be more degraded in the progressive cell line.
Animals↗