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A Zweibaum

Publications and source records attributed to A Zweibaum.

At least 37 records · Page 2Linked to original sources
PubMed1994

Modifications of the anti-oxidant metabolism during proliferation and differentiation of colon tumor cell lines.

The anti-oxidant metabolism was studied at different times after sub-culture in 2 colon cell lines previously characterized for their growth and differentiation properties. The HT29 cell line is mainly composed of proliferative and undifferentiative cells, while the derived 5-fluorouracil (FUra)-adapted cells undergo growth-dependent differentiation, which is complete at post-confluence. In the 2 cell lines, all the anti-oxidant parameters studied appeared to be related to proliferation, with increased activity of superoxide dismutase (SOD) 1 and 2, catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GSR), and glutathione transferase (GST), and decreased glucose-6-phosphate dehydrogenase (G6PD) activity and glutathione content, in parallel with slowing down of proliferation. At post-confluence, these metabolic parameters remained stable, except for GPX activity, which continued to increase, and CAT activity, which decreased. The amounts of SOD1, SOD2 and CAT immunoreactive proteins, estimated by Western blotting, appeared to be correlated to their respective enzymatic activities. SOD1, CAT and GST activity and glutathione content, which remained at similar levels in the 2 cell lines for all times studied, appeared unrelated to the differentiation process. GSR and GPX activity, which was lower in FUra-adapted than in parental cells only at post-confluence, could be considered as markers of differentiated cells. The higher SOD2 and lower G6PD activity observed in FUra-resistant cell in comparison with parental cells at all times after sub-culture could be characteristic both of differentiative and of differentiated cells. Interestingly, cytogenetics have previously indicated that deletions of the long arm of chromosome 6, which carry the gene for SOD2, were frequently observed in parental but not in FUra-adapted cells. These results demonstrate that modifications of the anti-oxidant metabolism occur in relation with proliferation and differentiation, and suggest a particular role for SOD2 in these cellular processes.

Antioxidants↗
PubMed1994

Expression of cytochrome P-450 3A in HT29-MTX cells and Caco-2 clone TC7.

HT-29 sublines and Caco-2 clones were analyzed for the expression of cytochrome P-450 3A. The enzyme was found to be expressed in differentiated HT-29 cells selected by resistance to methotrexate and in one of seven Caco-2 clones, TC7. Its expression parallels the differentiation process, with highest levels being observed at late confluency. P-450 3A mRNA and protein patterns, as well as subcellular distribution, are intermediate between those observed in human adult intestine and fetal liver.

Blotting, Northern↗
PubMed1994

Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption.

Seven clones from the Caco-2 cell line, three isolated from passage 29 (PD7, PD10, PF11) and four from passage 198 (TB10, TC7, TF3, TG6), all of them selected on the basis of differences in the levels of expression of sucrase-isomaltase and rates of glucose consumption, were analysed for the expression of hexose-transporter mRNAs (SGLT1, GLUT1-GLUT5) in relation to the phases of cell growth and the associated variations of the rates of glucose consumption. All clones showed a similar pattern of evolution of the rates of glucose consumption, which decreased from the exponential to the late-stationary phase, but differed, in a 1-40-fold range, in the values observed at late postconfluency. According to these values, clones could be divided into high- (PD10, PF11) and low-glucose-consuming cells (PD7, TB10, TC7, TF3 and TG6). GLUT1 and GLUT3 mRNAs were expressed in all clones and showed a similar pattern of evolution: their level decreased, from the exponential to the stationary phase, in close correlation with the decrease in rates of glucose consumption, with only high-glucose-consuming clones maintaining high levels in the stationary phase. In contrast, SGLT1, GLUT2 and GLUT5 mRNAs were only expressed, like sucrase-isomaltase mRNA, in the low-glucose-consuming clones, and their level increased from the exponential to the stationary phase, in parallel with the differentiation of the cells. GLUT4 was undetectable in all the clones. Glucose deprivation generally resulted in a discrete decrease in the levels of all transporter mRNAs in all clones, one exception being GLUT2, which in the high-glucose-consuming clones is only detectable when the cells are grown in low glucose. These clones should be ideal tools with which to study in vitro, at the single-cell level, how these transporters concur to the utilization and transport of hexoses and how their exclusive or co-ordinated expression is regulated.

Carrier Proteins↗
PubMed1994

Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation.

The expression of the brush border-associated hydrolase sucrase-isomaltase was shown to increase from early to late passages of Caco-2 cells, concomitant with a decrease in the rates of glucose consumption. Twenty-six clones were isolated from early (P29) and late (P198) passages of the cell line. These clones show considerable and inverse differences in the levels of sucrase activities and rates of glucose consumption, without marked changes in other features of enterocytic differentiation of the cells (presence of an apical brush border, levels of expression of other brush border-associated hydrolases). Clones with low sucrase-isomaltase expression show a mosaic expression of the enzyme and a 38-fold higher rate of glucose consumption than clones with high sucrase-isomaltase expression. The clones with high expression show an homogeneous apical distribution of the enzyme and 70-fold and 35-fold higher levels of sucrase activities and sucrase-isomaltase mRNA, respectively. In contrast no differences were found from one clone to another in the enrichment of sucrase activity in brush border-enriched fractions as compared to cell homogenates. Switch to low glucose-containing medium (1 mM versus 25 mM in standard culture conditions) of cells with low sucrase-isomaltase results in an increased and more homogeneous expression of the enzyme and a tenfold augmentation of the levels of sucrase-isomaltase mRNA and sucrase activity. These results show that glucose interferes with the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level.

Adenocarcinoma↗
PubMed1993

[Differentiation of human colon cancer cells: a new approach to colon cancer].

Our purpose is to demonstrate that some human colon cancer cells are able to express the same differentiation features as normal intestinal epithelial cells and to report experimental data which suggest that these particular cells are spontaneously resistant and adaptable to anticancer drugs. The concept of normal differentiation of colon cancer cells is supported by observations made with two cultured cell lines, Caco-2 and HT-29. The cell line Caco-2 expresses spontaneously and homogeneously an enterocytic differentiation. The cell line HT-29 is heterogeneous as it contains a small proportion (< 5%) of differentiated cells of either enterocytic or mucus-secreting type. Homogeneous populations of differentiated HT-29 cells of either type have been isolated by pressure selection. In order to investigate whether the pressure associated with anti-cancer drugs would result in the selection of differentiated populations, HT-29 cells were cultured in the presence of increasing concentrations of methotrexate and 5-fluorouracil. The resulting resistant populations were found to be totally differentiated. This supports the view that the small proportion of parental cells which are able to differentiate are also able to spontaneously resist to drug pressure. These results imply that the concept of cellular differentiation should be considered by pathologists. They also imply that basic research should be developed in order to unravel which mechanisms of drug resistance are specifically associated with the ability of the cells to differentiate.

Cell Differentiation↗
PubMed1993

cis-Golgi resident proteins and O-glycans are abnormally compartmentalized in the RER of colon cancer cells.

Neoplastic transformation is commonly associated with altered glycosylation of proteins and lipids. To understand the basis for altered mucin glycosylation, we have examined the distribution of RER markers, a cis-Golgi resident protein, and the GalNAc alpha-O-Ser/Thr epitope (Tn) in human colon cancer cells and in normal colon. In cultured mucin-producing colon cancer cells, Gal-NAc alpha-O-Ser/Thr was found in mucin droplets and in RER cisternae. In addition, the Golgi apparatus was disorganized in a proportion of cells and a 130 kDa cis-Golgi resident protein was also abnormally redistributed to the RER. The distribution of the MUC2 intestinal apomucin, protein disulphide isomerase, Gal-NAc alpha-O-Ser/Thr, and the 130 kDa cis-Golgi resident protein was analysed in normal colon and in colon cancer tissues. In normal colon, MUC2 apomucin and protein disulphide isomerase were located in the RER, whereas the cis-Golgi resident protein and GalNAc alpha-O-Ser/Thr were detected only in the cis-Golgi compartment. In contrast, the two Golgi markers colocalized with the MUC2 apomucin and protein disulphide isomerase in the RER of colon cancer cells. On the basis of these results, we propose that in colon cancer cells a redistribution of molecules normally present in the Golgi apparatus takes place; this alteration may contribute to the abnormal glycosylation of proteins and lipids associated with neoplastic transformation.

Cell Compartmentation↗
PubMed1993

Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations.

Mucin expression was analysed, in relation to cell growth, in parental HT-29 cells and in two populations of mucus-secreting HT-29 cells selected by adaptation to methotrexate (HT29-MTX) or 5-fluorouracil (HT29-FU). These two populations express mature mucins that differ in their immunoreactivity to antibodies against gastric (HT29-MTX) or colonic mucins (HT29-FU). In the parental population, at late confluency, only very few cells produce mucins or the MUC1 glycoprotein, this being consistent with the low level of expression of the mRNAs corresponding to the MUC1 to MUC5C mucin genes. In the HT29-MTX and HT29-FU populations, the appearance of mucus droplets, as shown by histochemistry and immunofluorescence, starts a few days after confluency, progressively involving a greater proportion of cells and reaching a steady state at late confluency. The MUC1 glycoprotein appears earlier, already being detectable in preconfluent cells. Its distribution is restricted to the apical surface of the cells and is distinct from that of the mucus droplets. In both populations the growth-related levels of MUC1 mRNA are concordant with the apparent levels of expression of the MUC1 glycoprotein. The levels of MUC2, MUC3, MUC4 and MUC5C mRNAs differ from one population to another and, within each population, according to the stage of the culture. The highest levels of MUC2 and MUC4 mRNAs are found in the HT29-FU cells, whereas the highest levels of MUC3 and MUC5C are found in the HT29-MTX cells, suggesting that the differences observed in the mature mucins expressed by either population may be related to which MUC genes are expressed. In both populations significant or even high levels of MUC mRNAs are already present in early cultures, i.e. at a stage when the mature mucins are not yet detectable, suggesting that mucin maturation is a later event.

Blotting, Northern↗
PubMed1993

[Differentiation of human colon cancer cells: a new approach to cancer of the colon].

Our purpose is to demonstrate that some human colon cancer cells are able to express the same differentiation features as normal intestinal epithelial cells and to report experimental data which suggest that these particular cells are spontaneously resistant and adaptable to anticancer drugs. The concept of normal differentiation of colon cancer cells is supported by observations made with two cultured cell lines, Caco-2 and HT-29. The cell line Caco-2 expresses spontaneously and homogeneously an enterocytic differentiation. The cell line HT-29 is heterogeneous as it contains a small proportion (< 5%) of differentiated cells of either enterocytic or mucus-secreting type. Homogeneous populations of differentiated HT-29 cells of either type have been isolated by pressure selection. In order to investigate whether the pressure associated with anti-cancer drugs would result in the selection of differentiated populations, HT-29 cells were cultured in the presence of increasing concentrations of methotrexate and 5-fluorouracil. The resulting resistant populations were found to be totally differentiated. This supports the view that the small proportion of parental cells which are able to differentiate are also able to spontaneously resist to drug pressure. These results imply that the concept of cellular differentiation should be considered by pathologists. They also imply that basic research should be developed in order to unravel which mechanisms of drug resistance are specifically associated with the ability of the cells to differentiate.

Animals↗
PubMed1992

Expression and characterization of mucins associated with the resistance to methotrexate of human colonic adenocarcinoma cell line HT29.

A series of experiments were conducted to study synthesis and secretion of mucin in mucus-secreting subpopulations of HT29 human colonic adenocarcinoma cells selected by resistance to methotrexate (MTX). Mucin was quantitated by [3H]glucosamine labeling and chromatography on Sepharose CL-4B. The mucinous nature of the labeled high molecular weight glycoprotein was verified by alkaline borohydride treatment, cesium chloride density gradient ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of these experiments demonstrated that MTX-treated cells have increased amounts of mucin in medium, cytosol, and membrane fractions. This was associated with the increase in the activities of polypeptidyl-N-acetylgalactosaminyltransferase and beta-1,3-galactosyltransferase compared to control cells. DEAE-Sephacel chromatography of [3H]glucosamine-labeled high molecular weight glycoproteins suggest that MTX-treated cells are less acidic compared to controls. Using complementary DNA probes for two distinct human intestinal mucins (MUC2 and MUC3) and one mammary mucin (MUC1), it was found that MTX-treated cells expressed more mucin messages compared to untreated cells. These results were consistent with immunoblots using anti-MRP (MUC2 repeat peptide), anti-M3P (MUC3 repeat peptide), 139H2 (MUC1 peptide), anti-T (peanut lectin), anti-Tn (91S8), and anti-sialosyl Tn (JT10e) antibodies. These data indicate that MTX-resistant HT29 cells show enhanced secretion and synthesis of mucin as well as expression of MUC1-, MUC2-, and MUC3-related mucin polypeptide epitopes.

Cell Differentiation↗
PubMed1992

Expression and localization of GLUT-5 in Caco-2 cells, human small intestine, and colon.

The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically in the gut. Northern blots indicate that Caco-2 cells express GLUT-1 and GLUT-5 mRNAs but not the mRNA coding for the basolateral glucose transporter GLUT-2. The level of GLUT-5 mRNA is growth dependent, being detectable only in postconfluent differentiated cells. In addition, the expression of GLUT-5 increases with the number of cell passages and is approximately 10 times higher in later passages (passage 184) than in early ones (passage 26). With the use of polyclonal antibodies directed against the COOH-terminus of GLUT-5, indirect immunofluorescence and Western blotting indicate that GLUT-5 is mainly localized to the brush border of Caco-2 cells. GLUT-5 is also found to be associated with the brush border of epithelial cells from fetal and normal adult human small intestine, but is absent from the colon.

Adult↗
PubMed1991

Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line.

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

Blotting, Western↗
PubMed1991

Adaptation to 5-fluorouracil of the heterogeneous human colon tumor cell line HT-29 results in the selection of cells committed to differentiation.

The HT-29 cell line contains a small proportion of differentiated polarized, enterocytic and mucus-secreting cell types (less than 95%) which can be selected under various pressure conditions, e.g., glucose deprivation or methotrexate. The purpose of the present work was to investigate whether this also applies to 5-fluorouracil (FUra). Stepwise adaptation of exponentially growing cells to 1, 5, 10 and 20 microM FUra results, after a phase of high mortality, in the emergence of adapted sub-populations with stable growth rates and curves, and IC50 6, 18, 37, and 110 times higher than in untreated cells respectively. FUra-adapted cells are all differentiated, according to 2 phenotypes: (I) polarized dome-forming cells which express carcinoembryonic antigen at their apical surface and (2) goblet cells which secrete a mucus of colonic immunoreactivity. These phenotypes are present in the parental population and are different from those selected e.g., by glucose deprivation or methotrexate. This differentiation pattern is maintained when the cells are subcultured in drug-free medium. Resistance to FUra is acquired through gene amplification as substantiated by a 4- to 6-fold increase of thymidylate synthase gene copies in cells stably adapted to the drug. Whether the same mechanism or others are responsible for the first steps of resistance to FUra remains to be elucidated. Altogether, these results support the hypothesis that some of the cells which are present in the parental line and are committed to differentiation possess advantages which allow them to immediately resist and secondarily adapt to FUra. Comparison of the differentiation characteristics of FUra-adapted cells with those from cells selected under other pressure conditions suggests that resistance and adaptation to either type of pressure may depend on the differentiated phenotype to which the cells are committed.

Blotting, Northern↗
PubMed1991

Increased growth adaptability to 5-fluorouracil and methotrexate of HT-29 sub-populations selected for their commitment to differentiation.

Adaptation of the heterogeneous human colon carcinoma cell line HT-29 to lethal concentrations of methotrexate (MTX) and 5-fluorouracil (FUra) was shown to result in the emergence of sub-populations of cells all stably committed to differentiation. It was postulated that these populations result from selection of a few cells present in the parental line which possess, associated with their ability to differentiate, particular advantages allowing them to adapt to adverse conditions such as MTX or FUra. The purpose of the present study was to further verify this hypothesis by investigating whether HT-29 sub-populations selected for the commitment of all cells to differentiation would spontaneously be more resistant and adaptable than the parental cells to MTX and FUra. This study included a mucus-secreting clone (HT29-16E), a transporting clone (HT29-19A), and an enterocytic population selected by glucose deprivation (HT29-Glc-/+). Although all 3 populations show only a slight increase in their spontaneous resistance to both drugs, as substantiated by the values of IC50 which are only less than 2-fold higher than in parental cells, they are more adaptable as judged by growth curves, over a 50-day culture period, under exposure to 1 microM FUra and 0.1 microM MTX. In sharp contrast to parental cells, which, at these concentrations, show a high rate of mortality, all 3 populations, although growing slowly, reach densities more or less close, depending on the drug and population concerned, to that of control untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗
PubMed1991

Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells.

Postconfluent cultures of HT-29 cells form a heterogeneous multilayer of which greater than 95% of the cells are undifferentiated. In contrast, when stably adapted to normally lethal concentrations of methotrexate (10(-6)-10(-5) M), they form a monolayer of gobletlike cells (Lesuffleur et al., 1990) which secrete large quantities of mucins and display a discrete brush border with the presence of villin, dipeptidylpeptidase-IV, and carcinoembryonic antigen. When adapted to even higher concentrations of methotrexate (10(-4) and 10(-3) M) there is a shift in the pattern of differentiation from gobletlike to dome-forming absorptive-like cells. These cells still display an apical brush border which expresses villin and dipeptidylpeptidase-IV, but no longer express significant levels of mucins and carcinoembryonic antigen. This shift of differentiation coincides with a sudden amplification of the gene coding for dihydrofolate reductase and an increased activity of the enzyme.

Blotting, Southern↗
PubMed1990

Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells.

The purpose of this work was to investigate whether the phenomenon of metabolic adaptation of HT-29 cells to glucose deprivation and subsequent emergence of differentiated subpopulations (A. Zweibaum et al., J. Cell. Physiol., 122: 21-29, 1985) also applies to anticancer drugs that act at a metabolic level like methotrexate (MTX). Stepwise adaptation of exponentially growing HT-29 cells to increasing concentrations of MTX (10(-7), 10(-6), and 10(-5) mol) results, after a phase of high mortality, in the emergence of subpopulations with stable growth rates and curves close to those of untreated control cells. In contrast to control cells which are heterogenous and contain, after confluency, only a small proportion of differentiated cell types (less than 4%), postconfluent cultures of MTX-adapted cells are totally differentiated. Cells adapted to 10(-7) M MTX form a mixed population of columnar absorptive and mucus cells; at higher concentrations cells are almost exclusively of the mucus-secreting type. All cells, whether mucus-secreting or not, develop an apical brush border which strongly expresses dipeptidylpeptidase IV, carcinoembryonic antigen, and villin. These differentiation features, which resemble those of fetal colon, are associated with decreased rates of glucose consumption and lactic acid production. Both differentiation characteristics and metabolic changes are stably maintained when the cells are subcultured in the absence of the drug. Like the original population, MTX-adapted cells are tumorigenic in nude mice. We propose that cells which are able to differentiate and which are the origin of the small proportion of differentiated cell types found in postconfluent cultures of the original cell line possess an advantage which allows them to be adaptable to "metabolic stress" conditions.

Carcinoma↗
PubMed1990

N-glycosylation modification of proteins is an early marker of the enterocytic differentiation process of HT-29 cells.

The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.

Cell Differentiation↗
PubMed1989

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↗