Multicentre cohort study of antihypertensive and lipid-lowering therapy cessation after bariatric surgery.

BACKGROUND: Few studies have assessed changes in antihypertensive and lipid-lowering therapy after bariatric surgery. The aim of this study was to assess the 6-year rates of continuation, discontinuation or initiation of antihypertensive and lipid-lowering therapy after bariatric surgery compared with those in a matched control group of obese patients. METHODS: This nationwide observational population-based cohort study used data extracted from the French national health insurance database. All patients undergoing gastric bypass or sleeve gastrectomy in France in 2009 were matched with control patients. Mixed-effect logistic regression models were used to analyse factors that influenced discontinuation or initiation of treatment over a 6-year interval. RESULTS: In 2009, 8199 patients underwent primary gastric bypass (55·2 per cent) or sleeve gastrectomy (44·8 per cent). After 6 years, the proportion of patients receiving antihypertensive and lipid-lowering therapy had decreased more in the bariatric group than in the control group (antihypertensives: -40·7 versus -11·7 per cent respectively; lipid-lowering therapy: -53·6 versus -20·2 per cent; both P < 0·001). Gastric bypass was the main predictive factor for discontinuation of therapy for hypertension (odds ratio (OR) 9·07, 95 per cent c.i. 7·72 to 10·65) and hyperlipidaemia (OR 11·91, 9·65 to 14·71). The proportion of patients not receiving treatment at baseline who were subsequently started on medication was lower after bariatric surgery than in controls for hypertension (5·6 versus 15·8 per cent respectively; P < 0·001) and hyperlipidaemia (2·2 versus 9·1 per cent; P < 0·001). Gastric bypass was the main protective factor for antihypertensives (OR 0·22, 0·18 to 0·26) and lipid-lowering medication (OR 0·12, 0·09 to 0·15). CONCLUSION: Bariatric surgery is associated with a good discontinuation of antihypertensive and lipid-lowering therapy, with gastric bypass being more effective than sleeve gastrectomy.

Health care utilization by men with prostate cancer during the year before their death: A 2015 population-based study.

INTRODUCTION: To study the characteristics and health care utilization of men with prostate cancer (PCa) during their last year and last month of life, as these data have been rarely reported to date. SUBJECTS AND METHOD: Men covered by the national health Insurance general scheme (77% of the French population) treated for PCa (2014-2015), who died in 2015 were identified in the national health data system, including reimbursed hospital and outpatient care, and their causes of death. RESULTS: A total of 11,193 men (mean age: 81 years, SD: 9.6) were included. Almost 58% of these men died in a short-stay hospital (SSH), 4% died in hospital-at-home, 9% died in Rehab, 9% died in skilled nursing homes and 21% died at home. During the last year of life, almost all men were hospitalised at least once in SSH and 47% received hospital palliative care (HPC), immediately prior to death in 8% of cases. During the last month of life, 76% of men were hospitalised at least once in SSH, 43% attended an emergency department and 14% were admitted to intensive care, 7% received a chemotherapy session, and 24% received an antineoplastic agent dispensed by a retail pharmacy. Cancer was the main cause of death for 63% of men, corresponding to PCa in 40% of cases, and cardiovascular disease was the main cause of death for 13% of men with marked variations according to age, place of death, and use of HPC. The mean cost reimbursed per man during the last year of life was €38,750 (€48,601 including HPC). CONCLUSIONS: In France, end-of-life management of men with PCa, regardless of the cause of death, is centered on SSH and HPC, essentially at the time of death. Certain indicators of end-of-life management were particular high. LEVEL OF EVIDENCE: 4.

Long-term follow-up after bariatric surgery in a national cohort.

BACKGROUND: Lifelong medical follow-up is mandatory after bariatric surgery. The aim of this study was to assess the 5-year follow-up after bariatric surgery in a nationwide cohort of patients. METHODS: All adult obese patients who had undergone primary bariatric surgery in 2009 in France were included. Data were extracted from the French national health insurance database. Medical follow-up (medical visits, micronutrient supplementation and blood tests) during the first 5 years after bariatric surgery was assessed, and compared with national and international guidelines. RESULTS: Some 16 620 patients were included in the study. The percentage of patients with at least one reimbursement for micronutrient supplements decreased between the first and fifth years for iron (from 27.7 to 24.5 per cent; P < 0.001) and calcium (from 14·4 to 7·7 per cent; P < 0·001), but increased for vitamin D (from 33·1 to 34·7 per cent; P < 0·001). The percentage of patients with one or more visits to a surgeon decreased between the first and fifth years, from 87·1 to 29·6 per cent (P < 0·001); similar decreases were observed for visits to a nutritionist/endocrinologist (from 22·8 to 12·4 per cent; P < 0·001) or general practitioner (from 92·6 to 83·4 per cent; P < 0·001). The mean number of visits to a general practitioner was 7·0 and 6·1 in the first and the fifth years respectively. In multivariable analyses, male sex, younger age, absence of type 2 diabetes and poor 1-year follow-up were predictors of poor 5-year follow-up. CONCLUSION: Despite clear national and international guidelines, long-term follow-up after bariatric surgery is poor, especially for young men with poor early follow-up.

Classic and emergent psychosocial work factors and mental health.

BACKGROUND: Little is known about associations between emergent psychosocial work factors and mental health. AIMS: To explore associations between classical and emergent psychosocial work factors and depression and anxiety symptoms in employees in France. METHODS: A national cross-sectional study (the SUrveillance Médicale des Expositions aux Risques professionnels (SUMER) survey) assessed psychosocial work factors including psychological demands, decision latitude, social support, reward and its sub-dimensions (esteem, job security and job promotion), bullying, verbal abuse, physical violence and sexual assault, long working hours, shift and night work, unsociable work days, predictability and demands for responsibility. We also measured depression and anxiety symptoms using the Hospital Anxiety and Depression scale. We used gender-stratified generalized linear models to adjust for age, occupation and economic activity. RESULTS: A total of 26883 men and 20079 women participated (response rate 87%). Low decision latitude, high psychological demands, low social support, low reward, bullying and verbal abuse were associated with depression and anxiety in both genders (ß coefficients from 0.14 to 1.40). In men, low predictability was associated with both depression and anxiety (ß = 0.12 [95% confidence interval (CI) 0.01, 0.24] and 0.19 [95% CI 0.06, 0.32]) and long working hours were associated with anxiety (ß = 0.48 [95% CI 0.27, 0.69]). The strongest associations were observed for bullying, reward (especially esteem) and psychological demands. Using a less conservative approach, we found more factors to be significantly associated with mental health symptoms. CONCLUSIONS: Most psychosocial work factors studied are associated with depression and/or anxiety symptoms. Comprehensive prevention policies may help to reduce exposure to psychosocial work factors, including emergent ones, and improve mental health at work.

Long-term chronic diseases and 1-year use of healthcare services by children under 18 years of age during 2018-2019: A French nationwide observational study.

OBJECTIVE: Among children younger than 18 years, the prevalence of long-term chronic diseases (LTDs) is not well known in France, nor the frequency of the use of healthcare services. This nationwide observational study focused on both topics over a 1-year period following the birth or birthday of French children in 2018 and compared the LTD status and use of healthcare. MATERIALS AND METHODS: We selected children living in mainland France from the national health data system (SNDS). It includes data concerning the LTD status, which guarantees 100% reimbursement for related healthcare expenditures. We calculated the median and interquartile range (IQR) for the prevalence of LTDs and the rate of children using healthcare services at least once during the year. RESULTS: We included 13.211 million children (51.2% boys), of whom 4% had at least one LTD (boys: 4.6%, girls: 3.3%). Mental and behavioral disorders were the most frequent cause (1.6%). At least one visit to a general practitioner (GP) or pediatrician was found for 88% of children (median: 3, IQR: 2-6): 98% for children under 1 year of age and 81% for children aged 14-17 years. A pediatrician was visited by 17% of children, another specialist by 39%, a dentist by 37%, with peaks of about 60% at the ages of 6, 9, and 12 years; 8% visited a nurse and 7% visited a physiotherapist. At least one emergency department visit was recorded for 24% of children (42% <1 year) and one short-stay hospitalization (SSH) for 9%. Regional variations were observed. Children with LTDs more frequently used all services, such as specialist visits (50% vs. 40%), ED visits (32% vs. 23%), SSHs (26% vs. 8% and 15% vs. 4.0% for one night or more), and psychiatric hospital admissions (5% vs. 0.1%). CONCLUSION: Most children saw a GP or pediatrician during the year and children with an LTD showed more frequent use. Nevertheless, outpatient visits appeared to be underutilized with respect to recommendations or free-of-charge prevention visits, such as for dentists. More detailed studies are required to identify factors associated with the use of healthcare services in France, for example, studies including the deprivation index and regional variations.

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.

GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

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.

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.

Specific secretion of gel-forming mucins and TFF peptides in HT-29 cells of mucin-secreting phenotype.

Trefoil factor family (TFF) peptides are typical secretory products of mucin-producing cells, e.g. of the gastrointestinal tract. Here, the expression and secretion of mucins and TFF peptides was studied in the HT-29 cell line throughout cellular growth and differentiation in relation to a mucin-secreting (HT-29 MTX) or an enterocyte-like (HT-29 G(-)) phenotype. mRNAs of several MUC and TFF genes were expressed in both cell subpopulations. However, for most MUC and TFF genes, the expression appeared strongly induced with the differentiation into the mucin-secreting phenotype. On the other hand, TFF2 was specifically expressed in the mucin-secreting HT-29 MTX cells. The differentiation of HT-29 MTX cells into the mucin-secreting phenotype was characterised by secretion of the gel-forming mucins MUC2, MUC5AC, and MUC5B, however, according to a different pattern in the course of differentiation. A significant amount of TFF1 and TFF3 was secreted after differentiation, also according to a different pattern, whereas TFF2 was only faintly detected. Secretagogues, known to induce the secretion of mucus, increased the secretion of all three TFF peptides. In contrast, neither a secretory mucin nor a TFF peptide was found in the culture medium of HT-29 G(-) cells. Overlay assays indicated that HT-29 MTX mucins bound to secretory peptides of HT-29 MTX cells with relative molecular mass similar to TFF peptides. TFF1 and TFF3 were specifically localised in the mucus layer of HT-29 MTX cells by confocal microscopy. Finally, the secretion of TFF peptides and mucins appears as a co-ordinated process which only occurs after differentiation into goblet cell-like phenotype.

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.

Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations.

Previous studies from our laboratory have shown that HT29 cells selected by adaptation to methotrexate (HT29-MTX) express mature mucins that differ in their immunoreactivity to antibodies against gastric mucin and in the level of one of two major gastric mucin MUC5AC (MUC5) mRNA compared with parental HT29 cells. In this study, we examined the expression of another major gastric mucin, MUC6 mRNA, as well as that of MUC2, -3 and -5 mRNAs in HT29-MTX cells. We also examined their relationship to mucin-related antigen expression and biological properties of the cells such as adhesion to matrigel and E-selectin and in vitro invasiveness, liver colonising activity and degree of differentiation of nude mouse xenograft. Slot blot and Northern analysis revealed markedly increased levels of MUC5 mRNA but no change in MUC6 mRNA level in HT29-MTX cells compared with parental HT29 cells which express barely detectable levels of MUC6 mRNA. A nuclear run-on study showed that MUC5 mRNA was up-regulated at the transcriptional level. The marked increase in MUC5 mRNA was associated with a significant increase in the expression of human gastric mucin and apomucin antigens in HT29-MTX cells. When the adhesive capacity of two cell lines was compared, HT29-MTX cells showed significantly lower adhesion to E-selectin consistent with their lower expression of sialyl Le(x) and sialyl Le(a) antigens compared with HT29 cells. HT29-MTX cells also showed lower adhesive capacity to matrigel than HT29 cells. Interestingly, HT29-MTX cells exhibited significantly decreased liver colonisation capacity in nude mice following splenic vein injection. Furthermore, nude mouse xenograft tumours produced by HT29-MTX cells exhibited a significantly greater degree of differentiation, consisting of mucin-secreting glands than those produced by HT29 cells. In conclusion, these results indicate a shift of predominantly colonic-type mucins to the gastric type, specifically the surface epithelial cell type (MUC5) but not the mucous neck cell or antral gland type (MUC6) in HT29-MTX cells and strongly suggest that altered regulation of mucin genes and the degree of differentiation in cancer cells may be responsible for the altered biological behaviour of these cells.

Inhibition of the glycosylation and alteration in the intracellular trafficking of mucins and other glycoproteins by GalNAcalpha-O-bn in mucosal cell lines: an effect mediated through the intracellular synthesis of complex GalNAcalpha-O-bn oligosaccharides.

To address the function of carbohydrates in mucins, GalNAcalpha-O-bn has been used in in vivo experiments on several human mucosal cultured cells as a potential competitor of the glycosylation of N-acetylgalactosamine residues. GalNAcalpha-O-bn is metabolized by glycosyltransferases expressed in the cell, and give rise to different internal derivatives starting in particular from the formation of the disaccharide Galalpha1-3GalNAcalpha-O-bn. In this line, GalNAcalpha-O-bn exposure inhibits peripheral glycosylation according a cell-type specific manner. The metabolic alterations are very important in HT-29 cell line, leading to a massive accumulation of GalNAcalpha-O-bn oligosaccharide derivatives and to a strong inhibition of the terminal elongation of O-glycans by alpha2,3 sialyltransferase ST3Gal I. GalNAcalpha-O-bn treatment also induced alterations at the cellular level, exhibiting a large scale in HT-29 cells, i.e. 1) an inhibition of mucin secretion, 2) a blockade in the targeting of some membrane glycoproteins (brush border glycoproteins such as dipeptidylpeptidase IV, carcinoembryonic antigen and the mucin-like glycoprotein MUC1, and the basolateral cell adhesion molecule CD44), 3) an inhibition in the processing of lysosomal enzymes. Morphological abnormalities have been evidenced in GalNAcalpha-O-bn treated cells, in particular the accumulation of numerous intracellular vesicles in HT-29 cells. Taken together, these data suggest that O-glycosylation might be involved in the regulation of the targeting of O-glycosylproteins through carrier vesicles.

[Expression of laminin is correlated to the differentiation of human colonic cancer cells]. / L'expression de la laminine est corrélée à la différenciation des cellules cancéreuses coliques humaines.

The level and molecular composition of laminin, a major basement membrane glycoprotein formed of three chains (A, B1 and B2) have been analyzed in various human colonic cancer cells (Caco-2 and HT29). The synthesis of laminin over a 24 h period, corresponding to cellular and secreted molecules purified by affinity chromatography, was the highest in the more differentiated cells. Immunocytochemical detection of the constituent chains of laminin in permeabilized cells as well as after separation on polyacrylamide gels showed that A, B1 and B2 chains were expressed in Caco-2 cells, whereas A chain was not detected in HT29 cells. When cancer cells were cultured on a monolayer of confluent fibroblastic cells, laminin was deposited at the basement membrane level only in the case of the most differentiated cells. In an attempt to define the role of laminin-A chain, transfection of Caco-2 cells with A chain antisense cDNA was performed; among the clones obtained, 3 were deficient for the target polypeptide. The consequences of the absence of laminin-A chain on basement membrane formation, cellular differentiation and tumor invasion will be currently determined.

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.

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)

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.

Resistance of the human colon carcinoma cell line HCT-8 to methotrexate results in selection of cells with features of enterocytic differentiation.

Results obtained previously with the human colon carcinoma cell line HT-29 have shown that the ability of the cells to develop resistance against methotrexate (MTX) or 5-fluorouracil is restricted to cells committed to differentiate. With the aim of investigating whether this observation is cell type-specific or more general, we have extended our studies to another colon cell line, HCT-8. We have compared HCT-8 parental cells and the MTX-resistant subline HCT8-MTX using transmission electron microscopy and immuno-fluorescence detection of markers of cell polarity and differentiation. Post-confluent parental HCT-8 cells appeared highly heterogeneous and occurred in clusters of piled-up cells in which the majority were unpolarized and undifferentiated, with a minority exhibiting features of enterocyte-like cells. In contrast, HCT8-MTX cells formed domes and appeared as a monolayer of polarized cells with tight junctions and a discrete apical brush border which expressed villin, dipeptidylpeptidase-IV, CEA and the epithelial mucin MUC1. Together, our results suggest that, as in HT-29 cells, induction of resistance to MTX of HCT-8 cells results in the selection of differentiated cell types.