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.

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.

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.

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.

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.

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.

Differential effect of GalNAcalpha-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern.

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcalpha-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G(-) and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G(-) cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcalpha-O-bn metabolites. We observed a strong inhibition of alpha2,3-sialylation of glycoproteins in HT-29 G(-) cells correlated to the high expression of alpha2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galbeta1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of alpha1,2-fucosylation was observed, in correlation with the high expression of alpha1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G(-) cells, GalNAcalpha-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcalpha-O-bn metabolites.

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis.

BACKGROUND & AIMS: Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. METHODS: A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. RESULTS: In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. CONCLUSIONS: 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.

Postmitotic differentiation of colon carcinoma caco-2 cells does not prevent reentry in the cell cycle and tumorigenicity.

Our purpose was to analyze whether postmitotic Caco-2 colon cancer cells, although they express most of the differentiation characteristics of terminally differentiated intestinal epithelial cells, still maintain, unlike normal cells, a proliferation potential. Experiments were performed with clone TC7 of the Caco-2 cell line. Dividing TC7 cells are undifferentiated and express detectable levels of thymidylate synthase (TS) and cytochrome P450 1A1 (CYP1A1) mRNAs. When reaching confluence TS and CYP1A1 are downregulated, mitosis is no longer detectable, and differentiation takes place, as demonstrated by appearance and increasing levels of differentiation-associated marker mRNAs (e.g., sucrase-isomaltase (SI), dipeptidylpeptidase-IV (DPP-IV) or GLUT5), increasing activities of sucrase and DPP-IV, and increasing expression, on immunofluorescence analysis, of SI on the surface of the cell layer. Trypsinization and seeding of late postconfluent cells (day 30) expressing complete differentiation results within 24 h in upregulation of TS and CYP1A1, a concomitant and dramatic disappearance of differentiation marker mRNAs associated with a decrease in sucrase and DPP-IV activities, and delayed resumption of cell division. This is followed, after the cells have reached confluence again, by downregulation of TS and CYP1A1 and resumption of cell differentiation. The ability of differentiated cells to dedifferentiate was further confirmed by wounding the cell layer of late postconfluent differentiated cultures: within 24 h following the wound, cells migrate from the wound edge and dedifferentiate, as demonstrated by transmission electron microscopy and disappearance of SI from the cell surface of migrating cells. Late postconfluent differentiated cells were tumorigenic in nude mice. These results raise the question of the validity of the concept of differentiation therapy when applied to colon cancer cells.

Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.

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.

Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype.

Adaptation of HT-29 cells to increasing concentrations of methotrexate (MTX) results in the selection of differentiated populations which show sequential dose-dependent changes of their differentiated phenotype with, at the highest concentrations (0.1 and 1 mM), a shift of differentiation from a mucus-secreting to an enterocytic phenotype coinciding with an amplification of the DHFR gene. We show here that DHFR gene amplification itself does not play a role in the shift of differentiation. An alternative explanation is the presence, within the mucus-secreting population, of an undetectable minor population of cells committed to enterocytic differentiation and able to develop resistance to higher concentrations of MTX. This was confirmed by cloning the population of cells resistant to 10 microM MTX. Out of 19 isolated clones, 17 were found to be mucus-secreting and 2 enterocytic. We tested 9 of these clones for their ability to develop resistance to 0.1 mM MTX: only 1 of enterocytic phenotype, was found to develop resistance to this higher concentration and to amplify the DHFR gene. The ability of enterocytic cells to develop resistance to elevated MTX concentration through amplification of the DHFR gene was demonstrated in another enterocytic HT-29 population selected by glucose deprivation. Enterocytic cells resistant to 10 microM MTX were also found, unlike mucus-secreting cells, to be readily adaptable to 5-fluorouracil, this occurring without amplification of the thymidylate synthase gene. Together these results highlight a previously uncharacterized relationship between commitment to enterocytic differentiation of colon-cancer cells and their ability to develop resistance to MTX and 5-fluorouracil.

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.

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.

Characterization of a mucin cDNA clone isolated from HT-29 mucus-secreting cells. The 3' end of MUC5AC?

HT-29 cells resistant to 10(-6) M methotrexate (HT29-MTX) secrete mucins with gastric immunoreactivity (Lesuffleur, T., Barbat, A., Dussaulx, E., and Zweibaum, A. (1990) Cancer Res. 50, 6334-6343). A 3310-base pair mucin cDNA clone (L31) was isolated from an HT29-MTX expression library using a polyclonal serum specific for normal gastric mucosa. It shows a high level of identity (98.6%) to clone NP3a isolated from a nasal polyp cDNA library (Meerzaman, D., Charles, P., Daskal, E., Polymeropoulos, M. H., Martin, B. M., and Rose, M. C. (1994) J. Biol. Chem. 269, 12932-12939). However, as a result of changes in reading frame, the 1042-amino acid deduced peptide contains four regions of a low similarity to the NP3a peptide. The amino acid sequence shows 36.3% similarity to part of the carboxyl-terminal sequence of MUC2 including the so-called D4 domain and 21.3% to the pro von Willebrand factor. A short amino acid sequence is similar to cysteine-rich sequences repeated in tracheobronchial, gastric, and colonic mucin cDNAs. The gene corresponding to L31 is located in the mucin gene cluster on chromosome 11p15.5. The patterns of mRNA expression were indistinguishable from those revealed with the JER58 probe (MUC5AC). Southern blot analysis indicates that the L31 and JER 58 sequences are within 20 kilobase pairs of each other. Together, these results suggest that L31 clone is the 3' end of MUC5AC.

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.

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.