Circulating plasma phospholipid fatty acids and risk of pancreatic cancer in a large European cohort.

There are both limited and conflicting data on the role of dietary fat and specific fatty acids in the development of pancreatic cancer. In this study, we investigated the association between plasma phospholipid fatty acids and pancreatic cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. The fatty acid composition was measured by gas chromatography in plasma samples collected at recruitment from375 incident pancreatic cancer cases and375 matched controls. Associations of specific fatty acids with pancreatic cancer risk were evaluated using multivariable conditional logistic regression models with adjustment for established pancreatic cancer risk factors. Statistically significant inverse associations were found between pancreatic cancer incidence and levels of heptadecanoic acid (ORT3-T1 [odds ratio for highest versus lowest tertile] =0.63; 95%CI[confidence interval] = 0.41-0.98; ptrend = 0.036), n-3 polyunsaturated α-linolenic acid (ORT3-T1 = 0.60; 95%CI = 0.39-0.92; ptrend = 0.02) and docosapentaenoic acid (ORT3-T1 = 0.52; 95%CI = 0.32-0.85; ptrend = 0.008). Industrial trans-fatty acids were positively associated with pancreatic cancer risk among men (ORT3-T1 = 3.00; 95%CI = 1.13-7.99; ptrend = 0.029), while conjugated linoleic acids were inversely related to pancreatic cancer among women only (ORT3-T1 = 0.37; 95%CI = 0.17-0.81; ptrend = 0.008). Among current smokers, the long-chain n-6/n-3 polyunsaturated fatty acids ratio was positively associated with pancreatic cancer risk (ORT3-T1 = 3.40; 95%CI = 1.39-8.34; ptrend = 0.007). Results were robust to a range of sensitivity analyses. Our findings suggest that higher circulating levels of saturated fatty acids with an odd number of carbon atoms and n-3 polyunsaturated fatty acids may be related to lower risk of pancreatic cancer. The influence of some fatty acids on the development of pancreatic cancer may be sex-specific and modulated by smoking.

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro.

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1. MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 x 10(-6) M and 10(-5) M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes.

Characterization of two pathological tau protein, variants in Alzheimer brain cortices.

Tau proteins were detected in brain tissue homogenates from 10 patients with Alzheimer's disease versus 10 age-matched controls using the immunoblot technique and 2 polyclonal antibodies: anti-paired helical filaments (PHF) and anti-human native Tau proteins. In control brains, both antisera detected identically the normal set of Tau proteins, with molecular weight (MW) ranging from 45 to 62 kDa. Moreover, in association areas of neocortex from Alzheimer brains, the antisera detected 2 additional Tau variants of 64 and 69 kDa. Tau 64 and 69 were not found in regions of Alzheimer brains where the Alzheimer pathology was absent (caudate nucleus or cerebellum for example). The heavy MW of Tau 64 and 69 is due to their phosphorylation state as shown by the decrease of their MW after alkaline phosphatase treatment. Therefore, Tau 64 and Tau 69 are specific markers of the Alzheimer's disease neuronal degenerating process and their characterization demonstrates that an abnormal phosphorylation of Tau really occurs during the disease. Tau 64 and 69 were isolated with normal Tau proteins while the PHF were insoluble. Therefore, Tau proteins are likely to be abnormally phosphorylated prior to their incorporation in the PHF structure. Consequently, they might appear before the lesions and might be instrumental for the search of biochemical deregulations that precede the neurofibrillary degeneration.

[Direct demonstration of abnormal phosphorylation of Tau microtubular proteins in Alzheimer's disease]. / Démonstration directe d'une phosphorylation anormale des protéines microtubulaires Tau au cours de la maladie d'Alzheimer.

Two polyclonal antibodies, the first raised against Alzheimer's disease PHF and the second raised against human native Tau proteins, led us to find two Tau proteins with an abnormal molecular weight of 64 and 69 kDa in Alzheimer brain cortices. Tau 64 and Tau 69 were never detected in control brains. The molecular weight of Tau 64 and 69 dramatically decreased after dephosphorylation by the alkaline phosphatase, showing that they are abnormally phosphorylated. This is the first report demonstrating their specific presence in brain regions having the Alzheimer pathology. They could be a very useful tool for the study of the early events that lead to neuronal death.

Chemical modification of thiol group(s) in protein: application to the study of anti-microtubular drugs binding.

1. Different chemical procedures such as performic oxidation, carboxymethylation, carboxyethylation, aminoethylation, cyanylation, acylation, arylation etc. and addition of thiols to activated double bonds, titration of thiols with DTNB (Dithiobis-Nitro-Benzoate) and the reaction of thiols with organomercurials and the titration with p-chloro-mercuri-benzoate (PCMB) etc. are cited and discussed. Their chemical reactions are shown in the figures. 2. We describe in this paper that several chemicals interfere with microtubule assembly by combining with sulfhydryl residues. Reagents such as Cytochalasin-A and B, ethylacetylacrylate, FDNB (fluorodinitrobenzene), NEM (N-ethyl-maleimide), diamide, EBI (ethylene-bis-iodoacetamide, ethacrynic acid, methal ions, methylmercury, triethyllead ion and CDDP (cis-dichlorodiammine-platinum-II) are cited and their mechanisms are discussed.

Phylogenetic studies of cardiac myosins from amphibia to mammals.

Comparison between pig atrial and ventricular myosins was performed on the light chains (using SDS-PAGE) and on the heavy chains (using Ca2+-ATPase measurements and NTCBA peptide mapping). Light chain composition of pig cardiac myosins was compared to three other species ones (frog, chicken and human). Up to birds, atrial and ventricular myosin light chain composition was identical whereas in mammals atrial and ventricular myosin light chain composition was different; likewise the heavy chains. Six cardiac myosin isoenzymes have been thus characterized. No correlation can be established between cardiac myosin light chain pattern and species evolution.

Biochemical and immunological studies of bovine and porcine neurofilament triplet proteins by peptide mapping after cyanogen bromide cleavage.

Peptide mapping of the three bovine and porcine neurofilament protein subunits ("L", "M" and "H") with apparent mol. wts of 70, 160 and 210 kDa were performed with CNBr, leading to the cleavage of methionyl bonds. We have obtained two characteristic large fragments with molecular weights of 85 kDa for the "M" bovine subunit and 135 kDa for the "H" subunit of bovine neurofilament. A comparison of the electrophoretic patterns of CNBr generated polypeptides of "L" subunit from beef and pig showed that they are highly related structures. The peptide mappings of CNBr peptides of "M" and "H" subunits from beef and pig were significantly different. Antibodies were raised against the 85 kDa and 135 kDa CNBr fragments. Immunoblotting results with anti-85 kDa and anti-135 kDa of beef are in favour of large differences of structure between the "M" subunits from pig and beef. The "H" proteins were very similar and they also showed that the C-terminal part of bovine "H" and "M" proteins share common antigenic determinants.

[Alzheimer's disease: study of the distribution of tau proteins constituting helical filament pairs in human central nervous tissue]. / Maladie d'Alzheimer: Etude de la distribution des protéines tau constitutives des paires de filaments en hélice dans le tissu nerveux central humain.

Tau proteins are the major components of Paired Helical Filaments (PHF) of Alzheimer's disease. Using the immunoblot technique and an antiserum against PHF, we have studied the distribution of Tau proteins in the different areas of normal human brains and Alzheimer brains. Tau proteins were clearly present in cortical grey matter but were difficult to detect in the white matter. In Alzheimer brains, we observed two differences: first, there is an important background due to the partial dissociation of the lesions containing Tau aggregates. Second, the profile of Tau proteins is modified, due to abnormal phosphorylation. Thus, Tau proteins are found in large amounts in the grey matter of the cortical areas and are not exclusively distributed in the axonal domain. The normal cortical distribution of Tau in the human brain correlates well with the distribution of histological lesions that contain PHF (neurofibrillary tangles and neuritic plaques) in the Alzheimer cortex.

A case-control study of gastric cancer and nutritional factors in Marseille, France.

A case-control study was conducted in Marseille (France) to investigate the relationship between usual diet and risk of gastric cancer. Patients with histologically confirmed gastric adenocarcinoma were identified in 8 major centres for gastric surgery. Controls were selected in specialized medical centres from patients undergoing functional reeducation for injuries or trauma, according to the age and sex distributions of the cases. The study involved 92 cases and 128 controls who were interviewed with a dietary history questionnaire on their usual diet during the year preceding first symptoms for cases, or preceding interview for controls. Odds ratios for specific foods were calculated after adjustment for age, sex, occupation and energy intake. A reduced risk was observed for consumption of raw vegetables (OR2: 0.55; OR3: 0.41 for the second and third tertiles, respectively), fresh fruit (OR2: 0.63; OR3: 0.50), vegetable oil (OR2: 0.60; OR3: 0.52), pasta and rice (OR2: 1.06; OR3: 0.50) whereas consumption of cakes and pastries (OR2: 1.02; OR3: 2.96), sugar and confectionery (OR2: 0.96; OR3: 1.68) was associated with an increased risk. An increased risk was found for intake of saturated fat (OR2: 1.49; OR3: 1.67), simple sugars (OR2: 1.18; OR3: 1.78) and calcium (OR2: 1.84; OR3: 2.57). A decreased risk was observed with intake of fiber (OR2: 0.49; OR3: 0.59), fibre from vegetables and fruit (OR2: 0.83; OR3: 0.53) and iron (OR2: 0.70; OR3: 0.41).

Nitrosamine, nitrate and nitrite in relation to gastric cancer: a case-control study in Marseille, France.

A case-control study on gastric cancer and diet was conducted in Marseille (France). Ninety-two patients with histologically confirmed adenocarcinoma and 128 controls undergoing functional reeducation for injuries or trauma were interviewed by a trained dietician using a dietary history questionnaire on their usual diet during the year preceding the first symptoms for cases, or preceding interview for controls. Intake of nitrite, nitrate and pre-formed N-nitrosodimethylamine (NDMA) from food was estimated using a food composition table compiled ad hoc. Odds ratios (ORs) were calculated after adjustment for age, sex, occupation and calorie intake. The results indicated that high intake of NDMA was associated with increased risk for gastric cancer. The ORs for the second and third tertile of NDMA intake were: OR2 = 4.13 (95% CI = 0.93 18.27) and OR3 = 7.00 (95% CI = 1.85 to 26.46). Intake of nitrate and nitrite was not associated with increased risk of stomach cancer. Consumption of vegetables was protective in general and independent of their estimated nitrate content.

Comparative studies of the neurofilament triplet protein peptide mapping by chemical cleavage.

Peptide mapping of the three neurofilament protein subunits with apparent mol. weights of 210 kDa, 160 kDa and 70 kDa was performed with two different reagents: CNBr, BNPS-Skatole leading to the cleavage of methionyl and tryptophanyl bonds respectively. With BrCN we obtained two large fragments resistant to the cleavage, with mol. wts of 85 kDa for the 160 kDa and 135 kDa for the 210 kDa neurofilament proteins respectively. These fragments were located on the C-terminal part of the proteins (the tails) and correspond to specific regions responsible for their physiological identity. On the other hand, the cleavage with BNPS-Skatole at the tryptophanyl bonds gave similar patterns. The 210 kDa and 160 kDa neurofilament proteins gave a doublet of high mol. wt resistant to the cleavage, corresponding very likely to the C-terminal part and 4 fragments of mol. wt between 30 and 40 kDa corresponding to the N-terminal part. The neurofilament triplet share a common 30.5 kDa fragment located on the N-terminal part. From these peptide mapping studies, we conclude that the two neurofilament subunit proteins with mol. wts of 160 kDa and 210 kDa are different but related structures and that the CNBr characterized cleavage fragments of mol. wt 85,000 and 135 kDa are suitable polypeptides for sequence and immunological studies of the C-terminal part of these proteins.

Isolation and characterization of ATP-dependent proteolytically active ubiquitin in cock testis.

1. We have successfully isolated and purified ubiquitin from cock testis by using an inhibitor, p-CMB (p-chloromercuribenzoate), which is one of the inhibitors specific for thiol-proteases and with the following procedures: heating up to 85 degrees C, ammonium sulfate fractionation, gel filtration on Sephadex G-75, chromatography on DE-52 and CM-11 and lyophilization. 2. Amino-acid analysis showed that Ub isolated from cock testis has 76 residues including 6 glycines. 3. Hydrazinolysis and carboxypeptidase digestion were also performed: the C-terminal residue is glycine. 4. The purity was checked by analytical SDS-PAGE and the isolated Ub exhibited only one band. 5. The Ub-dependent proteolysis experiment showed that this Ub was ATP-dependently proteolytically active. 6. In this paper we present evidence that a thiol enzyme is present during the purification procedure.

Massive in vitro synthesis of tagged oligosaccharides in 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside treated HT-29 cells.

Permanent exposure of differentiated HT-29 cells to the sugar analogue, 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha- O -bn) leads to marked effects upon the phenotypic properties of mucin-secreting or enterocyte-like HT-29 cells: an inhibition in the secretion of mucins, a blockade in the apical targeting of membrane brush border glycoproteins and a swelling of cells with intracellular accumulation of numerous vesicles. Folch extraction and partition of treated enterocyte-like HT-29 cells revealed a very important accumulation of orcinol and/or resorcinol reactive material in the upper phase (usually containing gangliosides), as compared with untreated HT-29 cells and with treated and untreated Caco-2 cells. Structural analysis indicated the accumulation of a series of GalNAcalpha- O -bn derived oligosaccharides, most of them with the common core Galbeta1-3GalNAcalpha- O -bn. These oligosaccharides contained residues of GlcNAc, Gal, Neu5Ac, or Fuc. In particular, the tagged sialyl-Lewis(x)was identified, as well as more complex sialylated derivatives, including the sialyl-Lewis(x)substituted by an additional Neu5Ac residue. The benzylated oligosaccharides were not significantly detected in the culture medium except for Galbeta1-3GalNAcalpha- O -bn. Upon reversion of the treatment, these derivatives dis-appeared from the cells within few days, however were not recovered as such in the culture medium. Intracellular degradation occurred with desialylation and defucosylation as the first steps. The spectacular accumulation of benzylated oligosaccharides in HT-29 cell, permanently exposed to GalNAcalpha- O -bn very likely plays an important role in the alterations of cellular processes previously described in this cell line. The HT-29 cell culture system also appears to be an efficient source of several tagged oligosaccharides.

Pathological proteins Tau 64 and 69 are specifically expressed in the somatodendritic domain of the degenerating cortical neurons during Alzheimer's disease. Demonstration with a panel of antibodies against Tau proteins.

Bundles of paired helical filaments (PHF) accumulate in the pyramidal neurons that degenerate during Alzheimer's disease. This neurofibrillary degeneration is highly correlated with clinical signs of dementia. During the degenerating process, Tau proteins, which are the major antigenic components of PHF, are abnormally phosphorylated and two pathological isoforms named Tau 64 and 69 are expressed. We have studied their immunoblot distribution in the cortical gray and white matter from different regions of normal and Alzheimer brains, to determine if the degenerating process preferentially affects the somatodendritic or the axonal domain. Two categories of antibodies were used. The first category consisted of anti-human native Tau, anti-Tau proteins from different vertebrates, anti-PHF, monoclonal antibody Alz-50 and an anti-C terminal repeated region of Tau. In control brains, these antibodies strongly detected normal Tau proteins in the gray matter while Tau immunodetection was weak in the white matter. In Alzheimer brain cortices, each antibody detected Tau 64 and 69 in gray matter extracts but not at all in white matter extracts. The second category of anti-Tau consisted of the anti-PHF saturated with normal brain protein extracts. This antiserum only probed the abnormally phosphorylated Tau proteins. It detected Tau 64 and 69 exclusively in the cortical gray matter of Alzheimer brains. Moreover, a 55-kDa Tau protein was also immunolabelled, which might be an intermediary form between normal Tau and Tau 64 and 69. Our results demonstrate that Tau proteins are normal and major components of the somatodendritic domain and that Tau pathology, reflected by the presence of Tau 64 and 69, affects preferentially this domain during Alzheimer's disease.

Stimulation of the secretion of latent cysteine proteinase activity by tumor necrosis factor alpha and interleukin-1.

OBJECTIVE: Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1). METHODS: Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti-cathepsin B antiserum. Immunolocalization of cathepsin B was studied on cell monolayers. RESULTS: Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells. This activity was increased after treatment with either TNF alpha or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B. By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide. An intracellular increase in cathepsin B after treatment with TNF alpha was also seen with immunohistochemical studies. CONCLUSION: TNF alpha (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.

The state of differentiation of HT-29 colon carcinoma cells alters the secretion of cathepsin D and of plasminogen activator.

We have studied the cellular content and the extracellular release of cathepsins B and D, and of plasminogen activator, in 2 different tumor cell populations before confluence and after late confluence: the HT-29 colon carcinoma cell line, which contains primarily undifferentiated cells, and a subpopulation derived from this cell line, which contains cells committed to differentiation into mucus-secreting goblet cells after confluence. In both populations, cellular cathepsin-B activity increased after confluence, and latent cathepsin B was found in all culture media. In the parental cell line, cellular cathepsin D activity decreased after confluence; however, cathepsin D was secreted at high levels into the extracellular medium. In contrast, in the subpopulation of cells committed to differentiation, cellular cathepsin D activity increased after confluence, and cathepsin D was not secreted into the extracellular medium, but was immunolocalized in the apical brush border of the differentiated cells. Plasminogen activator of urokinase type was identified by immunocytochemistry. Both subconfluent cell populations, and the post-confluent undifferentiated cell population, produced plasminogen activator activity at similar levels. In contrast, in the differentiated postconfluent cells, the production of plasminogen activator activity was markedly lower. Our data show that the differentiation of HT-29 colon carcinoma cells into mucus-secreting cells impairs the secretion of plasminogen activator and cathepsin D, but does not affect cathepsin B.

Diet and bladder cancer in Spain: a multi-centre case-control study.

A multi-centre case-control study on bladder cancer and diet was carried out in 5 regions of Spain. We report results on 432 male cases and 792 matched controls. Usual dietary habits were investigated by means of an interview-based dietary history questionnaire. Bladder-cancer cases were selected from the registers of 12 hospitals located in the study areas. Each case was matched by sex, age and area of residence to 2 controls, one identified in the same hospital and one drawn from population lists. Descriptive analyses indicated that the average dietary pattern was typical of Mediterranean populations: a high P/S ratio, high intake of fish, fruits and vegetables and moderate or low intake of meat and dairy products. Relative risks for specific foods and nutrients were adjusted for tobacco smoking and energy intake. Subjects in the highest quarter of intake of saturated fat had a significantly increased risk of bladder cancer (RR for highest quarter = 2.25; 95% CI = 1.42 to 3.55). Moderate increases in risk for high intake of mono-unsaturated fats and calcium, and a slight decrease for iron were also found, but these disappeared after adjustment for saturated fat. Intake of vitamin E was related to slightly reduced risk (RR for highest quarter = 0.72; 95% CI = 0.48 to 1.09) which was not modified by adjustment for fat. No association was found with intake of retinol or carotene. These results, along with those of previous studies, suggest that saturated fat intake may influence the occurrence of bladder cancer.

Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation.

HT-29 cells selected by adaptation to 10(-5) M methotrexate (HT-29 MTX) are a homogeneous cell population producing high amounts of mucin. Intracellular mucins and proteoglycans were isolated from these cells by ultracentrifugation of cell lysates on a cesium bromide gradient and further separated by anion-exchange high performance liquid chromatography. The major mucin fraction isolated was characterized by a high hydroxy amino acid content (40%), a Thr/Ser ratio of 1.52, a high sialic acid content, and a low sulfate content. When the same procedure was applied to undifferentiated HT-29 cells, a minor mucin fraction was isolated which appeared less sialylated and more sulfated. The major proteoglycan species identified in HT-29 MTX cells showed less acidic behavior than the proteoglycan isolated from HT-29 cells. The effect of brefeldin A and the sugar analog GalNAc-alpha-O-benzyl on the synthesis and biochemical properties of mucins synthesized by HT-29 MTX cells was examined. Brefeldin A induced the synthesis of more-sulfated mucins. GalNAc-alpha-O-benzyl treatment resulted in mucins with an increased content of T antigen and a 13-fold lower sialic acid content. We show that GalNAc-alpha-O-benzyl was metabolized by the cells to Gal beta 1-3GalNAc-alpha-O-benzyl, which, in turn, was a potent competitive inhibitor of the O-glycan alpha-2,3-sialyltransferase. These results illustrate the suitability of HT-29 MTX cells as a model to analyse mucin synthesis and sialylation.

Regulation of cathepsin D dependent on the phenotype of colon carcinoma cells.

We have studied the intracellular trafficking of cathepsin D in different colon carcinoma cell populations: the HT-29 cell line, composed of >95% undifferentiated cells; 2 subpopulations derived from this cell line, containing cells committed to differentiation into mucin-secreting cells (HT-29 MTX) or enterocyte-like cells (HT-29 G-) after confluence; and the Caco-2 cell line, which spontaneously differentiates into enterocyte-like cells after confluence. Post-confluent undifferentiated HT-29 cells and differentiated enterocyte-like HT-29 G- and Caco-2 cells secrete significant levels of cathepsin D in culture medium, in contrast to post-confluent differentiated mucin-secreting HT-29 MTX cells, which secrete this enzyme at a very low level. The intracellular content and the mRNA level of cathepsin D increase after confluence in the different cell types, particularly in Caco-2 cells, which intensify the secretion of cathepsin D along with the differentiation process post-confluence. Membrane-associated mature cathepsin D was detected in HT-29 cells but not in Caco-2 cells. In the different types of cell, pro-cathepsin D associates with the membrane concomitantly to its binding to an Mr 72,000 protein. Membrane association persists after dissociation of the complex in HT-29 cells but not in Caco-2 cells. In the mucin-secreting HT-29 MTX cells, cathepsin D was immunolocalised to the membrane of mucin vacuoles localised under the brush border. Our results show that cathepsin D can be regulated differently in colon carcinoma cells, and this finding might have specific functional implications for each cell type.

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.