[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.

Organic compounds from diesel exhaust particles elicit a proinflammatory response in human airway epithelial cells and induce cytochrome p450 1A1 expression.

Diesel exhaust particles (DEP) are known to enhance inflammatory responses in human volunteers. In cultured human bronchial epithelial (16HBE) cells, they induce the release of proinflammatory cytokines after triggering transduction pathways, including nuclear factor (NF)-kappaB activation and mitogen-activated protein kinase (MAPK) phosphorylation. This study compares the effects of native DEP (nDEP), organic extracts of DEP (OE-DEP), and carbonaceous particles, represented by stripped DEP (sDEP) and carbon black particles (CB), in order to clarify their respective roles. OE-DEP and nDEP induce granulocyte macrophage colony-stimulating factor (GM-CSF) release, NF-kappaB activation, and MAPK phosphorylation. The carbonaceous core generally induces less intense effects. Reactive oxygen species are produced in 16HBE cells and are involved in GM-CSF release and in the stimulation of NF-kappaB DNA binding by nDEP and OE-DEP. We demonstrate, for the first time, in airway epithelial cells in vitro that nDEP induce the expression of the CYP1A1, a cytochrome P450 specifically involved in polycyclic aromatic hydrocarbons metabolism, thereby demonstrating the critical role of organic compounds in the DEP-induced proinflammatory response. Understanding the respective contributions of DEP components in these effects is important for vehicle manufacturers in order to improve their exhaust gas post-treatment technologies. In conclusion, the DEP-induced inflammatory response in airway epithelial cells mainly involves organic compounds such as PAH, which induce CYP1A1 gene expression.

Fluorescence in situ hybridization and immunohistochemical assays for HER-2/neu status determination: application to node-negative breast cancer.

BACKGROUND: HER-2/neu (ERBB2) gene amplification and/or overexpression is a major event in human breast tumorigenesis. HER-2/neu gene alterations have been the most frequently assessed prognostic factors during the last 10 years in breast cancer and have recently emerged as a management decision tool and a therapeutic target. There is still controversy over the best method to determine whether a tumor is HER-2/neu positive. Because of the increasing demand for HER-2/neu gene status determination in clinical practice, we compared HER-2/neu gene alterations at the DNA level (gene amplification) and the protein level (overexpression) in a panel of patients with lymph node-negative breast cancer who had received local radiotherapy alone, with no adjuvant therapy. METHODS: We tested 100 excised lymph node-negative breast tumors, using fluorescence in situ hybridization (FISH) with a biotinylated HER-2/neu DNA probe and immunohistochemical assays (IHC) with 2 different antibodies. RESULTS: The FISH frequency of HER-2/neu gene amplification was 15%, and the IHC frequency of overexpression was 21%. CONCLUSION: Although HER-2/neu amplification by FISH and HER-2/neu overexpression by IHC correlated well in this panel of lymph node-negative breast carcinomas, there were a number of discordant cases, pointing to the important need for determining HER-2/neu alteration for the future management of HER-2/neu-based clinical applications.

p53 abnormalities in adenocarcinoma of the gastric cardia and antrum.

AIM: To compare the frequency and type of p53 alterations (gene mutation and/or protein overexpression) in a consecutive series of surgically resected adenocarcinomas arising in the gastric cardia and gastric antrum, and to evaluate associations with clinicopathological findings (age, sex, and tumour histology, grade, and stage). METHODS: The series comprised 50 patients with adenocarcinoma of the cardia and 20 patients with adenocarcinoma of the antrum. p53 gene mutations (exons 5-8) were detected by denaturing gradient gel analysis and DNA sequencing. Nuclear p53 overexpression was detected by immunohistochemistry with the DO7 antibody. RESULTS: p53 gene mutations were found in 21 of 50 and five of 20 adenocarcinomas of the cardia and the antrum, respectively. Base transitions occurring at CpG dinucleotides were frequent in both types of tumour. p53 protein overexpression was seen in 32 of 50 and seven of 20 adenocarcinomas of the cardia and of the antrum, respectively. p53 gene mutation and/or protein overexpression were significantly more frequent in adenocarcinomas of the cardia (37 of 50) than in adenocarcinomas of the antrum (seven of 20). There were no differences in the clinicopathological characteristics of the tumours between p53 positive and p53 negative cases in both types of cancer. CONCLUSIONS: This study shows that p53 alterations are more frequent in adenocarcinoma of the cardia than in adenocarcinoma of the antrum. This feature is consistent with the clinical and epidemiological characteristics of these cancers, which suggest that adenocarcinoma arising in the gastric cardia might be related to oesophageal adenocarcinoma, and unrelated to adenocarcinomas of the gastric body and antrum.

[Gene p53 mutations and overexpression of p53 protein in tumors of the Vater's ampulla]. / Mutations du gène p53 et surexpression de la protéine p53 dans les ampullomes vatériens.

OBJECTIVES AND METHODS: New prognostic markers for tumors of the ampulla of Vater which could distinguish aggressive lesions would be highly useful. The aim of this study was to evaluate the abnormal expression of p53 protein in a series of 34 ampullomas with the monoclonal antibody DO7 directed against p53 protein. Mutations of exons 5 to 8 of p53 gene were also detected by PCR followed by denaturating gel electrophoresis and sequencing. RESULTS: One of 5 adenomas (20%) and 16 of 29 adenocarcinomas (55%) were p53 positive by immunohistochemistry. A constant feature was the presence of p53 positive glands in the surrounding dysplastic mucosa of p53 positive infiltrative lesions. There was no correlation between p53 immunostaining and tumor stage. Among the 19 tumors analyzed, 6 mutations were detected. CONCLUSION: p53 gene mutations and p53 protein immunoreactivity are frequently present in ampullomas. Because these alterations are not correlated to tumor extension and occur at an early stage in the carcinogenesis, their detection does not appear to be contributive for diagnosis of ampullary tumors.

PTEN/MMAC1/TEP1 involvement in primary prostate cancers.

The PTEN/MMAC1/TEP1 gene, located at 10q23.3, is a tumor suppressor gene responsible for the familial cancer syndromes Cowden disease and Bannayan-Zonana syndrome, and is commonly somatically mutated in several types of cancers. Mutations of the PTEN gene have been found in prostate cancer cell lines and LOH at 10q22-24 in prostate tumors have also been described with a high frequency. To determine the role of this gene in prostate tumorigenesis, we therefore analysed 22 primary tumors for loss of heterozygosity (LOH) within the 10q22-23 region such that tumors hemizygous at those loci may be examined for somatic PTEN mutations. Losses of heterozygosity of at least one locus was found in 12 (55%) of the 22 tumors DNAs. Among these, six tumors exhibited allele loss in the interval between D10S1765 and D10S541 wherein lies the PTEN gene. We searched the entire coding region of PTEN for somatic mutations in these six tumors. One somatic mutation (17%), a 1 bp deletion, was detected in exon 7 of the gene, in one tumor, indicating that somatic mutations of the PTEN gene may occur in primary prostate tumors.

Determination of the replication error phenotype in human tumors without the requirement for matching normal DNA by analysis of mononucleotide repeat microsatellites.

Microsatellite instability (MI) characterizing tumors with replication errors (RER+ tumors) was first described in colorectal tumors from hereditary non-polyposis colorectal cancer (HNPCC) patients as well as in sporadic cases. It has also been observed in subgroups of extracolonic sporadic tumors, but there is no consensus as to the number of microsatellite loci to examine, and the threshold percentage of unstable loci required to classify a tumor as RER+. We have recently shown that BAT-26, a mononucleotide repeat microsatellite, was quasi-monomorphic in DNA from normal individuals and from colorectal RER- samples, and showed important size variations in RER+ samples. In the present work, we analyzed BAT-26 allelic profiles in tumors of the breast (n = 107), brain (n = 78), stomach (n = 59), prostate (n = 49), esophagus (n = 36), thyroid (n = 31), endometrium (n = 12), and cervix (n = 10) whose RER status was already known, thus extending BAT-26 analysis to a total of 542 human solid tumors. BAT-26 alleles were quasi-monomorphic in RER- samples (475/481) and shortened in RER+ tumors (57/61), including four tumors shown to have been misclassified on the basis of dinucleotide repeat microsatellite analysis. In 3/481 RER- and 4/61 RER+ cases, BAT-26 size variation was important enough to attract attention, but not sufficient to establish the RER status of the corresponding tumors. In these cases, the analysis of BAT-25 and BAT-34C4, two other mononucleotide repeat microsatellites, was necessary to resolve the ambiguity. There were only 3 false positive cases. In conclusion, BAT-26 was able to identify the RER status of 539 out of 542 tumors from various origins (99.5% efficiency) in a single-step experiment without the requirement for matching normal DNA.

Alternative genetic pathways in colorectal carcinogenesis.

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER- subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER- and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor beta receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER- tumors may be more different than previously thought.

Loss of heterozygosity on chromosome 9 and p16 (MTS1, CDKN2) gene mutations in esophageal cancers.

Loss of heterozygosity on chromosome 9 has been reported in a variety of human cancers. The cyclin-dependent kinase inhibitor p16 gene, mapped on chromosome 9p21, is presumed to be the tumor-suppressor gene localized in this chromosome. The aim of our study was to determine, in 26 Barrett's adenocarcinomas and 20 squamous-cell carcinomas of the esophagus, the prevalence of loss of heterozygosity on chromosome 9 by typing of microsatellite loci and mutation of p16 by direct sequencing of exons 1 and 2. Allelic losses were found in 69% of adenocarcinomas, but only a microdeletion in exon 1 of p16 occurred in 1 tumor. Among squamous-cell carcinomas, 65% had allelic losses and 5 tumors were mutated on the p16 gene (1 deletion, 3 nucleotide substitutions and 1 insertion). The relatively low rate of p16 mutation observed here coupled with the high frequency of loss of heterozygosity on chromosome 9 suggests that one or several tumor-suppressor gene(s) distinct from p16 may be the target(s) of allelic deletion in most esophageal cancers or that p16 is inactivated in another way.

Infrequent microsatellite instability in oesophageal cancers.

Alterations of microsatellites have been found at relatively high frequency in hereditary and sporadic colorectal cancer and gastric and pancreatic cancers and at lower frequency in some other cancers. We determined the frequency of instability at 39 poly-CA microsatellite loci in 20 squamous cell carcinomas and 26 Barrett's adenocarcinomas of the oesophagus. None of the tumours presented instability for a high percentage of the tested loci. Four squamous cell carcinomas and six Barrett's adenocarcinomas showed microsatellite instability at one locus, and three Barrett's adenocarcinomas showed microsatellite instability at two loci. The presence of few loci showing microsatellite instability could be due to an instability background. We conclude that genetic defects in the DNA mismatch repair system do not play an important role in oesophageal cancers.

Inverse correlation between RER+ status and p53 mutation in colorectal cancer cell lines.

The high point mutation rate of replication error-prone (RER+) cells could theoretically lead to inactivation of the p53 gene by polyclonal mutations, which might explain the conflicting results that have been published on the p53 status of RER+ colon cancers. To address this issue, we tested the p53 status of 21 human colorectal cancer cell lines, including four showing microsatellite instability (RER+ phenotype). Denaturing gradient gel electrophoresis (DGGE) followed by sequencing showed that all four RER+ cell lines were wild type for p53 while 15 of the 17 RER- cell lines contained p53 mutations (P=0.001). Eight cell lines (four RER+ and four RER-) were analysed using three complementary methods to test more rigorously the polyclonal mutation hypothesis. (i) Of 87 single-cell clones (seven to 14 per cell line) examined by DGGE, only those derived from known p53 mutant cell lines showed altered profiles. (ii) Antibody DO-7 stained more than 80% of nuclei from the p53 mutant cell lines, but only 15% of nuclei from the RER+ cell lines. (iii) A yeast functional assay which can simultaneously detect polyclonal mutations at over 500 different sites in the p53 cDNA scored all four RER+ cell lines as containing only transcriptionally active p53. These data thus do not support the polyclonal mutation hypothesis and instead suggest that mismatch repair deficiency provides a p53-independent pathway for development of colorectal cancers.

[Profile of p53 mutations and abnormal expression of P53 protein in 2 forms of esophageal cancer]. / Profil des mutations du gène p53 et expression anormale de la protéine p53 dans les deux formes de cancer de l'oesophage.

AIMS AND METHODS: p53 tumor suppressor gene is involved in the development of esophageal cancer. However, its role is not precisely defined in the two types of cancer, squamous cell carcinoma and adenocarcinoma developed in Barrett's esophagus. The aim of this work was to compare the frequency and type of mutation of the p53 gene and the expression of the p53 protein in a series of 21 squamous cell carcinomas and 27 adenocarcinomas of the esophagus in a single institution. p53 protein accumulation was assessed by immunohistochemistry with the monoclonal antibody PAb 1801. The mutations of exons 5 to 8 of p53 gene were detected by denaturating gradient gel electrophoresis, and sequenced. RESULTS: A mutation of the p53 gene and/or an accumulation of the p53 protein were found in 85% of squamous cell carcinomas and 92% of adenocarcinomas, respectively. The profile of mutations differed with the type of tumor; large predominance of transitions on CpG dinucleotides in adenocarcinomas suggesting an endogenous mechanism, high percentage of transversions in squamous cell carcinomas suggesting a direct effect of carcinogens present in tobacco and alcohol. CONCLUSION: Mutation of the p53 gene is a very frequent event in the two types of esophageal cancer. The mechanism responsible for these mutations is different in squamous cell carcinomas and adenocarcinomas developed in Barrett's esophagus.

Absence of c-Ki-ras gene mutation in malignant and premalignant Barrett's oesophagus.

Aims-To establish the prevalence of c-Ki-ras gene mutations in codons 12 and 13 in 28 surgically resected Barrett's adenocarcinomas and 18 associated preneoplastic lesions in Barrett's oesophagus.Methods-Mutations were detected using the polymerase chain reaction followed by restriction fragment length polymorphism analysis. Human colon carcinoma cell lines with well characterised mutations in codons 12 and 13 were used as positive controls and to test the sensitivity of the method.Results-c-Ki-ras gene mutations were not detected in any of the 28 specimens of Barrett's adenocarcinoma or in the 18 specimens of Barrett's oesophagus (nine non-dysplastic cases, three cases with low and six with high grade dysplasia).Conclusions-These results suggest that the c-Ki-ras gene is not involved in the development of cancer in Barrett's oesophagus.

Overexpression of the p53 tumor suppressor gene product in esophageal and gastric carcinomas.

PURPOSE: p53 protein has been reported as frequently overexpressed in esophageal and gastric carcinomas. However, the correlation between p53 protein expression and clinico-pathological features of the tumors is debated in this heterogeneous group of cancers. The aim of this study was to establish the prevalence of p53 protein overexpression in a series of resected esophageal squamous carcinomas (n = 78), adenocarcinomas developed on Barrett's esophagus (n = 20), adenocarcinomas of the cardia (n = 36), and adenocarcinomas of the antrum (n = 30), and to correlate this expression with the clinico-pathological and flow-cytometric characteristics of the tumors. METHODS: Immunohistochemical staining was performed on frozen sections with a monoclonal antibody directed against wild type and mutated p53 protein (Pab 1801). An adjacent frozen specimen was used for flow cytometric determination of the DNA-ploidy and S phase fraction. RESULTS: p53 protein nuclear expression was detected in 76% of esophageal squamous carcinomas, in 75% of adenocarcinomas developed in Barretts esophagus, in 56% of adenocarcinomas of the cardia, and in 27% of adenocarcinomas of the antrum. Only the number of positive adenocarcinomas of the antrum was significantly lower when compared to the other three types of tumors (p = 0.001). No significant correlation was observed between p53 protein expression and most of the clinico-pathological and flow-cytometric parameters (sex, age, tobacco smoking, chronic alcohol consumption, size of the tumor, grade of differentiation, depth of infiltration, presence of lymph node metastases, UICC stage, DNA-ploidy, S phase fraction). p53 protein expression was more frequent in Lauren's intestinal adenocarcinomas (67%) when compared to the diffuse type tumors (24%) (p = 0.002). CONCLUSIONS: Our results confirm that overexpression of p53 protein is a common feature of esophageal and gastric carcinomas. The high prevalence of p53 protein overexpression found in cardiac adenocarcinoma when compared to antral adenocarcinoma reinforces the hypothesis of distinct carcinogenetic mechanisms in these two cancers. In particular the lack of correlation between p53 expression and tumor stage suggests that p53 protein overexpression is an early event in these tumors.

TP53 gene mutations and p53 protein immunoreactivity in malignant and premalignant Barrett's esophagus.

BACKGROUND/AIMS: Limited data are available regarding TP53 gene alterations in Barrett's esophagus. This study was undertaken to characterize TP53 mutations and p53 protein immunoreactivity in cancers and preinvasive lesions of Barrett's esophageal mucosa. METHODS: Seventeen Barrett's adenocarcinomas were examined by polymerase chain reaction amplification, denaturant gradient gel electrophoresis, and sequencing for the presence of TP53 mutations in exons 5-8. In 9 cases, Barrett's epithelium adjacent to the cancer was investigated. p53 protein immunoreactivity was studied with PAb 1801. RESULTS: Sixteen mutations were found in 15 adenocarcinomas, including 10 missense, 3 nonsense, 1 frameshift, and 2 mutations located within consensus splice donor and acceptor sequences. All nucleotide substitutions were transitions. Eight of the 12 transitions involving a GC base pair occurred within the context of a CpG dinucleotide. p53 immunostaining was present in all 10 cases with missense mutations and in 1 case without a detectable mutation. The surrounding Barrett's mucosa showed TP53 mutations identical to that observed in the carcinoma in only 3 of 5 specimens showing high-grade dysplasia. CONCLUSIONS: TP53 gene mutations and p53 protein immunostaining are present in a majority of Barrett's adenocarcinomas. Our results suggest that these mutations are involved at an early stage during malignant transformation of Barrett's esophagus.

Expression of c-erbB-2 oncogene product in Barrett's adenocarcinoma: pathological and prognostic correlations.

AIMS: To establish the prevalence of c-erbB-2 protein expression in a surgical series of Barrett's adenocarcinomas; and to correlate this expression with clinicopathological data and prognosis. METHODS: Sixty six surgical specimens of Barrett's adenocarcinomas were included in this retrospective study. Blocks of the tumour and of non-dysplastic Barrett's mucosa were stained with a polyclonal antibody specific for the intracytoplasmic domain of the c-erbB-2 protein. RESULTS: Seven of 66 tumours showed membrane staining for the c-erbB-2 protein. The non-dysplastic Barrett's mucosa was negative in all cases. There was no difference between c-erbB-2 positive and negative tumours with regard to mean age, sex ratio, percentage of alcohol misusers, percentage of smokers, tumour differentiation, depth of invasion, lymph node response, and proliferative activity, assessed by the percentage of tumour cells positive with the MIB-1 antibody directed against the Ki-67 antigen. All c-erb B2 positive tumours were of Lauren's intestinal type compared with negative c-erbB-2 tumours. Patients with c-erbB-2 positive tumours had a significantly poorer prognosis than patients with negative tumours. CONCLUSIONS: The prevalence of Barrett's adenocarcinomas expressing c-erbB-2 found in this study (11%) was similar to that observed in published series of gastric adenocarcinomas. c-erbB-2 protein expression could be an important prognostic indicator in Barrett's adenocarcinoma.

Overexpression of p53 protein in Barrett's syndrome with malignant transformation.

AIMS: To study the overexpression of p53 protein in Barrett's oesophagus with adenocarcinoma, and to correlate this expression with the pathological features of Barrett's syndrome. METHODS: Immunohistochemical staining was performed on frozen sections with a monoclonal antibody directed against wild type and mutated p53 protein (Pab 1801). Eleven cases of Barrett's adenocarcinoma were studied, seven of which had extensive sampling of benign Barrett's mucosa. RESULTS: Eight of 11 adenocarcinomas overexpressed the p53 protein. Both early and advanced tumours were positive. In Barrett's mucosa around the p53 positive tumours, high grade dysplasia was positive; low grade dysplasia and non-dysplastic mucosa were negative. CONCLUSIONS: P53 gene mutation with ensuing p53 protein overexpression is a common feature of Barrett's adenocarcinoma, both at early and advanced stages. This mutation appears as a relatively late event during the neoplastic transformation of Barrett's oesophagus.

Mucin exocytosis: a major target for Helicobacter pylori.

AIMS: To determine whether Helicobacter pylori impairs the secretory function of mucous cells. METHODS: The mucus secreting human cell line CL. 16E, maintained as confluent monolayers on nitrocellulose filters, was infected with H pylori strain CIP 101260. After three hours of incubation with H pylori the monolayers were washed and reincubated with fresh culture medium for various time periods (24, 48, or 72 hours) before evaluating both the morphology and function of mucous cells. For morphological studies, epithelial monolayers were fixed in situ and processed for both standard histochemistry on paraffin wax sections, and electron microscopy. To measure mucins secreted from cultured cells, the cells were metabolically labelled with 3H-glucosamine. Undegraded mucins were quantitated as the radioactive glycoproteins blocked at the stacker gel interface after sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the secretory glycoproteins. RESULTS: Control cultures of CL. 16E cells grew on filters as homogeneous monolayers of polarised mucous cells secreting a visco-elastic gel of mucins at the apical surface. In infected monolayers H pylori was in close contact with the apical surface of mucous cells. Cell counts and histological evaluation of the monolayers did not reveal any significant deleterious effect of H pylori on the mucous cells. H pylori induced only a modest inhibition of baseline mucus secretion from CL. 16E cells, this inhibition being significant only at 24 hours. In contrast, the mucus secretory response to two agents that raise intracellular cAMP and calcium--forskolin and ionophore A23187--was strongly inhibited. The inhibitory effect of H pylori on the exocytotic response was not paralleled by an inhibition of glycoprotein synthesis. CONCLUSION: Considering the fact that the exocytotic response to a variety of secretagogues constitutes the primary line of defence of the gastric mucosa in an emergency, it is suggested that H pylori exerts its deleterious effects by weakening this important physiological defence.

Cytokine effects in a human colonic goblet cell line. Cellular damage and its partial prevention by 5 aminosalicylic acid.

The aim of the present study was to investigate the effects of cytokines on intestinal goblet cells in vitro. For this purpose, we examined the effects of recombinant interferon gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on the human colonic goblet cell line Cl.16E by morphological and kinetic studies, and by the assessment of mucus production during IFN-gamma/TNF-alpha treatment. Control cultures of Cl.16E cells grown on nitrocellulose filters formed monolayers of polarized goblet cells, which had kinetic characteristics similar to those of a differentiated epithelium in steady state. The combined action of IFN-gamma and TNF-alpha caused a dose-related cellular exfoliation, leading to the formation of a mucoid cap made of mucus and cellular debris. The remaining viable cells underlying the mucoid cap were cuboidal and devoid of mucus granules. A dose-related increase in cellular incorporation of [3H]thymidine was reactive to the cytokine-induced cell loss. The synergistic effects of IFN-gamm and TNF-alpha were found to be reversible when the cells were reincubated in a culture medium without cytokines. Furthermore, 5-aminosalicylic acid partially protected Cl.16E cells against cellular injury caused by IFN-gamma and TNF-alpha. On the whole, these morphological and kinetic findings argue that the changes induced in Cl.16E cells by IFN-gamma and TNF-alpha closely parallel those observed during the acute phase of ulcerative colitis, and show that these cytokines can regulate intestinal mucus production by modulating cellular exfoliation, thus leading probably to a reinforced protection of the damaged mucosa.