Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mR A in leucocyte sensitised CaCO-2 cells. TNF-was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria. Conclusion-The diVerential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions. (Gut 2000;47:79-87)

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types.In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal–regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane–targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14–MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a ilimited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was cytes were transformed with adenovirus or adenovirus DNA, whereas transformation of rat fetal hepatocytes with 3'-methyl-4-dimethylaminoazobenzene led to cell lines that lack expression of albumin and P2-macroglobulin (7, 8).Metabolic activation of environmental carcinogens from several chemical classes have been studied in human liver tissue explants or microsomes and isolated human hepatocytes (4, 9-19). Furthermore, observed animal speciesspecific differences in aflatoxin B1 (AFB1) and 2-acetylaminofluorene metabolism indicate the need for studying human liver or hepatocytes (17, 18, 20). However, because tissue availability is limited, individuals vary in their propensity for xenobiotic metabolism, and standardized in vitro conditions are difficult to establish, a reproducible system with human liver cells for pharmacotoxicological studies has not been established.In this report, the immortalization of adult human liver epithelial cells from two different nondiseased donors with the SV40 T antigen is described. These cell lines are shown to be nontumorigenic, express hepatocyte differentiation markers, and possess enzymatic pathways responsible for xenobiotic metabolism. For example, AFB1, a known risk factor for human hepatocellular carcinoma (21), requires metabolic activation to exert its carcinogenic effects and is associated with a mutational hotspot in the p53 tumor-suppressor gene at the codon for 23). Thus, THLE-2 and -3 liver cell lines will be beneficial for multiple applications: standardized toxicological in vitro tests; investigations of species-specific nmechanisms of carcinogens and anticarcinogens; assessment ofthe mode ofaction ofbiologically active compounds; in vitro studies to assay factors involved in liver cancer, such as hepatitis infection; and protooncogene activation or tumorsuppressor gene inactivation. -7,t-8-dihydroxy-c-9,10epoxy-7,8,9,10tetrahydrobenzo[a]py- MATERIALS AND METHODS

No abstract

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.