A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly ...differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver‐specific mRNAs and DNA‐binding activity of ubiquitous and liver‐enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3‐methylcholanthrene‐inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA‐binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF‐1 and HNF‐4‐like DNA‐binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1‐like DNA‐binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer‐binding protein and HNF‐3‐like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis‐B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis‐B virus replication and virion production, in agreement with the notion that HNF‐1 activity is necessary for viral replication.
We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis‐B virus replication.
Background: Gastric cancer is one of the most common cancers in the world. The ‘‘economically developed countries’’ life style, including diet, constitutes a risk factor favoring this cancer. Diet ...modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate. br/Methodology/Principal Findings: A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy. br/Conclusions/Significance: Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.
Background: Gastric cancer is one of the most common cancers in the world. The ‘‘economically developed countries’’ life style, including diet, constitutes a risk factor favoring this cancer. Diet ...modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate. br/Methodology/Principal Findings: A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy. br/Conclusions/Significance: Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.
Background and objectives.br/ Dairy propionibacteria are isolated from various ecological niches, including soil, grass, silage, rumen, raw milk and dairy plants, showing great adaptability and ...robustness. Used as ripening starters in Swiss type cheeses, these food grade bacteria are described as nutraceutical producers and they release into the external medium short chain fatty acids (SCFA), folic acid, cobalamin and the bifidogenic 1,4- dihydroxy-2-naphthoic acid (DHNA). These compounds are known to play a pivotal role in the modulation of intestinal physiology through diet. Considering the pro-apoptotic potential of some of these metabolites, we investigated the potential of dairy products fermented by the main species, Propionibacterium freudenreichii, to prevent colon carcinogenesis. Methods.br/ We developed a new fermented milk. Taking advantage of milk fractions, high and stable populations of P. freudenreichii were obtained in this product. It was tested in vitro on gastric HGT-1 and colic HT-29 cells with respect to the typical hallmarks of apoptosis. In vivo, the fermented milk was evaluated in piglets with respect to general health parameters and in human microbiota-associated rats, treated or not with dimethylhydrazine (DMH), with respect to intestinal proliferation and apoptosis. Results. Fermented milk induced cell death in both gastric and colon cancer cells. Cellular and molecular characterization of cells dying in response to SCFA treatment revealed cell cycle arrest, drop in intracellular ATP, depolarization of mitochondria, translocation of key apoptosis proteins, processing of caspases and fragmentation of the nucleus. Interestingly, the new fermented milk was shown to potentiate the cytotoxic effect of molecules used in cancer chemotherapy. In accordance, it modulated expression of key apoptotic proteins. In vivo, the fermented milk was evaluated in piglets. Daily oral gavage led to high colic P. freudenreichii populations, to modulation of the intestinal microbiota and modulation of intestinal cytokines. Promotion of piglets’ growth also revealed general probiotic effects of fermented milk. Considering that propionibacteria were metabolically active in the intestine, we further investigated their impact on colon carcinogenesis (induced by dimethylhydrazine, DMH) in human microbiota-associated rats. Consumption of selected strains of propionibacteria resulted in enhanced apoptotic depletion of DMH damaged epithelial cells, yet had no effect on neither proliferation nor apoptosis in healthy rats. Conclusions and perspectives.br/ These results open new perspectives in the field of colon cancer cells prevention and/or treatment. The synergy with pro-apoptotic chemotherapy molecules suggests that such a fermented product may be proposed as a food supplement to enhance the effects of anticancer treatments.
We have developed a method to select for rat hepatoma cells that fail to express hepatocyte-specific functions. Well-differentiated cells descended from the H4IIEC3 hepatoma line express aldrin ...epoxidase (AE) activity, an indicator of the liver-specific forms of cytochromes P450 and, concurrently, are able to activate the procarcinogen aflatoxin B1 (AFB1) into highly toxic metabolites. Thus, differentiated hepatoma cells are highly sensitive to AFB1, while dedifferentiated derivatives, which fail to express AE activity, are resistant. Exposure of differentiated Fao cells to 10 microM AFB1 for 24 h permits the isolation, at a frequency of 5 x 10(-5), of resistant colonies that exhibit strongly reduced AE activity. Strikingly, various morphological types can be observed. In more than 90% of the colonies, cells are morphologically similar to the original differentiated cells and accumulate all liver-specific mRNAs examined in amounts comparable to Fao cells. Moreover, they are able to carry out gluconeogenesis, as judged by their capacity to grow in glucose-free medium. For a minor fraction of colonies, the cells exhibit nonhepatic morphology. These cells fail to express three or more of the liver functions and are not able to proliferate in glucose-free medium. Our results demonstrate that the use of AFB1 constitutes a simple and efficient single-step selective method for obtaining variant hepatoma cells of a wide variety of phenotypes.
A 23-kilobase-pair segment of DNA containing the entire mouse serum albumin gene as well as 2.2 kilobase pairs of 5′and 4.3 kilobase pairs of 3′flanking sequences has been introduced into pSV2dhfr, a ...plasmid in which expression of the mouse dihydrofolate reductase cDNA is under the control of simian virus 40 sequences. This vector, pSV2dhfr-alb, was used to transfect differentiated and variant dedifferentiated rat hepatoma cells. Nine independent clones of transfected differentiated cells secrete considerable amounts of mouse albumin, while the expression of the normal rat albumin is the same as in nontransfected cells. In contrast, only small amounts of mouse and rat albumin are produced by transfected dedifferentiated cells. The amounts of albumin mRNA present in the cells are consistent with the amounts of albumin produced. These results show that a transfected gene can be regulated in a fashion consistent with the overall differentiation profile of the cell.
We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a ...procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.
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