Immortalized human hepatocytes that can retain functions of drug-metabolizing enzymes would be useful for medical and pharmacological studies and for constructing an artificial liver. The aim of this study was to establish immortalized human hepatocyte lines having differentiated liver-specific functions. pSVneo deoxyribonucleic acid, which contains large and small T genes in the early region of simian virus 40, was introduced into hepatocytes that had been obtained from the liver of a 21-wk-old fetus. Neomycin-resistant immortalized colonies were cloned and expanded to mass cultures to examine hepatic functions. Cells were cultured in a chemically defined serum-free medium, ASF104, which contains no peptides other than recombinant human transferrin and insulin. As a result, an immortal human hepatocyte cell line (OUMS-29) having liver-specific functions was established from one of the 13 clones. Expression of CYP 1A1 and 1A2 messenger ribonucleic acid by the cells was induced by treatment with benz[a]pyrene, 3-methylcholanthrene, and benz[a]anthracene. OUMS-29 cells had both the polycyclic aromatic hydrocarbon receptor (AhR) and AhR nuclear translocator. Consequently, 7-ethoxyresorufin deethylase activity of the cells was induced time- and dose-dependently by these polycyclic aromatic hydrocarbons. This cell line is expected to be instrumental as an alternative method in animal experiments for studying hepatocarcinogenesis, drug metabolisms of liver cells, and hepatic toxicology.

We examined the effect of mechanical fluid flow and surfactant on macrophage aggregation as the indication of macrophage activation. Mechanical fluid flow enhanced aggregation in phorbol myristate acetate (PMA)-treated human myeloid leukemic cell lines (HL-60 cells), but had no effect on differentiation of PMA-treated HL-60 cells. Surfactant-TA (an artificial surfactant) inhibited fluid flow–induced aggregation, but had no effect on differentiation of PMA-treated HL-60 cells. Human alveolar macrophages spontaneously formed small aggregates without stimulation. This aggregation was enhanced by fluid flow and inhibited by surfactant-TA. Taken together, these data suggest that macrophage activation is affected by fluid flow and surfactants.

In the present study, we evaluated the individual and combined effects of environmental estrogens and flavonoids on the proliferation of human breast carcinoma MCF-7 cells. These compounds are as follows: (1) pharmaceutical chemicals such as diethylstilbestrol, 17α-ethynylestradiol (17ES), tamoxifen, mestranol, and clomiphene, (2) industrial chemicals such as bisphenol A (BisA), 4-octylphenol (OP), 4-nonylphenol (NP), and p,p′-biphenol, and (3) flavonoids such as daidzein (D), genistein (G), quercetin (Q), and luteolin (L). We found that nanomolar concentrations of 17ES, BisA, OP, and NP were sufficient to stimulate the proliferation of MCF-7 cells. Among them, 1 μM BisA exhibited cell proliferation–stimulating activity as strong as 10 nM 17β-estradiol; and D and G exhibited cell proliferation–stimulating activity at 10 nM. On the other hand, Q and L exhibited cell proliferation–inhibiting activity. We also found that 10 nM flavonoids, such as D, G, Q, and L, were able to inhibit the proliferation-stimulating activity in MCF-7 cells by 1 μM environmental estrogens.

During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5 mM) calcium (Ca² ) in the absence or presence of thyroxine (10⁻⁷ M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5 mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca² concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca² leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca² concentrations (0.5 mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation in Rana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.

The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D–induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.

The binding characteristics of tumor necrosis factor-α receptors (TNFRs) in primary stromal–vascular cultures from fat tissue of 7-d-old pigs were analyzed. Cells were plated and maintained in 10% fetal bovine serum from day 0 to day 3 and then switched to serum-free medium from day 3 to day 6 to induce lipid filling. On days 3 and 6 of culture, some of the cells were lysed for ligand and immunoblotting and the remainder subjected to competitive and inhibitory-binding assays. Media from day 6 of culture were subjected to ligand and immunoblotting. Competitive binding analysis showed one-site bindings, with IC₅₀s in the nanomolar and K_ds in the picomolar ranges, that were not significantly different at both time-points of measurement. However, the B_max decreased significantly with differentiation. Preincubation with antibody against TNF receptor type 1 (TNFR1) or TNF receptor type 2 reduced the specific binding by 95 and 15%, respectively, suggesting a dominating role of TNFR1 in ¹²⁵I-labeled TNFα (¹²⁵I-TNFα) binding. This was further supported by ligand blotting of cell lysates. Ligand and immunoblotting of cell lysates indicated that TNFα utilizes both types of surface receptors and their isoforms which were not modified during differentiation. Ligand blotting of media revealed soluble receptors with high M_r implying the formation of multimers. Immunoblotting suggested the presence of both types of TNFRs, but a greater abundance of soluble TNFR1. Also, it indicated the additional formation of smaller oligomers from both types of soluble receptors suggesting higher affinity of larger multimers for ¹²⁵I-TNFα.

Transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF-I) can attenuate drug-induced cell death in epithelial cells. Since milk whey contains a mixture of these and other growth factors, we evaluated mitogenic bovine whey extract (MBWE) for protective activity against chemotherapy drug damage in cultured epithelial cells (mink lung, Mv1Lu). Etoposide and vinblastine reduced cell survival by up to 90%. This was attenuated by the addition of MBWE before and during drug exposure, but not following drug removal. MBWE was compared with individual growth factors known to be present in the mixture. IGF-I and platelet-derived growth factor were ineffective, whereas TGF-β2 induced growth inhibition and cell survival, with a maximum response at 3 ng/ml. TGF-β2 bioactivity was also demonstrated by showing that acidification of MBWE (A-MBWE), to activate TGF-β2, enhanced its growth inhibitory and chemoprotective activities 60- and 12-fold, respectively. However, MBWE contained additional protective factors. When TGF-β2 and the MBWE preparations were compared, on the basis of growth inhibition equivalents, MBWE protected cells against drug toxicity at concentrations an order of magnitude lower than with TGF-β2 or A-MBWE. Immunoneutralization of the TGF-β present in MBWE and A-MBWE eliminated all growth inhibitory activity but not all cell survival activity. We conclude that the MBWE preparations are cytoprotective against two chemotherapy drugs when added before and during drug exposure. TGF-β contributes to this activity, but the extracts contain other factors that promote the survival of epithelial cells after chemotherapy drug exposure.