M. Emura

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction.

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.

A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters.

SummaryUsing repeated cloning and treatment with cis-HPL (200 μg/ml), an analogue of a procollagen precursor inhibitory to the growth of collagen-synthesizing cells of mesenchymal origin, clonally premature epithelial cell lines were isolated from fetal SGH lungs cultured on the 15th day of gestation. One of the cell lines, M3E3/C3, which has been extensively studied for biological characterization, developed poorly differentiated carcinomas in injected hamsters after transformation by MNNG. Moreover, when grown on collagen gel, this cell line indicated an obvious potency for in vitro differentiation in response to vitamin A by developing activated Golgi regions, well developed rER and a number of mucus-like granules. Since such a differentiative responses is expected to be definable in the light of respiratory epithelium developing in utero, this cell line may be useful for studying mechanisms of differentiation-dependent sensitivity of fetal organs to transplacental carcinogen exposure.

New functional cell-culture approach to pulmonary carcinogenesis and toxicology

SummaryModern pulmonary toxicology (including lung carcinogenesis) has, to assist its rapid development, constantly incorporated the knowledge obtained through cell and tissue-culture studies. While this has been carried out in rather a passive manner until quite recently, the currently necessary multi-disciplinary approach increasingly requires more active involvement of cell/tissue-culture techniques in this area.Our understanding in this regard is that one of such requirements is to establish a cell-culture system consisting of a single population of possible target cells for certain classes of hazardous inhalants. In addition, such target cells in culture should be able to function in a manner as closely resembling the situation in vivo as possible. In view of the culture techniques presently available, this requirement is probably too ideal to be met immediately. Nevertheless, efforts have been made in the last decade to achieve functioning cultures of Clara cells, type II pneumocytes or small mucus granule cells (SMGC), using undifferentiated cells obtained from animal and human fetuses. This attempt forms a sharp contrast to the usual approach, in that while the latter tries to keep the functions of adult cells in an already differentiated state, the former aims at inducing functional differentiation in undifferentiated cells by manipulating culture conditions. In carrying out these efforts, we have shown clear evidence that the type II pneumocytes and Clara cells induced in vitro are closely cognate and share a common precursor cell in culture, and that SMGC are at a prestage of differentiation to Clara cells. We have also shown an induced capacity for xenobiotic activation and conjugation in SMGC in culture. Our next plan is to prove similar activity (of mixed-function oxidase) in Clara cells and type II pneumocytes induced to differentiate in culture.

Regulation of growth and differentiation by vitamin a in a cloned fetal lung epithelial cell line cultured on collagen gell in hormone-supplemented medium

SummaryProliferative and differentiative responses to various doses of vitamin A (VA) were studied in the predifferentiated cells of a fetal Syrian hamster pulmonary epithelial line (M3E3/C3), which were cultured on a collagen gel in a hormone-supplemented medium. These predifferentiated cells possessed well-developed endoplasmic reticulum (ER) and Golgi apparatus. At VA doses higher than 8 μg/ml, periodic acid Schiff and slightly alcian blue positive mucuslike granules were produced, which were also detectable electron microscopically. These mucuslike products were rich in sialic acid and resembled quite well those from primary cultures of tracheal epithelial cells of Syrian hamster sucklings when analyzed by column chromatography on various types of gel. At all VA doses studied (2.4, 8, 24 μg/ml), cells grew exponentially with an average population doubling time of around 74 h, whereas in the absence of VA they had a linear growth rate and a population doubling time of 158 h between Days 4 and 11. The uptake of [3H]glucosamine into the whole cell homogenates showed a peak at Day 8, irrespective of VA doses (0 to 24 μg/ml), and at the highest VA dose (24 μg/ml) it exceeded by twofold the control (0 μg/ml) level. At the same time, [14C]thymidine demonstrated a high peak of uptake on Day 8 at 8 and 24 μg/ml VA. There was virtually no difference between 0 and 2.4 μg/ml VA, with both doses yielding much lower peaks. Based on the results currently presented and previously reported, three successive stages were hypothesized for the mucous differentiation processes in M3E3/C3. The process from the first undifferentiated stage to the second predifferentiated stage with well-developed ER and Golgi apparatus requires both collagen gels and hormones. Differentiationn from the second stage to the third secretory stage with mucous granules is stimulated by VA. These observations indicate that the cell line M3E3/C3 could provide a new system for investigating the mechanisms of mucus differentiation by VA.

Comparison of chrysene metabolism in epithelial human bronchial and Syrian hamster lung cells

Chrysene is metabolized to 1-, 2-, 3-, and 4-hydroxychrysene and trans-1,2- as well as trans-3,4-dihydroxydihydrochrysene in human and Syrian hamster epithelial lung cells as indicated by GC/MS analysis, whereas K-region oxidation is at most a very minor pathway. Cells of a permanent clonal line of fetal hamster lung metabolized 97% of the chrysene whereas fetal human bronchial epithelial cells converted 24% of the substrate within 8 days incubation. In human cells oxidation at the 3,4-position predominates, whereas oxidation at the 1,2-position is the major pathway in hamster cells. Indication for a bay-region oxidation of chrysene in hamster cells has been obtained.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE)

SummaryA human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibro-blastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 μg/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glyco-proteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. More-over, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

A new computer-controlled air-liquid interface cultivation system for the generation of differentiated cell cultures of the airway epithelium.

The increased application of in vitro systems in pharmacology and toxicology requires cell culture systems that facilitate the cultivation process and ensure stable, reproducible and controllable cultivation conditions. Up to now, some devices have been developed for the cultivation of cells under submersed conditions. However, systems meeting the requirements of an air-liquid interface (ALI) cultivation for the special needs of bronchial epithelial cells for example are still lacking. In order to obtain in vivo like organization and differentiation of these cells they need to be cultivated under ALI conditions on microporous membranes in direct contact with the environmental atmosphere. For this purpose, a Long-Term-Cultivation system was developed (CULTEX(®) LTC-C system) for the computer-controlled cultivation of such cells. The transwell inserts are placed in an incubator module (24 inserts), which can be adjusted for the medium level (ultrasonic pulse-echosensor), time and volume-dependent medium exchange, and frequency for mixing the medium with a rotating disc for homogeneous distribution of medium and secretion components. Normal primary freshly isolated bronchial epithelial cells were cultivated for up to 38 days to show the efficiency of such a cultivation procedure for generating 3D cultures exhibiting in vivo-like pseudostratified organization of the cells as well as differentiation characteristics like mucus-producing and cilia-forming cells.

Neuroendocrine phenotype differentiation in a hamster lung epithelial cell line under low oxygen pressure or after transformation by diethylnitrosamine

Oxygen pressure as low as 5% in the gas phase or 87 mmHg in the liquid phase induced various neuroendocrine cell (NEC) phenotypes in more than 80% of cells of a cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3). Further, cells from a number of colonies transformed in an anchorage-independent manner by diethylnitrosamine (DEN) demonstrated a NEC phenotype. Since the cell line used is of a pluripotent stem cell type, both hypoxia and DEN appear to possess a potency for NEC phenotype induction.

Secretory differentiation of human fetal bronchial epithelial cells in culture. A study by histochemistry and electron microscopy.

Human fetal bronchial epithelial (HFBE) cells at 6-8 passages were cultivated on a collagen gel for 10 days. A basal differentiative medium (BDM), consisting of RPMI 1640 supplemented with hormones and growth factors, was employed. Histochemistry, scanning electron microscopy and transmission electron microscopy revealed that HFBE cells developed secretory granules when cultivated on collagen gel in BDM. They were electron-dense and stained positive for PAS but negative for alcian blue. On additional treatment with 8 micrograms/ml vitamin A (VA), the number of secretory granules was increased. Moreover, the HFBE cells lost their surface microvilli, and dilation of rough endoplasmic reticulum was more marked than in culture without VA.

An in Vitro Approach for Interspecies Extrapolation Using Animal and Human Airway Epithelial Cell Culture

To assess the risk of cancer development we usually need to extrapolate data from laboratory experiments to the human situation. There is, however, a wide variety of both qualitative and quantitative results from studies with different mammalian species. To cite but a few examples, incubation of benzo(a)pyrene (BaP) with the microsomal preparations from respiratory organs produced predominantly trans4,5-dihydro-4,5-dihydroxybenzo(a)pyrene (trans-4,5-diol) in rats and rhesus monkeys, while in Syrian hamsters and humans trans-7,8-diol was produced (see review by Cohen and Ashurst, 1983). Polycyclic aromatic hydrocarbons (PAHs) and cigarette smoke induce tumors of the respiratory organs in the pulmonary peripheral region in rats, in the pulmonary hilar region in humans and in the upper respiratory tract in Syrian hamsters (see review by Mohr and Dungworth, 1988). In the respiratory tract cell cultures, 12-0-tetradecanoylphorbol-13-acetate (TPA) enhanced colony formation of rat cells, while it was inhibitory to hamster and human cells (Beeman et al., 1987). We should, therefore, recognize such species-dependent variations and try to understand the mechanisms underlying their occurrence to make the extrapolation more accurate.