Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells.

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha-(difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF-stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.

Effects of nickel sulfate on growth and differentiation of normal human bronchial epithelial cells.

Epidemiological studies have shown that inhalation of nickel compounds enhances the risk of human respiratory cancer. Cultures of normal human bronchial epithelial cells were continuously exposed to a dose (5-20 micrograms/ml) of NiSO4 that reduced their colony forming efficiency by 30-80%. After 40 days of incubation, the cultures consisted of large, squamous cells; mitotic cells were not observed. The cells were then maintained in medium without NiSO4. After 40-75 total days of incubation, colonies of mitotic cells appeared at a rate of 1 colony per 100 000 cells originally at risk; no colonies appeared in control cultures or in cultures exposed to less than 5 micrograms NiSO4/ml for 90 days. Twelve NiSO4-altered cell cultures isolated from five experiments have been expanded into mass cultures. Most of the cell lines have an increased population doubling potential (greater than 50 divisions). Some exhibit aberrations in the squamous (terminal) differentiation process whereas others have lost the requirement for epidermal growth factor for clonal growth. Aneuploidy and marker chromosomes have also been noted. However, none of these NiSO4-altered cell cultures are anchorage independent nor do they produce tumors upon injection into athymic nude mice.

Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum.

Recently we reported a low calcium (110 microM) serum-free medium (LHC-1) for clonal growth of normal human bronchial epithelial (NHBE) cells. NHBE cells within colonies are small (mean surface area = 1,250 mu2) rarely migratory, have few tonofilaments, and multiply with an average population doubling time of 28 h. We have also noted that adding small amounts of blood-derived serum to LHC-1 medium (as little as 2%) significantly decreased the clonal growth rate. We have now found that the growth inhibiting effect of serum is due to the induction of squamous (terminal) differentiation. Serum quickly increases the size of the cells (mean surface area = 4,900 mu2). In addition, the cells acquire numerous desmosomal junctions and an extensive network of keratin bundles. In contrast, human lung carcinoma cells multiply rapidly at clonal density in LHC-1 medium containing as much as 8% serum. Although high concentrations of calcium ions in the medium are known to induce squamous differentiation of epidermal keratinocytes in the absence of serum, high levels of Ca2+ (up to 1,000 microM) increased the number of desmosomal junctions, but did not significantly affect the clonal growth rate or size of the NHBE cells. However, high concentrations of calcium (above 450 microM) were found to potentiate serum differentiation-inducing activity. On the other hand, cholera toxin (10 ng/ml) inhibited the differentiation-inducing activity of serum. These results show that squamous differentiation of NHBE cells can be induced by serum and the potency of these serum factors can be modulated. In addition, the data show that lung carcinoma cells differ from their normal counterparts by not undergoing differentiation in the presence of serum.

Differential control by platelet factors of squamous differentiation in normal and malignant human bronchial epithelial cells.

Recently, we developed a nutritionally optimal medium for rapid clonal growth (greater than 1 population doubling/day) of normal human bronchial epithelial (NHBE) cells. Adding fetal bovine or adult human blood-derived serum to this medium depresses the clonal growth rate of NHBE cells in a dose-dependent fashion. In contrast, 10 representative lines of human lung carcinomas either replicate poorly or fail to grow at all when inoculated at clonal density in serum-free medium, and their rates of multiplication increase in direct proportion to the amount of blood-divided serum added to the optimized medium. Thus, the growth factor requirements of these lung carcinoma cell lines are significantly different from those of their normal counterparts. Blood-divided serum reduces the clonal growth rate of NHBE cells by specifically inducing the normal cells, but not lung carcinoma cells, to undergo squamous differentiation. The differentiation-inducing activity was found in platelet lysates. In addition, a growth-inhibiting activity that did not induce squamous differentiation of NHBE cells was also identified in partially purified commercial preparations of platelet-derived growth factor. This observation was in marked contrast to results using human bronchial fibroblasts and human lung carcinoma cell lines; the growth rate of the former was significantly stimulated by commercial preparations of platelet-derived growth factor, whereas the growth rates of the tumor cell lines were unaffected. These results indicate that an aberration in the cellular differentiation as assayed in vitro is positively correlated with cancer and suggests that decreased responsiveness to inducer(s) of differentiation may be a major aspect of bronchial cell carcinogenesis.

Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium.

Defined culture conditions for routine clonal growth of normal human adult bronchial epithelial cells have been developed. Serum and feeder cell requirements were abrogated by: (a) optimizing the calcium concentration in nutrient medium, MCDB 151; (b) supplementing with purified factors (epidermal growth factor, 5 ng/ml; insulin, 5 micrograms/ml; transferrin, 10 microgram/ml; hydrocortisone, phosphoethanolamine and ethanolamine, each at 5 x 10(-7) M; and trace elements); and (c) coating the surface of the culture dish with a mixture of fibronectin, collagen, and bovine serum albumin. Endothelial cell growth supplement (100 micrograms/ml) and retinoic acid (3 x 10(-10) M) further enchanced growth, whereas cholera toxin was nonmitogenic and serum supplementation (greater than 2%) markedly reduced the growth rate. Using the defined system, dissociated cultures of bronchial epithelial cells, obtained from more than 15 donors, have been subcultured at clonal densities with a colony forming efficiency of 3 to 4%. In addition, high density cultures have been subcultured more than five times with four to six population doublings per passage. The features of this system permit pathobiologic investigations of bronchial epithelial cells, e.g., aging, differentiation, and carcinogenesis using conditions that isolate the results from the influence of serum, feeder cells, and other undefined factors.

Clonal growth of epithelial cells from normal adult human bronchus.

Normal primary epithelial cell cultures devoid of fibroblastic cells have been developed from tissue explants of adult human bronchi. Conditions for clonal growth of secondary cultures of bronchial epithelial cells were optimized by coculturing the human cells with mitomycin C growth-arrested Swiss 3T3 mouse feeder cells, lowering the calcium concentration of medium M199, and supplementing it with hydrocortisone, insulin, cholera toxin, epidermal growth factor, and 1.25% fetal bovine serum. The epithelial cells grew for an average of 35 population doublings and had the normal human karyotype, expressed keratin and blood group antigen epithelial cell markers, metabolized benzo(a)pyrene, and were capable of differentiating into both ciliated and squamous cells. This culture system makes it potentially possible to investigate various aspects of differentiation and carcinogenesis in human bronchial epithelial cells.