Dharam P. Chopra

PRIMARY AND LONG TERM EPITHELIAL CELL CULTURES FROM HUMAN FETAL NORMAL COLONIC MUCOSA

SummaryPrimary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbeccos minimum essential medium and 50% Hams F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO2: 95% air.The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiffs reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.

Long-term culture of epithelial cells from the normal rat colon

SummarySerial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagles minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures.

Effects of Insulin, Transferrin, Cholera Toxin, and Epidermal Growth Factor on Growth and Morphology of Human Fetal Normal Colon Epithelial Cells

Primary and serially passaged human fetal normal colon epithelial cells have been propagated and characterized with regard to their nature and origin. The cells exhibited many characteristics of colonic epithelial cells including the presence of mucopolysaccharides and carcinoembryonic antigen. Serial passaging of the cultures required supplementing the medium with insulin, transferrin, epidermal growth factor, and cholera toxin. This study also shows that these factors have specific roles in the regulation of growth and morphologic differentiation of the cell cultures. Insulin apparently is mainly associated with cell multiplication, whereas transferrin, epidermal growth factor, and cholera toxin are associated with the maintenance of morphologic differentiation status of the cell cultures.

Epithelial cell cultures from the colon of the suckling rat

SummaryEpithelial cells from the colon of suckling rats have been propagated in vitro. The colons were excised and cut longitudinally. The epithelial sheets were peeled off and dissociated in 0.1% trypsin solution at 25°C for 10 min. The first cell suspension was discarded and the remaining fragments trypsinized again for an additional 20 min. The dissociated cells were washed and cultured. Forty-eight hours later, several epithelial colonies consisting of closely packed polygonal cells were formed. Transmission and scanning electron microscope examination of the colonies showed numerous regularly spaced microvilli on the surface and tight junctions and desmosomes between adjacent cells. Immunocytochemical studies with antiserum prepared against the brush-border membrane of the colonic epithelium showed specific staining of the epithelial colonies.Epithelial colonies were subcultured by the penicylinder method. Although the subcultured cells retained their epithelial characteristics, the proliferative activity of the cells gradually decreased. Currently, efforts are being made to determine the optimum nutritional requirements of the primary and low-passage cultures.

Effects of theophylline and dibutyryl cyclic AMP on proliferation and keratinization of human keratinocytes.

Recent evidence suggested that cyclic adenosine 3′,5′‐monophosphate (cAMP) may be involved in the regulation of cell proliferation and differentiation. The effects of theophylline and dibutyryl cAMP on cell division and keratinization of human epidermal cultures were examined. Nine to 12‐dayold cultures were treated with these agents, separately and in combination, for various intervals. Both agents, either singly or in combination, depressed mitoses. The maximum mitotic inhibition was obtained in cultures treated with theophylline or theophylline plus dibutyryl cAMP. Tritiated thymidine studies showed that the test agents had no effect on labelling index (LI) at 4 h, but a 74% inhibition of LI was observed at 24 h. The maximum inhibition of LI (93%) occurred at 96 hours. In contrast to the control cultures, which rarely contain keratohyaline granules (KG), a marked increase in the production of these granules occurred in cultures treated with dibutyryl cAMP plus theophylline. The KG were present over the whole outgrowth. Theophylline alone also stimulated the production of KG, whereas dibutyryl cAMP had no effect. These data show that these agents inhibit cell division and this inhibition may be accompanied by an increased production of KG.

Retinoid reversal of squamous metaplasia in organ cultures of tracheas derived from hamsters fed on vitamin A-deficient diet

Cytokinetic and ultrastructural studies were carried out to elucidate mechanisms involved in the reversal of squamous metaplasia (SM) by beta-retinoic acid in organ cultures of tracheas derived from vitamin A-deficient hamsters. Tracheal cultures exhibiting focal areas of SM were treated with the retinoid for up to 7 days. The retinoid significantly inhibited [3H]-thymidine labeling indices in the basal cells and stimulated the labeling indices in mucous cells. At the ultrastructural level the retinoid induced marked remodeling alterations in the metaplastic epithelium that included: (a) disruption of desmosomes and widening of intercellular spaces; (b) extensive vacuolation and degeneration of the metaplastic cells; (c) extrusion of the degenerated cells; (d) aggregation of keratin filaments; and (e) differentiation of certain basal cells into secretory cells. Consequently most degenerated metaplastic cells were extruded and the epithelium repopulated as a result of differentiation of basal cells into mucous cells and hyperplasia of the pre-existing mucous cells. The degenerative effects of the retinoid were limited to the metaplastic foci since the uninvolved epithelium adjoining metaplastic foci were not significantly altered. The results suggest that the restoration of normal tracheal epithelium following the retinoid treatment of explants exhibiting focal areas of squamous metaplasia is associated with the enhanced proliferation of the mucous cells. The inhibition of proliferation of basal cells further prevented hyperplasia and restored cell replication within the normal range.

Activity of retinoids against benzo(a)pyrene-induced hyperplasia in mouse prostate organ cultures

Abstract The antihyperplastic activity of several retinoids (with ring, side chain or polar group modifications) against benzo(a)pyrene (BP)-induced hyperplasia in mouse prostate cultures was examined. Prostate explants were made hyperplastic by treatment with BP for 8 days , followed by simultaneous treatment with BP and different concentrations of a retinoid. The anti-mitotic activity of retinoids was compared with β-retinoic acid (RA). Different retinoids produced various degrees of mitotic inhibition in the hyperplastic prostate epithelium. Five retinoids: 13-cis -retinoic acid, the methylketo cyclopentenyl analog of retinoic acid, the 1 -methoxyethyl cyclopentenyl analog of retinoic acid, N -retinoylglycine, and the 14 -fluoro derivative of the trimethylmethoxyphenyl analog of retinoic acid ethyl ester, showed greater activity than RA. Seven other retinoids were as active as RA. Two retinoids were less active than RA, and one retinoid produced no mitotic inhibitory effect.

Viability of cultured Lewis lung cell populations exposed to beta-retinoic acid.

Summary β-Retinoic acid inhibited the proliferation of cultured Lewis lung cells without decreasing the viability (as determined by cloning in semisolid medium) of the cell populations. We would like to thank Mrs. Carolyn M. Andrews and Dr. W. R. Laster for the Lewis lung tumors and Mrs. Carol S. Eldridge for excellent technical assistance with the cell culture experiments.

Histogenesis of benzo(a)pyrene-induced lesions in tracheal explants

SummaryCytokinetic and histogenic alterations associated with the development of benzo(a)-pyrene (BP) induced epidermoid metaplasia were studied in tracheal explants derived from normal hamsters. Treatment of the expiants with BP induced hyperplasia in both the basal and mucous cells. The hyperplasia of the basal cells persisted throughout the duration of the experiment whereas the hyperplasia of the mucous cells subsided between 7 and 10 days after treatment. This was accompanied by stimulation of ciliated cell differentiation and aberrant ciliogenesis which was not limited to the surface cells since some basal cells were observed differentiating into ciliated cells. Subsequently, the differentiation of basal cells into mucous cells was inhibited. Instead, the basal cells differentiated into metaplastic cells. With the progression of the lesions, the mucociliary surface layer was sloughed into the lumen due to the population pressure from the underlying actively proliferating metaplastic cells and their subsequent epidermoid differentiation. Approximately 50% of the expiants exhibited focal areas of squamous metaplasia at 7 days after the treatment and extensive epidermoid metaplasia was present in approximately 90% of the expiants at 10 days. These results support the hypothesis that BP induced epidermoid metaplasia of tracheal explants originates from the basal cells.

Effect of retinoids and estrogens on testosterone-induced hyperplasia of mouse prostate explants in organ culture.

Summary Testosterone produced epithelial hyperplasia in mouse prostate explants in organ culture. Simultaneous treatment of ex-plants with testosterone and 13-cis-retinoic acid inhibited the induction of hyperplasia. When explants were first made hyperplastic by treatment with testosterone for 8 days and followed by simultaneous treatment with testosterone and a retinoid for an additional 96 hr, the following retinoids reversed the hy-perplasia: 13-cis-retinoic acid, the 1-methox-yethylcyclopentenyl analog of retinoic acid, the trimethylmethoxyphenyl analog of retinoic acid, the trimethylphenyl analog of retinoic acid ethylamide, and the trimethylmethoxyphenyl analog of retinoic acid ethyl ester. The data indicate that these retinoids prevent androgen-induced hyperplasia in prostatic tissue in this system by a direct influence on the tissue. Since estrone or 17β-estradiol had no effect on testosterone-induced hyperplasia in the prostate explants, it is concluded that these estrogens may act by way of an indirect mechanism (pituitary-tesis axis) in preventing androgen-induced hyperplasia of the prostate.