Squamous metaplasia in organ cultures of vitamin A-deficient hamster trachea: cytokinetic and ultrastructural alterations.

Cytokinetic and morphologic studies were performed to elucidate the origin of epidermoid metaplasia in organ cultures of tracheas derived from vitamin A-deficient Syrian golden hamsters. Focal areas of squamous metaplasia were observed 2 days after explantation, and extensive epidermoid metaplasia and cornification were present in approximately 89% of the explants at day 10. During the first 2 days, both basal and mucous cells proliferated actively. By day 3, the total mucous cell [3H]thymidine labeling index (LI) had declined and remained at very low levels during the remaining culture period. The basal cell LI also declined from its higher level, but it remained at relatively higher levels than those of the mucous cells between days 3 and 9 after culture. In the lesions, labeled cells were generally confined to the basal layer, and the LI was about thirtyfold greater than the total basal cell LI. As the lesions progressed, the surface layer containing the mucous and ciliated cells was exfoliated as a result of population pressure from the underlying actively proliferating basal cells and subsequent epidermoid differentiation of the daughter cells. These data support the hypothesis that epidermoid metaplasia of tracheobronchial epithelium caused by vitamin A deficiency originates from the generative or basal cells.

Inhibition and reversal by beta-retinoic acid of hyperplasia induced in cultured mouse prostate tissue by 3-methylcholanthrene or N-methyl-N'-nitro-N-nitrosoguanidine.

The effect of beta-retinoic acid (RA) on carcinogen-induced hyperplasia was studied in organ cultures of mouse prostate gland. 3-Methylcholanthrene (MCA), requiring metabolic activation, or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), not requiring activation, were used to induce hyperplastic changes. Treatment of cultures with MCA or MNNG stimulated cell proliferation and caused the alveolar epithelium to become hyperplastic. The development of this hyperplasia was inhibited when RA was added simultaneously with MCA or MNNG. However, RA had no significant effect on cell proliferation in untreated control cultures. Elimination of carcinogen from the hyperplastic cultures after 8 days of treatment did not reverse hyperplasia of the alveolar epithelium. When the withdrawal of MCA or MNNG was followed by treatment of the cultures with RA, hyperplasia was markedly reversed within 96 hours. Thus RA actively inhibited and reversed the effect of MCA and MNNG, two carcinogens that may have different mechanisms of action.

Reversal by vitamin A analogues (retinoids) of hyperplasia induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse prostate organ cultures.

The antihyperplastic activity of beta-retinoic acid (RA) and nine synthetic analogues (retinoids) was examined in organ cultures of mouse prostate made hyperplastic by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). After 8 or 10 days, when most explants developed hyperplasia, the carcinogen was withdrawn and explants were incubated in control medium and medium containing different concentrations of a retinoid. The antimitotic activity of retinoids was compared with that of RA. Different retinoids produced variable degrees of mitotic inhibition in the hyperplastic prostate epithelium. The methylketo cyclopentenyl and 1-methoxyethyl cyclopentenyl analogues of RA were at least 50-fold more active than RA in reversing MNNG-induced hyperplasia. The trimethylmethoxyphenyl analogue of RA and retinyl methyl ether were significantly more active than RA. Three analogues, N-acetyiretinylamine, retinal acetyl hydrazone, and retinal oxime, were as active as RA. The chlorotrimethylphenyl analogue showed less activity than RA, and alpha-retinyl acetate was completely devoid of mitotic inhibitory activity.

Isolation and characterization of epithelial cell types from the normal rat colon.

Populations enriched in proliferative, mucous goblet, and absorptive cells were isolated by a method of repeated time dissociation of the colonic mucosa from the adult rat. Five sequential cell suspensions were obtained by rotating the everted colon on 0.2% trypsin solution in Eagle's minimum essential medium at 30 degrees for 20 min each time. Approximately 3.7 x 10(7) cells were obtained from each colon, and 87% of the cells were found viable. The results, based on the [3H]-thymidine-labeling index, [3H]thymidine incorporation, thymidine kinase activity, and histochemical and ultrastructural observations, indicate the Cell Suspension I, separated from the luminal surface, contains 82 +/- 9% (S.D.) absorptive cells; Suspension III, separated from the middle level of the crypts, contains 80 +/- 7% mucous cells. Suspension V, isolated from the crypt base, contains the majority of proliferative epithelial cells (85 +/- 10%). This method provides a suitable tool for a variety of studies in colon carcinogenesis.

Extended survivability of prostate cancer cells in the absence of trophic factors: increased proliferation, evasion of apoptosis, and the role of apoptosis proteins.

This project was undertaken to study the survival properties of various prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells (LNCaP, PC3, and Du145), in the absence of trophic factors. Cell proliferation and cell death were quantitated by enumerating the number of live cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole dihydrochloride nuclear staining. These cells demonstrated an overall survivability in the order of BPH < NHP < LNCaP < PC3 < PCA < Du145. Upon growth factor deprivation, NHP/BPH cells rapidly underwent apoptosis, leading to a decreased number of live cells. PCA/PC3/Du145 cells, in contrast, demonstrated an initial phase of aggressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell proliferation, followed by a later phase in which apoptosis exceeded the cell proliferation. LNCaP cells demonstrated survival characteristics between those of NHP/BPH and PCA/PC3/Du145 cells. We concluded that the increased survivability in prostate cancer cells results from enhanced cell proliferation as well as decreased apoptosis. The molecular mechanisms for evasion of apoptosis in prostate cancer cells were subsequently investigated. Quantitative Western blotting was used to examine the protein expression of P53 and P21WAF-1, Bcl-2 and Bcl-X(L) (anti-apoptotic proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and down-regulated the expression of P21. Furthermore, NHP and BPH cells endogenously expressed little or no Bcl-2. In sharp contrast, prostate cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 and/or BclX(L), lost response to withdrawal-induced up-regulation of Bax/Bad/Bak or decreased or even completely lost Bax expression and expressed some novel proteins such as P25 and P54/55 complex. These data together suggest that prostate cancer cells may use multiple molecular mechanisms to evade apoptosis, which, together with increased proliferation, contribute to extended survivability of prostate cancer cells in the absence trophic factors.

Growth of human nondiploid primary prostate tumor epithelial cells in vitro.

Research into molecular and cellular defects underlying prostate cancer would be advanced by in vitro models of prostate tumor cells representing patient tumors. We have propagated, in serum-free medium, epithelial cell cultures derived from nondiploid prostate tumors and normal human prostate. The serial passage tumor cells exhibited nondiploid karyotype and transformed phenotypes of focus formation and anchorage-independent growth. In contrast, the normal prostate cells showed diploid karyotype and lacked transformed phenotypes. Both the tumor and normal cells were positive for prostate-specific antigen and cytokeratins 18 and 19 and negative for keratin 15. These results demonstrate that the nondiploid prostate tumors and normal prostate epithelial cell cultures retained their respective in vivo properties and should allow studies to elucidate molecular alterations involved in human prostate cancer.

Evaluation of vitamin A analogs in modulating epithelial differentiation of 13-day chick embryo metatarsal skin explants.

Seventeen vitamin A compounds were evaluated in organ culture for activity in altering epithelial differentiation of metatarsal skin explants from 13-day-old chick embryos. The explants keratinized in 6 to 8 days and, when cultured in the presence of beta-retinoic acid (RA), inhibition of keratinization occurred and a mucous metaplasia developed. A cyclopentenyl analog of retinoic acid was approximately 10-fold more effective than RA in producing mucous metaplasia. Six other analogs exhibited about the same activity as RA: trimethylmethoxyphenyl analog of retinoic acids, alpha-retinoic acid, 13-cis-retinoic acid, methyl retinoate, ethyl retinoate, and N-ethylretinamide. The following 5 vitamin A compounds were about one-fourth as effective as RA: the trimethylmethoxyphenyl analog of ethylretinamide, the phenyl analog of retinoic acid, the trimethylmethoxyphenyl analog of ethyl retinoate, beta-retinyl acetate, and retinol. The furyl analog of retinoic acid and N,N-diethylretinamide were approximately one-tenth and one-fifteenth less effective than RA in inhibiting keratinization. The analog, alpha-retinyl acetate, was about one-hundredth as effective as RA and the pyridyl analog of retinoic acid (2.5 X 10(-5) M) did not inhibit keratinization. Since the property of altering epithelial differentiation may be a fundamental requirement for the prophylaxis and/or treatment of malignant epithelial lesions, this system can be used to determine whether the new synthetic analogs of vitamin A are active in modulating epithelial differentiation.

Ultraviolet light carcinogenesis in hairless mice: cell kinetics during induction and progression of squamous cell carcinoma as estimated by the double-labeling method.

The double-labeling method for studying cell cycle kinetics was applied to uninvolved epidermis of hairless mice (irradiated and unirradiated), and at various stages of ultraviolet light (UVL)-induced squamous cell carcinoma. The growth fraction (GF) was determined by continuous labeling with tritiated thymidine (3HTdR). The data indicate that the double-labeling technique is an acceptable method for studying cell kinetics in UVL-induced squamous cell carcinoma. The GF in normal epidermis (unirradiated and irradiated), hyperplastic epidermis, and lesions of squamous cell carcinoma is 1. The duration of cell generation (Tc) and DNA synthesis (Ts) periods decreased progressively with induction and progression of squamous cell carcinoma. For normal epidermis (irradiated mice) the Tc and Ts were 91 and 6.3 hr, respectively. In the hyperplastic epidermis, the Tc and Ts were reduced to 44 and 4.5 hr, respectively, while even lower values were obtained for the tumor (Tc = 16 hr, Ts = 3.1 hr). The labeling index (LI) in normal epidermis was 7% and increased progressively in hyperplastic epidermis (10%) and squamous cell carcinoma (19%). Epidermal cell differentiation in hyperplastic and tumor tissues appears to have been delayed (GF = 1), since cells above the basal layer, which in normal epidermis keratinize, retain the ability to proliferate, as evidenced by extensive incorporation of [3H]TdR in these cells. The results suggest that tumor production is associated with a progressive shortening of the cell cycle and delayed keratinization of epidermal cells.

The effect of vitamin A on growth and differentiation of human keratinocytes in vitro.

Epithelial outgrowths (keratinocytes) from normal human skin in vitro were exposed daily for 30 min to vitamin A alcohol for periods up to 5 weeks. There was a markedly decreased number of keratohyaline granules in treated cultures, indicating an effect on the differentiation process, but there was no evidence for mucous metaplasia. The area of vitamin A-treated outgrowths was greater than that of controls at all times. In addition, there was a higher mitotic index, higher labeling index, and larger growth fraction in treated cultures. The combination of altered differentiation and enhanced proliferation of keratinocytes would appear to account for the larger outgrowth area found in vitamin A-treated cultures.

Effect of retinoids on the differentiation of chick embryo metatarsal skin explants.

Twelve retinoids were evaluated in organ culture for activity in modulating epithelial differentiation of metatarsal skin explants from 13-day chick embryos. The epithelium differentiated into a squamous, keratinizing epidermis; but, in the presence of active retinoids, keratinization was inhibited, and a mucous metaplasia developed. The methyl-keto and 1-methoxyethyl cyclopentenyl analogs of retinoic acid were about tenfold more effective than retinoic acid in altering epithelial differentiation. The dichlorophenyl analog exhibited about the same activity as retinoic acid. The following analogs were one-half to one-third as effective as retinoic acid in inhibiting keratinization: the chlorotrimethylphenyl analog of retinoic acid and the 13-cis, 10-fluoro analog of trimethylmethoxyphenyl methyl retinoate. The other 7 retinoids were essentially not active at the concentration tested (1.4--2.0 x 10(-5) M). The activity of synthetic retinoids in altering epithelial differentiation may be related to their ability to affect or treat epithelial lesions provided that modification of the retinoid molecule can enhance its activity and decrease toxicity.

Reversal by retinoids of keratinization induced by benzo[alpha]pyrene in normal hamster tracheal explants: comparison with the assay involving organ culture of tracheas from vitamin A-deficient hamsters.

In a defined culture system for hamster tracheal explants, the activity of 12 different retinoids was evaluated for reversal of keratinization induced by exposure to the carcinogen, benzo[alpha]pyrene (BP-HTOC assay). The effects of retinoids in this system were compared to those in a defined culture system for tracheal explants from vitamin A-deficient hamsters (standard-HTOC assay). In both assays, all-trans-retinoic acid (RA) and 13-cis-RA were the most active retinoids. For RA and 13-cis-RA, the values of ED50 determined in the BP-HTOC bioassay were 4 x 10(-12) and 1 x 10(-11) M, respectively, whereas the corresponding values in the standard HTOC assay were 2 x 10(-11) and 3.3 x 10(-10) M. For all 12 retinoids, the ED50 values from the BP-HTOC were lower than those from the standard-HTOC assay, and there was also a statistically significant correlation between the rank-ordering of ED50 values from the 2 assays. Among 3 N-(retinoyl)amino acids examined in both assays, N-(retinoyl)leucine was the most active, N-(retinoyl)phenylalanine the least active, and N-(retinoyl)alanine intermediate. Among a novel series of bifunctional retinoic acid analogues, the dicarboxyl derivative was the most active. On the basis of these results, the BP-HTOC assay appears to be one of the most sensitive assays for retinoids yet developed. This assay is an appropriate model for evaluating the chemopreventive potential of new retinoids in vitro.

Successful in vitro growth of human respiratory epithelium on a tracheal prosthesis.

Reconstruction of tracheal defects may be necessary following trauma or oncologic surgery. Defects up to 8 cm can often be repaired using end-to-end anastomosis. Use of a tracheal prosthesis for larger defects has been complicated by recurrent stenosis and infection. Recent animal studies, utilizing a Dacron polyurethane prosthesis suggest that problems with anastomotic stenosis and infection can be controlled. Problems with a central stenosis within the prosthesis persist when used for defects greater than 6 cm. Establishment of a confluent lining of respiratory epithelium is believed to be necessary for successful prosthetic tracheal reconstruction. Using cell culture techniques, we report the first successful seeding and growth of human respiratory epithelium onto a Dacron polyurethane tracheal prosthesis.

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands.

Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Ad12-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca+2 and Mg(+2)-free Hanks', balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the delta F508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and cellular transport of [11-3H] all-trans-retinoic acid in the liver of vitamin A-deficient hamsters.

In this study, we examined, by ultrastructural autoradiography, the uptake and intracellular transport of [3H]all-trans-retinoic acid ([3H]RA) in the livers of vitamin A-deficient hamsters. Four-week-old animals were administered 25 microCi of [3H]RA by gavage, and, at different intervals thereafter, one animal was sacrificed. Their livers were excised and processed for autoradiography. Radioactive grains were observed to pass randomly through the plasma membrane by diffusion. No evidence of retinoid internalization by endocytosis was observed. Between 1 and 30 min after gavage, the radioactivity in parenchymal cells was associated mainly with rough endoplasmic reticulum (RER) and mitochondria. The labeling over nuclei was apparent at 1 min, remained relatively high up to 30 min, and subsequently decreased. At 2 and 5 hr, only a few grains were observed over nuclei, RER and mitochondria. At 24 hr, most of the labeling was associated with endothelial cells and sinusoidal spaces, indicating mobilization of [3H]RA from the liver. The results indicate that [3H]RA is transported through the plasma membrane by transmembrane diffusion without endocytosis and, after entering the cells, the ligand is rapidly translocated into nuclei.

Effects of theophylline, epidermal chalone and x-irradiation on proliferation and differentiation of human keratinocytes in vitro.

Outgrowth cultures of normal human epidermis were used to study a possible relationship between growth inhibition and differentiated function. The effects of theophylline, epidermal chalone and x-irradiation on mitoses and the characteristic production of epidermal keratohyaline granules (KG) were examined at various intervals after the treatment. Theophylline (an inhibitor of cyclic nucleotide phosphodiesterase) or epidermal chalone inhibited mitoses and enhanced KG production. X-irradiation inhibited mitoses but had no effect on KG formation. These results indicate that inhibition of proliferation per se is not sufficient to enhance keratinization of human epidermal cells.