The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts.

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.

Effects of 12-0-tetradecanoyl-phorbol-13-acetate on oral epithelium in vitro.

TPA, a tumor promotor whose initial site of action is the cell membrane, was examined for its actions on hamster oral mucosa using light and scanning electron-microscopic procedures. Hamster cheek-pouch explants were cultured in vitro, then treated for 72 h with 1.6 X 10(-8) M TPA. Treated cultures showed a higher mitotic index and more extensive growth than control cultures. Epithelial cells in the control cultures appeared polygonal, with thin, small to medium microvilli. The cells in the treated cultures showed variable shape and surface morphology. Some were interconnected by broad cytoplasmic extensions while others demonstrated long, thin processes that traversed great distances. The unusual surface morphology may be a manifestation of altered phospholipid metabolism that produces a more "fluid" cytoplasmic membrane.

Effects of calcium ions on hamster cheek pouch epithelium grown in vitro.

Epithelial outgrowths from hamster cheek pouch explants were cultured for 14 days in media containing calcium concentrations of 0.05 mM, 0.075 mM, 0.1 mM, 0.25 mM, 0.75 mM or 1.45 mM (control). Compared with controls, the cultures grown at lower calcium concentrations (0.25-0.75 mM), exhibited higher mitotic indices and an increase in outgrowth size. The mitotic index of cultures at 0.25 mM calcium concentration was 30 +/- 2.26, compared to 24 +/- 2.24 for controls. Increase in size of the outgrowths was also observed at 0.25 mM calcium concentration compared with those grown in control medium. The epithelial outgrowths grown in control media exhibited cell stratification not observed at lower calcium concentrations. These studies indicate that lower calcium concentration in the media (0.25-0.75 mM) increased epithelial cell proliferation and inhibited keratinization.

Effects of retinoic acid on the differentiation of hamster cheek pouch epithelium grown in vitro.

Recently our laboratories have successfully attained preferential epithelial outgrowths from hamster cheek pouch explants. This has allowed us to systematically analyze oral epithelial cell differentiation as weel las examine the modulatory effects of various physiologic and pharmacologic agents in this epithelial cell system. The objective of the current investigation was to study the effects of various doses of retinoic acid (RA) on the epithelial outgrowths of hamster cheek pouch. Light microscopic observations employing colcemid treatment indicated that 10 IU/ml RA enhanced mitotic activity of the epithelium up to 25%. Scanning electron microscopic observations of control cultures showed flat, polyhedral epithelial cells containing thin long microvilli connecting the adjacent cells. In contrast, cultures treated with 10 IU/ml RA showed round or spherical cells exhibiting short of medium sized thicker microvilli. These were covered with a fuzzy appearing layer probably representing glycocalyx. Many bleb-like formation were also observed. Transmission electron microscopic examination of the control cultures showed the presence of tonofilaments and desmosomes, characteristic components of the keratinizing epithelia. In contrast, the RA treated cultures included cells which apparently were either devoid of tonofilaments or contained few fine filaments and small intercellular functions. In addition many vacuoles as well as variable numbers of electron-dense and electron-lucent granules were observed. Thus, it appears that RA inhibited keratinization and altered the differentiation pattern of keratinization and altered the differentiation pattern of keratinizing epithelium.

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.