A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures.

PURPOSE: This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. METHODS: Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. RESULTS: Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. CONCLUSIONS: Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

Inhibition of Colon Cancer Cell Growth by Phosphodiesterase Inhibitors Is Independent of cGMP Signaling.

There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colorectal cancer (CRC) prevention and treatment. The present study has tested the idea that PDE inhibitors inhibit growth and viability of CRC cell lines by increasing cyclic guanosine monophosphate (cGMP) and activating cGMP-dependent protein kinase (PKG). Colon cancer cell lines and those with ectopic PKG2 expression were treated with membrane-permeable 8Br-cGMP or inhibitors of PDE5, PDE9, and PDE10a. Levels of cGMP capable of activating PKG were measured by immunoblotting for phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The effects of treatment on CRC cell proliferation and death were measured using hemocytometry with trypan blue. Treatment with 8Br-cGMP had no effect on CRC cell proliferation or death. Endogenous PKG activity was undetectable in any of the CRC cells, but expression of ectopic PKG2 conferred modest inhibition of proliferation but did not affect cell death. Extremely high concentrations of all the PDE inhibitors reduced proliferation in CRC cell lines, but none of them increased cGMP levels, and the effect was independent of PKG expression. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generating machinery. In conclusion, PDE inhibitors that target cGMP only reduce CRC growth at clinically unachievable concentrations, and do so independent of cGMP signaling through PKG. SIGNIFICANCE STATEMENT: A large number of in vitro studies have reported that PDE inhibitors block growth of colon cancer cells by activating cGMP signaling, and that these drugs might be useful for cancer treatment. Our results show that these drugs do not activate cGMP signaling in colon cancer cells due to a lack of endogenous guanylyl cyclase activity, and that growth inhibition is due to toxic effects of clinically unobtainable drug concentrations.

Effects of 1,25 and 24,25 Vitamin D on Corneal Epithelial Proliferation, Migration and Vitamin D Metabolizing and Catabolizing Enzymes.

This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.