A Comparative Study of Gene Array of Cell Cycle Modulators in Human Corneal Epithelial Cells Stimulated with Epidermal Growth Factor(EGF), Hepatocyte Growth Factor(HGF), or Keratinocyte Growth Factor(KGF)

ABSTRACT

PURPOSE: To identify and differentiate genes that are up-regulated or down-regulated in human corneal epithelial cells in response to epidermal growth factor(EGF), hepatocyte growth factor(HGF) or keratinocyte growth factor(KGF). METHODS: Primary cultures of human corneal epithelial cell(HCE) were treated with 25 ng/ml of EGF, 25 ng/ml HGF, 25 ng/ml KGF, or vehicle in serum-free medium for 8 hours. Total RNA was isolated with TRIZOL(GIBCO, NY), and treated with DNAse I.P 32-labeled complementary DNA(cDNA) probes were synthesized using 6 ug of total RNA made from HCE cells. Equivalent counts of P 32-labeled cDNA probes were hybridized with the membrane of Atlas human cell cycle array at 68degreesC overnight. After sequential washing, the membranes were exposed to X-ray film for three days. These results were analyzed using Atlas Image TM 1.1 Software. RNAse protection assay was used to confirm one of known genes on the array, which was up-regulated by EGF, KGF, and HGF in the human corneal epithelial cells. RESULTS: Autoradiographic analysis showed that out of 111 genes analyzed, 22 were up- or down-regulated in EGF, 26 in HGF and 7 in KGF compared to untreated corneal epithelial cell. After different signal intensity was normalized more than 2000 by Atlas Image TM 1.1 Software, 12 genes were up-regulated and 10 genes down-regulated in EGF. HGF have 6 up-regulated genes and 1 down-regulated gene and KGF had all up-regulated 7 genes. EGF, HGF and KGF all up-regulated the expression of cyclin D1(BCL-1 oncogene) and serine/threonine-protein kinase PITALRE in the primary cultured human corneal epithelial cells. EGF and KGF both up-regulated E2F-1 pRB-binding protein gene. HGF and KGF up-regulated cyclin D2 gene. Proto-oncogene raf was down-regulated by EGF and HGF. CONCLUSIONS: The three growth factors seemed to have similar effects on the genes that contribute to cell cycle control. Studies to analyze the significance of the differences among these growth factors are ongoing.