J Korean Ophthalmol Soc.  2001 Mar;42(3):473-482.

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)

• Lee JS
• Hong JW
• Shin DH
• Liu JL
• Wilson SE

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.

Keyword

Complementary DNA; Epidermal growth factor; Hepatocyte growth factor; Human corneal epithelial cell; Keratinicyte growth factors

MeSH Terms

Cell Cycle Checkpoints
Cell Cycle*
Cyclin D2
Cyclins
Deoxyribonucleases
DNA, Complementary
Epidermal Growth Factor
Epithelial Cells*
Hepatocyte Growth Factor
Hepatocytes*
Humans*
Intercellular Signaling Peptides and Proteins
Keratinocytes*
Membranes
Phosphotransferases
Proto-Oncogenes
Ribonucleases
RNA
X-Ray Film
Cyclin D2
Cyclins
DNA, Complementary
Deoxyribonucleases
Epidermal Growth Factor
Hepatocyte Growth Factor
Intercellular Signaling Peptides and Proteins
Phosphotransferases
RNA
Ribonucleases