Acridine orange differential staining of total DNA and RNA in normal and galactosemic lens epithelial cells in culture using flow cytometry

Abstract

The lens epithelial cells are a primary site of involvement in galactosemia. Changes in their size, shape and proliferative capacity have been observed upon exposure to high galactose. In this report, changes in the cell cycle pattern of normal and galactosemic lens epithelial cells were examined by use of flow cytometry. Both changes in DNA and RNA were observed using the fluorochrome, acridine orange. Under the appropriate conditions acridine orange can be used to differentiate double-stranded DNA from single-stranded RNA. Using this approach, the DNA and RNA of normal and galactosemic (1, 4, or 7 days) lens epithelial cells can be compared. The results indicate that lens epithelial cells, when exposed to 40 mM galactose media or 30 mM glucose for 7 days, are induced to enter mitosis. Mannitol did not mimic these results. Changes in the cell cycle pattern were not observed when the cells were treated for 1 or 4 days. Although higher numbers of cells in mitosis were observed after 7 days exposure to 40 mM galactose, a correlation between proliferation, as measured by ³H-thymidine uptake, and mitosis was not possible. Apoptosis was evaluated as a possible explanation for these results. The changes in the DNA staining pattern could be use to monitor lens epithelial cells during galactosemia.

Key Words:

• acridine orange
• bovine (cattle)
• cell cycle
• galactosemia
• lens epithelial cells
• nucleic acids