Abstract
Abstract: :
Purpose: Aldehyde dehydrogenase 3A1 (ALDH3A1) is an NAD(P)+-dependent enzyme that is expressed in high concentrations in corneal epithelial cells, and metabolizes aldehydes produced during UV-induced lipid peroxidation. We have recently found that ALDH3A1 protects human corneal epithelial cells against UV-induced oxidative damage using genetically engineered cell lines expressing ALDH3A1. In the course of these experiments, we observed a decreased cell growth in ALDH3A1-expressing cells compared to mock-transfected cells. The aim of the present study was to investigate the effects of ALDH3A1 expression on cell proliferation. Methods: SV-40 transformed human corneal epithelial (HCE) and human keratinocyte (NCTC 2544) cell lines were genetically engineered to express human ALDH3A1 by stable transfection. The effects of the ALDH3A1 expression on cell growth and cell cycle were studied by: a) cell proliferation and colony formation assays, b) flow cytometry and BrdU incorporation assays, and c) Western blot analysis to assess the levels of the cell cycle-inhibitory protein p21. Results: Expression of ALDH3A1 in both HCE and NCTC 2544 cell lines decreased colony formation efficiency by more than 40%. In mock-transfected HCE and parental HCE cells the doubling time and length of the cell cycle were found to be 1.8 and 2.7 days, respectively. Expression of ALDH3A1 in HCE cells increased the doubling time to 3.7 days and the length of cell cycle to 5.5 days. Furthermore, mock-transfected NCTC 2544 and parental NCTC 2544 cells exhibited a doubling time of 1.4 days, while the length of cell cycle was 2.0 days. Expression of ALDH3A1 in NCTC 2544 cells increased the doubling time and the length of cell cycle to 3.8 and 5.4 days, respectively. Analysis for DNA content by flow cytometry in asynchronous cultures of both cell lines did not reveal cell accumulation in a particular phase of the cell cycle. However, BrdU incorporation was three-fold higher in mock-transfected HCE cells compared to ALDH3A1-expressing cells. Moreover, we found decreased p21 protein levels in asynchronous cultures of ALDH3A1-expressing cells compared to the mock-transfected cells. Conclusions: These results provide evidence that ALDH3A1 may be involved in the control of cell growth and the progression of cell cycle by modulating expression of cell cycle regulatory proteins.
Keywords: cornea: epithelium • proliferation • cornea: basic science