Korean J Ophthalmol.  2011 Jun;25(3):196-201. 10.3341/kjo.2011.25.3.196.

Mechanisms of Apoptosis on Human Lens Epithelium after Ultraviolet Light Exposure

• Kim ST
• Koh JW

• KMID: 1010028
• DOI: http://doi.org/10.3341/kjo.2011.25.3.196

Abstract

PURPOSE
The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes.
METHODS
Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway.
RESULTS
Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased.
CONCLUSIONS
Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.

Keyword

Caspase 3; Lens epithelium; NOXA; p53; Ultraviolet rays

MeSH Terms

Apoptosis/*physiology
Caspase 3/metabolism
Cell Line
Cell Survival/radiation effects
Epithelial Cells/radiation effects
Humans
Lens, Crystalline/cytology/*physiology/*radiation effects
Proto-Oncogene Proteins c-bcl-2/metabolism
Tumor Suppressor Protein p53/metabolism
*Ultraviolet Rays

Figure

• 

  Fig. 1 After human lens epithelial cells were exposed to ultraviolet light, the survival rate (mean±SD) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 0, 2, 4, 6, 12, and 24 post-exposure. The MTT reduction ratios among the control group, 6 hour post-exposure group, and 12 hour post-exposure group were 100, 72 ± 4.9, and 21.5 ± 6.7%, respectively (p < 0.05). Cell viability decreased with time. UV = ultraviolet.

• 

  Fig. 2 Hoechst staining. (A) The control group, which was not exposed to ultraviolet irradiation, showed normal nuclei appearance without nuclear fragmentation. (B) The experimental group (4 hours post-exposure) showed abnormal nuclei appearance with nuclear fragmentation. It appeared that the fragments moved to neighboring cells or that neighboring cells move to embrace the fragments.

• 

  Fig. 3 An immunochemical study using anti-caspase 3 antibodies (A). The control group showed no fluorescent signal corresponding to active caspase 3 protein. (B) The experimental group showed a significantly increased fluorescent signal corresponding to active caspase 3 protein. The fluorescent signal was significantly strong and distributed throughout cytoplasm and nuclei with a spotted character after ultraviolet exposure.

• 

  Fig. 4 Gene expressions were evaluated by Western blotting. We compared the control group with the experimental group (4 hours post-exposure). (A) An immune band was observed around 53kDa. The expression of p53, caspase 3 and NOXA relative to beta-actin (GeneTex Inc., used as an internal control) was checked. (B) p53 decreased from 86 to 40, caspase 3 increased from 25 to 103 and NOXA increased from 24 to 194. C = control; UV = ultraviolet.

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