PARP-1/PAR Activity in Cultured Human Lens Epithelial Cells Exposed to Two Levels of UVB Light

Abstract

This study investigated poly(ADP-ribose) polymerase-1 (PARP-1) activation in cultured human lens epithelial cells exposed to two levels of UVB light (312 nm peak wavelength), 0.014 and 0.14 J cm^-2 ("low" and "high" dose, respectively). At the low dose, PARP-1 and poly(ADP-ribose) (PAR) polymers acted to repair DNA strand breaks rapidly with no subsequent major effects on either cell morphology or viability. However, following the high UVB dose, there was a dramatic second phase of PARP-1 activation, 90 min later, which included a sudden reappearance of DNA strand breaks, bursts of reactive oxygen species (ROS) formation within both the mitochondria and nucleus, a translocation of PAR from the nucleus to the mitochondria and an ultimate 70% loss of cell viability occurring after 24 h. The results provide evidence for an important role for PARP-1 in protecting the human lens epithelium against low levels of UVB light, and possibly participating in the triggering of cell death following exposure to toxic levels of radiation.

Figure 1

Spectral output of the UV lamp used in the study. Note the peak intensity at 312 nm wavelength. Light below 280 nm (UVC) was removed with a filter.

Figure 2

Photographs of cultured human LECs at various times after exposure to either a low dose (0.09 mW/cm²) or high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 60 mm plates. a. 24 hr control; b. low dose UVB, 1 hr; c. low dose UVB, 3 hr; d. low dose UVB, 24 hr; e. high dose UVB, 1 hr; f. high dose UVB, 3 hr; g. high dose UVB, 24 hr; h. high dose UVB, 24 hr, stained for F-actin. Note the cell loss and abnormal threadlike structures between cells (arrows) in g and h. The results are representative of three experiments.

Figure 3

Viability of cultured human LECs at various times after exposure to either a low dose (0.09 mW/cm²) or high dose (0.9 mW/cm²) of UVB radiation for 2.5 min. Analysis was done using the MTT assay for cell viability. Results are expressed as percent of control cells not exposed to UVB light, means +/− S.D. for three experiments. Open bars: low UVB dose; solid bars: high UVB dose. **: p<0.01, relative to control.

Figure 4

DNA strand-breaks (TMR Red assay) and DAPI staining in cultured human LEC’s at various times after exposure to either a low dose (0.09 mW/cm²) or high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 4-well chamber slides. a. low dose; b. high dose; c. densitometric fluorescence scanning of the cell nuclei; average cell nucleus brightness minus control for 6 to 24 cells, normalized per cell. Dashed line: low dose; solid line: high dose; n=3; error bars: S.D. (some error bars were smaller than the markers). ***: p<0.001, relative to control.

Figure 5

Reactive oxygen species (ROS) detection (CellROX® Red and Green) in cultured human LEC’s at various times after exposure to either a low dose (0.09 mW/cm²) or high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 4-well chamber slides. Red fluorescence indicates ROS (mainly H₂O₂) in the cytoplasm. Green fluorescence indicates ROS (mainly H₂O₂) in the nucleus. a. low dose; b. high dose; c. densitometric fluorescence scanning of the cells; average cell brightness minus control for 6 to 40 cells, normalized per cell (red and green brightness added); Dashed line: low dose; solid line: high dose. n=3; error bars: S.D. ***: p<0.001, relative to control.

Figure 6

Mitochondrial O₂⁻ detection (MitoSOX Red reagent) and Hoechst nuclear staining in cultured human LEC’s at various times after exposure to a high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 4-well chamber slides. a. fluorescent images; b. densitometric fluorescence scanning of the cells; average cell brightness minus control for 12 to 46 cells, normalized per cell. n=3; error bars: S.D. (some error bars were smaller than the markers). ***: p<0.001, relative to control.

Figure 7

PARP-1 enzyme immunofluorescence and DAPI nuclear staining in cultured human LEC’s at 5 and 90 min after exposure to a high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 4-well chamber slides. a. the fluorescent images; b. densitometric fluorescence scanning of the cells; average cell nuclear brightness minus control for 27 to 41 cells. n=3; error bars: S.D. (some error bars were smaller than the markers).

Figure 8

PAR polymer immunofluorescence and DAPI nuclear staining in cultured human LEC’s at various times after exposure to either a low dose (0.09 mW/cm²) or high dose (0.9 mW/cm²) of UVB radiation for 2.5 min in 4-well chamber slides. a. low dose; b. high dose; c. densitometric fluorescence scanning of the cells; average cell brightness minus control for 6 to 59 cells, normalized per cell. Dashed line: low dose; solid line: high dose; n=3; error bars: S.D. (some error bars were smaller than the markers). ***: p<0.001, relative to control.

Figure 9

Higher magnification (b and d) of one cell each (shown in the rectangles of a and c) from the 5 min (a and b) and 90 min(c and d) merged results of the experiment conducted in Fig. 8b (PAR and DAPI following exposure to the high dose of UVB).