Figure 1.
UVB induced apoptosis in FHN, XP-C and CS-B fibroblasts:
a) Cells were synchronized in G1, irradiated with steadily increasing doses of UVB, and collected 72 hours after the irradiation. Apoptosis rates were determined by sub-G1 quantification through flow cytometry. b) Cytomorphological analysis of FHN fibroblasts was to determine the percentage of apoptotic cells.
Table 1.
UVB doses defined as low or high based on the levels of apoptosis (sub-G1) induce in each cell line (based on Figure 1a).
Figure 2.
Effect of the irradiation of FHN, XP-C and CS-B fibroblasts with low or high UVB doses during cell cycle progression:
Cells were synchronized in G1, irradiated with different doses of UVB, and collected at the indicated times after irradiation. Fluorescence histograms were obtained for each condition after PI staining and flow cytometry. The proportion of cells in the G1, S and G2 phases of the cell cycle were analyzed and quantified for each histogram using ModFit LT. The results obtained were combined to generate the graphs presented. UVB doses used were: upper panels – non-irradiated controls; middle panels – low doses (750, 375 and 125 J/m2 for FHN, XP-C and CS-B respectively); lower panels – high doses (2,000, 750 and 500 J/m2 for FHN, XP-C and CS-B respectively).
Figure 3.
Recovery of transcription in FHN, XP-C and CS-B fibroblasts after irradiation with low and high UVB doses:
Cells were synchronized in G1, irradiated with different UVB doses, and labeled with tritiated uridine for 1-irradiated controls for each time-point.
Figure 4.
Western blot detection of p53 and Mdm2 proteins:
FHN (a), XP-C (b) and CS-B (c) fibroblasts were irradiated with the indicated doses of UVB and collected at the time intervals shown. Beta-actin was used as loading control.
Figure 5.
Kinetics of protein levels of Mdm2 and p53 in FHN fibroblasts irradiated with increasing doses of UVB (as indicated).
Beta-tubulin was used as loading control.
Figure 6.
Proposed model for the different responses of human cells to UVB irradiation depending on their ability to remove DNA lesions.
Upon UVB-irradiation of human fibroblasts synchronized in the G1 phase of the cell cycle, the presence of lesions on transcribed regions of the genome elicits the DNA Damage Response (DDR), comprising DNA repair, cell cycle arrest, and general transcription inhibition. In CS-B cells, the lack of TCR leads to low levels of Mdm2 and apoptosis induction. When DNA repair removes transcription blocking lesions (TBLs), Mdm2 counteracts p53 activation and cells are released to go through S-phase, and survive higher levels of lesions. In the case of XP-C (GGR−) fibroblasts, however non-removed lesions lead to replication blockage and cell death, even in the presence of increased levels of Mdm2.