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. 2005 Oct 19:5:135.
doi: 10.1186/1471-2407-5-135.

Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

Hsin-Lung Lo  1 , Satoshi Nakajima, Lisa Ma, Barbara Walter, Akira Yasui, Douglas W Ethell, Laurie B Owen
Affiliations

Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

Hsin-Lung Lo et al. BMC Cancer. .

Abstract

Background: UV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations.

Methods: NER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m2 UVB exposure photoreactivation light (PR, UVA 60 kJ/m2) was provided for photolyase activation and DNA repair. Apoptosis was monitored 24 hours later by flow cytometric analysis of DNA content, using sub-G1 staining to indicate apoptotic cells. To confirm the effects observed with CPD lesions, the molecular mimic of CPD, pTpT, was also tested in vitro and in vivo for its effect on cell cycle and apoptosis.

Results: The specific repair of 6-4PP lesions after UVB exposure resulted in a dramatic reduction in apoptosis. These findings suggested that 6-4PP lesions may be the primary inducer of UVB-induced apoptosis. Repair of CPD lesions (despite their relative abundance in the UV-damaged cell) had little effect on the induction of apoptosis. Supporting these findings, the molecular mimic of CPD, (dinucleotide pTpT) could mimic the effects of UVB on cell cycle arrest, but were ineffective to induce apoptosis.

Conclusion: The primary response of the cell to UV-induced 6-4PP lesions is to trigger an apoptotic program whereas the response of the cell to CPD lesions appears to principally involve cell cycle arrest. These findings suggest that CPD and 6-4 PP may induce differential biological effects in the UV-damaged cell.

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Figures

Figure 1
Figure 1
Radio-immunoassay (RIA) of UV-induced 6-4PP and CPD. Twelve hours after UVB irradiation, genomic DNA was isolated from treated cells and assayed for UV-induced DNA damage using lesion-specific antibodies. An average of fifty-five CPD and twelve 6-4PP lesions per million bases were detected after exposure to 300 J/m2 UVB. Results are the average of duplicate assays from two independent experiments. Error bars show standard deviation.
Figure 2
Figure 2
Detection of photolyase expression and function. Photolyase expression was confirmed by western blots of whole cell lysates from stably transfected XPA cell lines containing empty vector or both photolyases. Blots were probed with antibodies specific for CPD-PL (A) and 6-4PP-PL (B). Photolyase repair of UV induced DNA damage in XPA cells was detected by immuno-dot-blot, using the same antibodies. Representative examples of 6-4PP (C) and CPD (D) lesion studies are shown.
Figure 3
Figure 3
Impact of photoreactivation on UVB-induced apoptosis. Apoptosis in XPA cells was monitored at a 24 hour time point following exposure to 300 J/m2 UV and photoreactivation-induced repair. Apoptosis was examined by PI staining and FACS, gating the sub-G1 population as apoptotic. Apoptotic values were normalized to empty vector containing cells (amount of apoptosis set as 100%). Bar graphs show the relative amount of apoptosis for control cells, cells expressing CPD-PL alone (40% rescue), cells expressing 6-4PP-PL alone (70% rescue), or cell expressing both-PLs (75% rescue).
Figure 4
Figure 4
Effects of pTpT on cell cycle and apoptosis in vitro. A. Jurkat cells were exposed to pTpT (100 μM) or pApA (100 μM) in vitro and cell cycle analysis performed after 1 and 24 hours by flow cytometry as described in Materials and Methods. Results show representative histograms from five independent experiments. Note that only pTpT stalls the cell cycle, not pApA, and no increase in apoptotic sub-G1 DNA content was observed with either pTpT or pApA. B. Bar graph showing percentage of cells in each phase of the cell cycle in cells treated with pTpT or pApA for 24 hrs.
Figure 5
Figure 5
CPD mimic, pTpT, does not induce apoptosis in vivo. TUNEL-stained sections from mouse dorsal skin coated with pTpT or pApA, or exposed to a single dose 4 kJ/m2 UVB as a positive control. (A) Green fluorescence indicates 3' DNA breaks, representative of apoptotic cells. (B) Numbers of TUNEL positive cells in skin sections from three separate experiments, consisting of three mice in each experimental group. Apoptotic values indicate the number of TUNEL-positive cells by tissue length. Pictures were taken on a Nikon ECLIPSE TE2000-U microscope under FITC illumination at 400×.
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