Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells

Abstract

Despite the predominance of ultraviolet A (UVA) relative to UVB in terrestrial sunlight, solar mutagenesis in humans and rodents is characterized by mutations specific for UVB. We have investigated the kinetics of repair of UVA- and UVB-induced DNA lesions in relation to mutagenicity in transgenic mouse fibroblasts irradiated with equilethal doses of UVA and UVB in comparison to simulated-sunlight UV (SSL). We have also analyzed mutagenesis-derived carcinogenesis in sunlight-associated human skin cancers by compiling the published data on mutation types found in crucial genes in nonmelanoma and melanoma skin cancers. Here, we demonstrate a resistance to repair of UVB-induced cis-syn cyclobutane pyrimidine-dimers (CPDs) together with rapid removal of UVA-induced oxidized purines in the genome overall and in the cII transgene of SSL-irradiated cells. The spectra of mutation induced by both UVB and SSL irradiation in this experimental system are characterized by significant increases in relative frequency of C-->T transitions at dipyrimidines, which are the established signature mutation of CPDs. This type of mutation is also the predominant mutation found in human nonmelanoma and melanoma tumor samples in the TP53, CDKN2, PTCH, and protein kinase genes. The prevailing role of UVB over UVA in solar mutagenesis in our test system can be ascribed to different kinetics of repair for lesions induced by the respective UV irradiation.

Figure 1. Assessment of cell proliferation capacity in UV-irradiated mouse embryonic fibroblasts

Second passage mouse embryonic fibroblasts were seeded at a density of 3 × 10⁴ cells per culture dish on the day prior to UV-irradiation (T = 0), and subsequently irradiated with equilethal (~75% cell survival) doses of UVA, UVB, and SSL in comparison with control as described in Materials and Methods. Eight hours post-irradiation (T = 1) and every 24 hours thereafter until day four (T = 2, T = 3, and T= 4), a subset of cell cultures from all treatment groups was harvested, and subsequently total cell counting was performed. The total number of viable cells was determined in triplicate cultures irradiated with respective types of UV-irradiation, and the results are averaged for all time points. Arrows indicate the defined times at which UV-irradiation and cell harvesting for mutagenicity experiments, respectively, were performed. Error bars = standard deviations.

Figure 2. Qualitative assessment of induced DNA damage in mouse genome

Mouse embryonic fibroblasts were irradiated with equilethal (~75% cell survival) doses of UVA, UVB, and SSL in comparison with control as described in Materials and Methods. Genomic DNA was isolated and digested with specialized DNA repair enzymes, including (I) UVDE for detecting CPDs and (6-4)PPs combined, (II) T4 Endo V for determining CPDs only, and (III) Fpg for detecting oxidized (ring-opened) purines. The DNA digests were subjected to alkaline agarose gel electrophoresis, followed by standardized visualization procedure. For brevity, results at select time points are shown. (+) and (−) represent the presence and absence, respectively, of the indicated treatment conditions. In this assay, the frequency of lesions is roughly estimated from the central location of the most intense part of the smear but not from the length of the smear. Digestion buffer only = No enzyme was added to the reaction mix. M = molecular size marker.

Figure 3. Mapping of induced DNA damage in the cII transgene

TD-PCR and LM-PCR footprinting of the full-length cII transgene was done using the genomic DNA of mouse embryonic fibroblasts irradiated with equilethal (~75% cell survival) doses of UVA, UVB, and SSL in comparison with control. (a) Footprinting of dipyrimidine photolesions (CPDs and (6-4)PPs combined) by TD-PCR. (b) Footprinting of CPDs by LM-PCR. The LM-PCR bands migrate approximately 3 bases faster than the corresponding TD-PCR bands due to the addition of three riboguanosine triphosphate to all primer extension products in the latter method. (c) Footprinting of oxidized (ring-opened) purines by LM-PCR. (+Fpg), with Fpg enzyme pre-digestion to quantify oxidized (ring-opened) purines; (−Fpg), without Fpg enzyme pre-digestion to quantify non-specific background bands, e.g., UV-induced single strand breaks or abasic sites and strand breaks, resulting form spontaneous depurination of DNA. For brevity, results at select time points are shown. bp = base pair; M = molecular size marker; nt = nucleotide position.

Figure 4. UV-induced and spontaneous cII mutation spectrometry

Detailed mutation spectra of the cII transgene in mouse embryonic fibroblasts irradiated with equilethal (~75% cell survival) doses of UVB, UVA, and SSL in comparison to control. Mutations were quantified using the lambda transgenic shuttle vector recovery kit for Big Blue® rodents (Stratagene). Randomly selected cII mutant plaques, including 110, 161, and 100 plaques induced by UVB-, UVA, and SSL-irradiation, respectively, in comparison to 154 control plaques were subjected to DNA sequencing. Of the respective number of plaques, 108, 152, 87, and 147 contained a minimum of one mutation in the cII transgene. The UVB- and SSL-induced mutations are typed above the reference cII sequence, whereas the UVA-induced mutations and spontaneous control mutations are shown below the reference cII sequence. The UVB-induced mutations and spontaneous control mutations are typed in capital letters, whereas the SSL- and UVA-induced mutations are typed in small letters, the two are separated from one another, respectively, by (--). Data on UVA mutation spectrometry are adapted from Ref. (15). Substituted bases are in bold. Deleted bases are underlined; multiple deletions are continuously underlined. Inserted bases are shown with an arrow. Numbers below the bases are the nucleotide positions.

Figure 5. Mutation spectra in human skin tumors

The pie charts show the percentage of particular types of mutations found in the TP53 gene of human non-melanoma skin tumors (basal cell and squamous cell carcinomas), in the TP53, CDKN2, and protein kinase genes of melanomas, and in the PTCH gene of basal cell carcinomas. Data for the TP53 gene (n = 94 for melanomas and n = 482 for non-melanoma skin cancers) were obtained from the International Agency for Research on Cancer (IARC) mutation database (R11 version, October 2007) (33). Data for the CDKN2 gene (n = 99) (–46) and PTCH gene (n = 183) (–58) were derived from the published literature. The mutations found in protein kinase genes (n = 146) are derived from high throughput sequencing of cancer genomes (59).

Figure 6. Qualitative assessment of induced DNA damage in the genome of human cells

Normal human skin fibroblasts were irradiated with equilethal (~75% cell survival) doses of UVA, UVB, and SSL in comparison with control as described in Materials and Methods. Genomic DNA was isolated and digested with Fpg for detecting oxidized (ring-opened) purines and T4 Endo V for determining CPDs. The DNA digests were subjected to alkaline agarose gel electrophoresis, followed by standardized visualization procedure. For brevity, results at select time points are shown. M = molecular size marker.