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Cancer Research 66, 11140, December 1, 2006. doi: 10.1158/0008-5472.CAN-06-0563
© 2006 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Activation of the Fanconi Anemia/BRCA Pathway and Recombination Repair in the Cellular Response to Solar Ultraviolet Light

Jessica Dunn, Marisa Potter, Adam Rees and Thomas M. Rünger

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts

Requests for reprints: Thomas M. Rünger, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118. Phone: 617-638-5551; Fax: 617-638-5515; E-mail: truenger{at}bu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recombination repair plays an important role in the processing of DNA double-strand breaks (DSB) and DNA cross-links, and has been suggested to be mediated by the activation of the Fanconi anemia (FA)/BRCA pathway. Unlike DNA damage generated by ionizing radiation or DNA cross-linking, UV light–induced DNA damage is not commonly thought to require recombination for processing, as UV light does not directly induce DSBs or DNA cross-links. To elucidate the role of recombination repair in the cellular response to UV, we studied the FA/BRCA pathway in primary skin cells exposed to solar–simulated light. UV-induced monoubiquitination of the FANCD2 protein and formation of FANCD2 nuclear foci confirmed the activation of the pathway by UV light. This was only observed when cells were irradiated during S phase and was not caused by directly UV-induced DSBs. UV-exposed cells did not exhibit FANCD2 nuclear foci once they entered mitosis or when growth-arrested. In addition, UV-induced nuclear foci of the recombination proteins, RAD51 and BRCA1, colocalized with FANCD2 foci. We suggest that in response to UV light, when nucleotide excision repair failed to repair, or when translesional DNA synthesis failed to bypass UV-induced DNA photoproducts, the FA/BRCA pathway mediates the recombination repair of replication forks stalled at DNA photoproducts as a third line of defense. (Cancer Res 2006; 66(23): 11140-7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, growth retardation, various congenital abnormalities, and a predisposition to cancer, in particular to acute myeloid leukemia and to solid tumors including squamous cell carcinomas of the head and neck and the anogenital region (1–7). Cells from patients with FA are characterized by a hypersensitivity to DNA cross-linking agents and oxygen (3, 7, 8), increased spontaneous and DNA cross-linker–induced chromosomal instability (7), and accelerated telomere shortening (3, 9). FA is genetically heterogeneous with at least 12 complementation groups (FANCA–FANCM), all of which, except FANCI, have been cloned. Most of the FA proteins have been shown to act in a common DNA damage response signaling pathway, which also involves BRCA1 and BRCA2 (mutated in hereditary breast cancer; refs. 8, 10–13). This FA/BRCA pathway is part of a complex genome stability network (3, 7, 8, 13). Germ line mutations in this network increase cancer risk and are related to a number of cancer-prone syndromes. In addition to FA, these are ataxia telangiectasia, Nijmegen breakage syndrome, ataxia telangiectasia–like disorders, Seckel syndrome, and xeroderma pigmentosum (14).

It has been suggested that the activation of the FA/BRCA pathway mediates DNA repair through homologous recombination (3, 7, 8, 10, 13, 15, 16). Central to this pathway is the monoubiquitination of the FANCD2 protein at lysine 561, upon which FANCD2 forms nuclear foci with BRCA1 and the MRE11-NBS1-RAD50 complex (ref. 10; and others). Monoubiquitination of FANCD2 requires the assembly of the FA complex, containing FANCA, FANCC, FANCE, FANCF, and FANCG proteins, and activity of the monoubiquitin ligase, FANCL (ref. 12; and others). Activation of the FA/BRCA pathway, which can be shown by monoubiquitination of FANCD2 and formation of FANCD2 nuclear foci, has been reported after the exposure of cells to DNA cross-linking agents and ionizing radiation (refs. 3, 11; and others). DNA cross-links and ionizing radiation–induced DNA double-strand breaks (DSB) are considered to be types of DNA damage that activate the FA/BRCA pathway, as they involve DNA recombination for repair.

Among the various types of exogenous or endogenous genotoxic stressors to which cells must respond to in order to maintain the integrity of their genome, solar UV light plays a pivotal role for skin cells (17). UV light generates DNA photoproducts and oxidative base modifications (18). These are processed by nucleotide and base excision repair pathways. UV light is not thought to directly induce DNA DSBs (19), and recombination repair is not commonly considered to be required for the repair of UV-induced DNA damage. Very high doses of shortwave UV light (UVC), which is not part of the solar UV spectrum reaching the earth's surface, have been reported to induce the monoubiquitination of FANCD2 and the formation of FANCD2 nuclear foci in transformed cells or tumor cells (11, 20). Except for a functional link with BLM, this response to UVC has not been further characterized. Therefore, we did a detailed analysis of the FA/BRCA pathway activation in response to physiologic doses of solar UV light and compared it with the response to ionizing radiation, a qualitatively very different type of physical mutagen, well established to induce DNA single-strand and double-strand breaks (19, 21). The immortalization of cells is known to often alter how cells respond to DNA damage. For example, p53, a pivotal, UV-activated regulator of apoptosis, cell cycle arrest, and DNA repair (22, 23), is commonly inactivated in immortalized cells (24–26). Therefore, in order to investigate DNA damage responses in cells with an intact network of DNA damage response pathways, we chose primary human skin fibroblasts as a model system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture conditions. Primary normal neonatal human skin fibroblasts were cultured from dermal explants of neonatal foreskins as described previously (27). Nontransformed, primary human skin fibroblasts from patients with FA complementation groups A (GM01309B) and C (GM00449), and from a patient with xeroderma pigmentosum complementation group A (GM00544C) were purchased from the Coriell Institute for Medical Research (Camden, NJ). Primary FANCA (PD872.F), FANCC (PD331.F), and FANCD2 (PD733.F) fibroblasts were also provided by the Fanconi Anemia Fund Cell Repository (Portland, OR). All cells were cultured in MEM-{alpha} (Life Technologies, Rockland, MA), supplemented with 10% calf serum at 37°C and 5% CO2, and used in exponential growth phase unless otherwise noted. For some experiments, cells were synchronized by serum starvation using medium with 0% calf serum for 1 day and 0.1% calf serum for 3 days.

Irradiations. After washing twice with PBS, cells were irradiated using a solar simulator (LH 153; Kratos Analytical, Ramsey, NJ). Cells were covered with a thin layer of PBS (1 mL in p60 dishes) to avoid drying, and culture dishes were placed on a water bath at room temperature to avoid overheating during irradiation. Irradiation was done through the lid of the tissue culture dish to ensure the sterility of the cultures. The tissue culture lid also removes contaminating UVC and only slightly reduces the irradiance of the shorter wavelengths in the UVB range. The resulting emission spectrum was published by Werninghaus et al. (28). After warming up, an irradiance level of ~0.3 W/m2 allowed irradiation with 200 J/m2 UVB in ~12 minutes. For most experiments, we used solar–simulated light (SSL) with a UVB dose of 200 J/m2, a solar available dose and easily accumulated during 10 to 15 minutes of midsummer midday sun exposure.

For some experiments, cells were irradiated with UVA, using a 2 kW metal halogenide UVA lamp (SELLAS Sunlight, Germany) with an emission spectrum ranging from 335 to 440 nm and an emission maximum at 375 nm (spectrum published in ref. 29).

Radiometric measurements were done for each experiment. For dosimetry, an IL-1700 Research Radiometer (International Light, Peabody, MA) was used, equipped with a UVB sensor (SED 240) or a UVA sensor (SEF 015). At a dose of 200 J/m2 UVB, the solar simulator emitted 2.5 kJ/m2 UVA. The UVA source does not emit UVB (minimum detection sensitivity of 1 x 10–6 W/cm2; ref. 29). A cesium-137 gamma irradiator was used to expose cells to 10 Gy of gamma radiation. Immediately after irradiation, the PBS cover was removed, and cells were fed with fresh prewarmed complete medium. For cell survival studies, exponentially growing cells were irradiated in p35 dishes 2 days after plating and counted at the indicated time points using a Coulter counter.

Immunoblotting. Whole cell extracts were prepared and Western blotting was carried out using standard procedures. The antibodies used were directed against FANCD2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and serine 15 phosphorylated p53 (1:1000; Cell Signaling, Beverly, MA). Densitometry of scanned autoradiograms was done using Quantity One software (Bio-Rad, Hercules, CA).

Immunostaining. At the indicated time points, cells grown on coverslips were washed thrice in PBS, fixed in 4% paraformaldehyde (10 minutes at room temperature), and permeabilized by 0.5% Triton X-100/PBS (7 minutes at room temperature). After blocking with 10% goat serum (Jackson ImmunoResearch, West Grove, PA) in 0.1% NP40/PBS (30 minutes at room temperature), cells were incubated with primary antibody in 1% bovine serum albumin/PBS (16 hours at 4°C). The primary antibodies used were directed against FANCD2 (1:200; Novus Biochemicals, Littleton, CO). After washing with 0.1% NP40/PBS (thrice for 10 minutes), cells were incubated with a secondary antibody (1:100; anti-rabbit IgG conjugated to FITC) for 1 hour at room temperature, washed thrice with 0.1% NP40/PBS and mounted on glass slides using a 4',6-diamidino-2-phenylindole (DAPI)–containing embedding medium (Vectorshield, Vector Laboratories, Burlingame, CA). Double staining was achieved by incubation of anti-BRCA1 (D-9; 1:100; Santa Cruz Biotechnology), anti-RAD51 (1:200; Abcam, Cambridge, MA), or anti-phospho-H2A.X (Ser139; 1:500; Upstate, Lake Placid, NY) mouse antibodies with the FANCD2 rabbit antibody. Secondary antibodies were also coincubated (1:200 rhodamine red-X-conjugated anti-mouse IgG; Jackson ImmunoResearch). Cells were inspected with an Eclipse E400 fluorescence microscope (Nikon, Japan), and images were acquired with a Spot RT digital camera and Spot Advanced RT software (Diagnostic Instruments, Sterling Heights, MI).

Cell cycle analysis. Subconfluent cells were collected by trypsinization, fixed in 24.5% ethanol, treated with RNase A, and stained with propidium iodide. Stained cells were analyzed for DNA content using a fluorescence-activated cell sorter (Becton Dickinson Biosciences, San Jose, CA) and CELLQUEST software.

Neutral comet assay. For the detection of DNA DSBs, a neutral single cell gel electrophoresis (neutral comet) assay was done following the protocol described by Wojewodzka et al. (30). Briefly, 5,000 trypsinized cells were suspended in 100 µL of low-melting agarose (Sigma-Aldrich; St. Louis, MO) to a final concentration of 0.75%. After solidification on comet slides (Trevigen, Gaithersburg, MD), cells were lysed for 1 hour at 4°C in prechilled buffer (2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris-HCl, 1% N-lauroylsarcosine, 0.5% Triton X-100, 10% DMSO; pH 9.5). After washing and equilibration in electrophoresis buffer (300 mmol/L sodium acetate, 100 mmol/L Tris-HCl; pH 8.3) horizontal gel electrophoresis was done for 1 hour at 4°C and 22 V (300 mAmp) followed by DNA staining with DAPI-methanol (1 µg/mL) for 15 minutes at 37°C. Comet inspection and image acquisition was done as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Following the exposure of normal human fibroblasts to various physiologic doses of SSL (containing 100, 200, and 300 J/m2 UVB), Western blot analysis of the FANCD2 protein revealed a shift from the inactive, nonubiquitinated S-isoform to the activated, monoubiquitinated L-isoform and an induction of the L-isoform as early as 2 hours after irradiation, which persisted for at least 12 hours, and was gone by 24 hours (Fig. 1A and B ; showing a representative example of several experiments). This shows the activation of the FA/BRCA pathway by UV at the 2-, 6-, and 12-hour time points after irradiation. Following activation, UV-induced degradation of the FANCD2 protein was observed at the later time points (24 > 12 hours; already discernible with the highest UV dose at the 6-hour time point). The parallel activation of p53, as shown by phosphorylation at serine 15 (Fig. 1A), was more prominent at the 6-hour than at the 2-hour time point, and more persistent than the monoubiquitination of FANCD2, as it was still detectable 24 hours after UV exposure. UV light also induced the formation of FANCD2 nuclear foci (Fig. 1C), further demonstrating the activation of the FA/BRCA pathway. The number of FANCD2 nuclear foci ranged from 0 to >50 per nucleus. Only nuclei that contained 10 or more foci were considered positive. Six hours after UV irradiation, almost as many cells as those irradiated with 10 Gy of ionizing radiation showed FANCD2 focus formation (Fig. 1D). Cells exposed to 200 kJ/m2 UVA did not show induction of FANCD2 nuclear foci.


Figure 1
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  		Figure 1. Exposure to SSL activates the FA/BRCA pathway in primary human skin fibroblasts. A shift from the nonubiquitinated S-isoform of FANCD2 to the monoubiquitinated L-isoform can be observed as early as 2 hours after exposure of normal fibroblasts (nonsynchronized) to various doses of SSL (containing 100, 200, or 300 J/m2 of UVB), parallel to the activation of p53 (serine 15 phosphorylation), but not as long-lasting. A, Western blot; B, densitometry of FANCD2 ubiquitination (BL, baseline; at time of irradiation). C, immunostaining for FANCD2 6 hours after exposure of normal fibroblasts with SSL (containing 200 J/m2 UVB) or 10 Gy of ionizing radiation reveals the formation of FANCD2 nuclear foci. D, the percentage of normal fibroblasts with FANCD2 nuclear foci (10 or more foci per cell) is increased 6 hours after irradiation with SSL (containing 200 J/m2 UVB) or ionizing radiation (10 Gy), as compared with sham-irradiated cells. Columns, means from four to seven experiments with cells from four to seven different donors; bars, ±SD. In each experiment, 3 x 100 cells were counted in three different fields and averaged. Cells irradiated with pure UVA (300 kJ/m2; one donor: column, mean from separate counts of the same sample; bar, SD) did not show an induction of FANCD2 nuclear foci. E, whereas normal fibroblasts show monoubiquitination of FANCD2 at 2 and 6 hours after exposure to SSL (containing 200 J/m2 UVB), primary fibroblasts from patients with FANCA (GM1309B) and FANCC (GM0449) do not show any monoubiquitination of FANCD2, either spontaneously, or following UV exposure (Western blot). F, in contrast, cells from patients with xeroderma pigmentosum complementation group A do exhibit increased monoubiquitination (shift to the L-isoform) of FANCD2 following UV exposure (SSL containing 100 and 200 J/m2 UVB; Western blot).

 
Cells from patients with FANCA or FANCC fibroblasts did not show any activated, monoubiquitinated L-isoform of FANCD2, either spontaneous or UV-induced (Fig. 1E). This shows that these two proteins of the FA complex are also required for the UV-induced monoubiquitination of FANCD2. Cells from patients with FANCA, FANCC, or FANCD2 fibroblasts did not exhibit any FANCD2 nuclear foci, either spontaneously, or following UV light or ionizing radiation exposure. In contrast, cells from patients with the nucleotide excision repair–deficient xeroderma pigmentosum complementation group A readily exhibited UV-induced monoubiquitination of FANCD2 (Fig. 1F).

The finding that in nonsynchronized cells, only a subset of cells show UV-induced formation of FANCD2 nuclear foci suggests that the activation of the FA/BRCA pathway by UV may be cell cycle–dependent, in line with previous findings with ionizing radiation or DNA cross-linkers (31–33). To analyze this, we synchronized normal primary skin fibroblasts by serum starvation and irradiated them at various time points after release from serum starvation. The cell cycle profiles at these time points are shown in Fig. 2A . The percentage of cells with FANCD2 nuclear foci (Fig. 2B, black columns) correlated with the fraction of cells in S phase (Fig. 2B, white columns), suggesting that FANCD2 nuclear foci are formed when cells are irradiated in S phase. The observation that at 32 hours after irradiation, there are more cells with FANCD2 nuclear foci than cells in S phase is likely due to the number of cells in late S phase with almost tetraploid chromosomes and subsequent underestimation of the S phase fraction. Consistent with previous reports describing spontaneous monoubiquitination of FANCD2 during S phase (11, 33), we also observed a spontaneous increase in the formation of FANCD2 nuclear foci during S phase (sham-irradiated cells; Fig. 2B, gray columns), but it was much smaller than that seen induced by UV light. When cells entered mitosis 6 hours after UV irradiation, none of the mitotic cells exhibited FANCD2 foci (Fig. 2C). In cells undergoing mitosis, FANCD2 is localized in the whole cell (most likely due to the physiologic dissolution of the nuclear membrane), whereas it is confined to the nucleus in interphase cells (Fig. 2C). Adjacent interphase cells readily show the UV-induced formation of FANCD2 nuclear foci.


Figure 2
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  		Figure 2. The FA/BRCA pathway is activated by SSL when cells are irradiated during the S phase and is inactivated when cells enter mitosis. Normal primary human skin fibroblasts were synchronized by serum starvation and irradiated at various time points after release from serum starvation (2, 20.5, 23, 28, and 32 hours) or irradiated just prior to release from serum starvation (0 hour). A, the cell cycle profiles at these time points. B, the percentage of cells in S phase (white columns, means of duplicate samples; bars, ±SD), as derived from the profiles in (A), together with the percentage of cells with nuclear foci of FANCD2 (≥10 foci per nucleus) 6 hours after irradiation with SSL (containing 200 J/m2 UVB; black columns, means from counting three fields of 100 cells; bars, ±SD) or sham (gray columns, not determined at 20.5- and 28-hour time points). C, 6 hours after UV exposure (SSL containing 200 J/m2 UVB), primary normal human skin fibroblasts in four different stages of mitosis, identified by the condensed chromosomes in the DAPI DNA stain (prometaphase, metaphase, anaphase, and late telophase/just completed mitosis; assignments of mitotic stage done as far as possible based on DAPI staining alone), showed only diffuse FANCD2 staining of the whole cells, and did not exhibit FANCD2 foci. This is in contrast with adjacent interphase cells, which do show the UV-induced FANCD2 nuclear foci and FANCD2 staining largely restricted to the nucleus.

 
FANCA and FANCC fibroblasts were not hypersensitive to UV light (Fig. 3A ), which confirms prior reports (34). In addition, they are characterized by an intact G1-S checkpoint, as they, similar to normal cells, did not exit into S phase when UV-irradiated just prior to release from serum starvation (Fig. 3B).


Figure 3
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  		Figure 3. Primary skin fibroblasts from patients with FANCA or FANCC fibroblasts were not hypersensitive to SSL (containing 200 J/m2 UVB), as the cell yield was not lower than with normal fibroblasts (A). Growth rates of sham-irradiated control cells were 0.9, 0.8, and 0.7 population doublings per day (normal; GM1309B, FANCA; GM0449, FANCC cells, respectively). Points, mean from triplicate samples; bars, ±SD. FANCA and FANCC fibroblasts also have an intact G1-S checkpoint (B), as they did not exit into S phase when synchronized by serum starvation and were irradiated with SSL (containing 200 J/m2 UVB) just prior to release from serum starvation (third row), whereas all cells did exit into S phase when sham-irradiated (second row).

 
UV light and ionizing radiation also induced nuclear foci of BRCA1, RAD51, and {gamma}-H2AX. These foci all colocalized with FANCD2 foci, at least to some degree (Fig. 4 ). Most BRCA1 foci colocalized with FANCD2 foci, and cells without FANCD2 foci did not exhibit BRCA1 foci. Although most of the RAD51 foci also colocalized with FANCD2, there were, in contrast to BRCA1, usually more FANCD2 foci than RAD51 foci in a nucleus. With sham- or UV-irradiated cells, most of the nuclei with many (≥10 foci/nucleus) FANCD2 foci also exhibited many (≥10 foci/nucleus) {gamma}-H2AX foci, and only 4% to 6% of cells with many {gamma}-H2AX foci exhibited no FANCD2 foci. In contrast, all of the ionizing radiation–exposed cells showed many {gamma}-H2AX foci, 43% of which showed no or few (<10) FANCD2 foci.


Figure 4
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  		Figure 4. In primary skin fibroblasts, FANCD2 nuclear foci induced by SSL (containing 200 J/m2 UVB) or ionizing radiation (10 Gy) colocalize with BRCA1, RAD51, and {gamma}-H2AX. With each antibody, photographs were taken at identical exposure settings. Cells were fixed 6 hours after irradiation.

 
To further analyze this difference in FANCD2/{gamma}-H2AX staining between UV light– and ionizing radiation–exposed cells, we determined the kinetics of the formation of FANCD2 and {gamma}-H2AX foci (Fig. 5 ). Just 10 minutes after exposure to ionizing radiation, all (100%) cells showed strong {gamma}-H2AX foci, which persisted for at least 6 hours (Fig. 5A and C). In contrast, only a subset of UV (SSL)–irradiated cells showed {gamma}-H2AX foci, and those were only observed 45 minutes after irradiation or later. Cells irradiated with 100 or 200 kJ/m2 UVA did not exhibit {gamma}-H2AX foci at any of those time points (10–360 minutes after irradiation; data not shown). A moderate increase in FANCD2 foci was seen 90 minutes after UV irradiation and was most prominent 6 hours after irradiation (Fig. 5B). Although a modest increase in ionizing radiation–induced FANCD2 foci was seen early, the most prominent induction was also seen at the 6-hour time point (Fig. 5B).


Figure 5
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  		Figure 5. Difference in formation of {gamma}-H2AX and FANCD2 nuclear foci in primary human skin fibroblasts after exposure to SSL (containing 200 J/m2 UVB) or ionizing radiation (10 Gy). A, after ionizing radiation, all cells exhibit {gamma}-H2AX foci at all studied time points (10 minutes–6 hours). After UV exposure, only a subset of cells exhibits {gamma}-H2AX foci, and only after 45 minutes or later [all photographs taken at identical exposure settings; counts in (C)]. E, the formation of UV- and ionizing radiation–induced {gamma}-H2AX foci is independent of cell proliferation, as it occurs in proliferating or growth-arrested cells at an equal frequency (6 hours after irradiation). In the same samples, the fraction of cells with many FANCD2 foci was determined (samples were double-stained for {gamma}-H2AX and FANCD2); B, time course; D, comparison of growth-arrested versus proliferating cells. Columns, means from counting 3 x 100 cells in three fields; bars, ±SD (B–E).

 
After UV exposure, growth-arrested cells failed to induce FANCD2 foci (Fig. 5D), but did induce {gamma}-H2AX foci (Fig. 5E). After ionizing radiation exposure, growth-arrested cells showed a reduced formation of FANCD2 foci (Fig. 5D) and a normal induction of {gamma}-H2AX foci (Fig. 5E).

The neutral single cell gel electrophoresis (neutral comet) assay was used to compare the amount of DNA double-strand breakage induced by these irradiations (200 J/m2 UVB from SSL versus 10 Gy of ionizing radiation). Whereas both sham- and UVB/SSL-irradiated cells showed no or only weak comets, ionizing radiation–irradiated cells exhibited clear and long comets (Fig. 6 ), demonstrating double-strand breakage with ionizing radiation, but not with UVB/SSL. This further indicates that UVB/SSL-induced FANCD2 and {gamma}-H2AX focus formation is not, or at least to a much lesser extent than with ionizing radiation, mediated by directly induced DSBs.


Figure 6
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  		Figure 6. Neutral single cell gel electrophoresis (neutral comet assay) shows that 10 Gy of ionizing radiation induces more DSBs (stronger comets) than SSL containing 200 J/m2 UVB or sham-irradiated fibroblasts. Examples of cells processed either immediately after irradiation (top row) or of cells that were incubated for 60 minutes following irradiation.

 
FANCA (PD872.F), FANCC (PD331.F), and FANCD2 (PD733.F) cells also exhibited {gamma}-H2AX foci after ionizing radiation or UV exposure (data not shown). Following ionizing radiation exposure, as seen with normal cells, all (100%) FA cells also showed {gamma}-H2AX foci. After UV irradiation, the fraction of cells with {gamma}-H2AX foci was 21% to 48% lower than in normal fibroblasts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that physiologic doses of SSL activate the FA/BRCA DNA damage response pathway in primary human fibroblasts, as they induce the monoubiquitination of FANCD2 and the formation of FANCD2 nuclear foci. This response can be attributed to the UVB in the SSL, as UVA doses 80 times higher than the UVA contained in the SSL did not activate the pathway.

It has been suggested that DNA damage–induced FANCD2 foci mark sites of DNA DSBs (31, 35), and that the FA/BRCA pathway mediates the repair of DSBs through recombination (3, 7, 8, 13, 15, 16). Ionizing radiation, well established to activate the FA/BRCA pathway, induces DSBs directly (19, 21). Our findings that all ionizing radiation–exposed cells exhibit many nuclear foci of H2AX phosphorylated at serine 139 ({gamma}-H2AX, widely accepted to mark DNA-DSBs; refs. 36–39) very early after irradiation, independent of cell proliferation, before the induction of FANCD2 foci, and that ionizing radiation–induced FANCD2 foci colocalize with {gamma}-H2AX are consistent with the current thinking that directly induced DSBs activate the FA/BRCA pathway after exposure to ionizing radiation. Our findings that ionizing radiation–induced FANCD2 foci occur after the formation of {gamma}-H2AX foci and that they colocalize with the recombination proteins, BRCA1 and RAD51, are consistent with the interpretation that the FA/BRCA pathway mediates the repair of DSBs through recombination. Much of the ionizing radiation–induced activation of the FA/BRCA pathway can apparently occur in the absence of DNA replication, as the formation of FANCD2 nuclear foci was also observed in growth-arrested cells exposed to ionizing radiation, albeit in a smaller cell fraction than in proliferating cells.

In contrast to ionizing radiation, UV light of the solar spectrum, UVB in particular, is not thought to induce DSBs directly (19), and UV-induced DNA photoproducts are not commonly considered to require recombination for repair. A significant contribution of DSBs to the activation of the FA/BRCA pathway by UV could be excluded by the much weaker induction of DSBs, if any, by SSL, as compared with ionizing radiation (as seen with the comet assay). Furthermore, the UVA portion of the SSL would be the most likely wavelength to generate DSBs through photosensitized reactions and the formation of reactive oxygen species, and we did not observe FANCD2 focus formation with high doses of pure UVA. This raises two questions: where are UV-induced FANCD2 foci located, and what is the function of the FA/BRCA pathway in the cellular response to UV? UV-induced DNA photoproducts are primarily repaired by nucleotide excision repair (14). We show here that the repair of DNA photoproducts by nucleotide excision repair is not the signal that mediates the activation of the FA/BRCA pathway, as cells with a severely deficient nucleotide excision repair (xeroderma pigmentosum complementation group A cells; <5% of unscheduled DNA synthesis remaining) readily ubiquitinate FANCD2 following UV exposure.

The formation of {gamma}-H2AX foci was also observed after UV irradiation. However, in contrast to ionizing radiation, they were not formed directly by UV, as their formation was delayed and limited to a subset of cells. This further excludes a significant contribution of UV-induced DSBs, as those should occur in all exposed cells and immediately after irradiation. UV-induced DSBs might be induced through a photosensitized reaction with some delay, in particular, following exposure to UVA. However, much higher doses of UVA than the ones contained in the SSL did not induce {gamma}-H2AX foci. One possible explanation for the UV-induced {gamma}-H2AX foci is that they might mark DSBs formed as repair intermediates. As DSBs are not intermediates of nucleotide or base excision repair (14), it is conceivable that UV-induced {gamma}-H2AX foci mark DSBs introduced by DNA recombination, e.g., at DNA photoproducts. If this were the case, this recombination would be independent of the FA/BRCA pathway activation, as we clearly show that UV first induces {gamma}-H2AX foci, and then FANCD2 foci much later. In addition, the observations that UV-mediated formation of {gamma}-H2AX foci is unimpaired in growth-arrested cells and also observed in FANCA, FANCC, and FANCD2 cells makes it even less likely that UV-induced {gamma}-H2AX foci mark DSBs generated as intermediates of recombination repair. Although {gamma}-H2AX foci are generally believed to mark DSBs with high sensitivity (36–39), it has not been convincingly shown that they are also highly specific for DSBs. In fact, a recent report by Ichijima et al. (40) describes the phosphorylation of histone H2AX even in the absence of DNA damage. We therefore believe that {gamma}-H2AX foci do not only form at DSBs, but at other types of DNA damage as well, including DNA photoproducts, and that the UV-induced {gamma}-H2AX foci are related to the processing of replication forks stalled at DNA photoproducts during S phase. The latter is supported by our observations that UV-induced {gamma}-H2AX foci were only observed in a subset of UV-irradiated cells, only with delay, independent of cell proliferation, and that they colocalized with FANCD2 foci (which we show to be formed when cells are irradiated in S phase). Because UV does not directly induce DSBs (or only very few), the question regarding the location and the function of UV-induced FANCD2 foci remains.

In the past, homologous recombination has been thought to be mainly involved in the repair of DSBs. Increasingly, however, it has been suggested that its role is not limited to the processing of DSBs, as many agents that are not thought to directly induce DSBs, including alkylating agents, cross-linkers, and UV light, also induce recombination (41, 42). What these agents have in common is that they induce DNA lesions that interfere with replication. The FA pathway is thought to be involved in mediating the resolution of stalled replication forks (16, 43, 44). Recent reports of the association between the FA complex and chromatin during the S phase further underlines the particular role of the FA/BRCA pathway for processing DNA damage during S phase (32).

If cells enter S phase with unrepaired DNA damage, the regular replicative DNA polymerase will stall at the damaged template (14). For these instances, cells are equipped with specialized translesional DNA polymerases than can bypass the damaged template. These polymerases provide damage tolerance and may prevent mutation formation (45). DNA polymerase {eta}, mutated in xeroderma pigmentosum variant, serves that function for DNA photoproducts (46, 47). Recent reports indicate that repair recombination might be required for the resolution of stalled replication forks at DNA photoproducts should they occur when nucleotide excision repair and translesional DNA synthesis have failed (48, 49). Replication forks stalled at DNA photoproducts have been suggested to degrade to DSBs, in particular, in xeroderma pigmentosum variant cells (49, 50). Although we excluded the role of directly UV-induced DSBs (see Discussion above), it remains a possibility that such degraded replication forks mediate the activation of the FA/BRCA pathway. If this was the case, it would be unlikely that replication fork degradation–induced DSBs are recombination repair intermediates, as the activation of the FA/BRCA pathway should precede, not follow, recombination repair.

Pichierri et al. (20) and Garcia-Higuera et al. (11) reported that FANCD2 ubiquitination and focus formation is induced by very high doses of UVC in transformed or malignant cell lines. They suggested that replication forks stalled at DNA photoproducts might activate the FA/BRCA pathway and that this mediates the resolution of those stalled replication forks by recombination repair, similar to its hypothesized function in processing DNA cross-links. Here, we provide a detailed analysis of the response of the FA/BRCA pathway to UV light (using primary cells instead of transformed or malignant cells, and SSL instead of UVC). Our data are indeed consistent with this interpretation, as we show that (a) the UV-induced activation of the FA/BRCA pathway predominantly, if not exclusively, occurs when cells are irradiated in S phase, (b) that it is replication-dependent, (c) nucleotide excision repair–independent, (d) directly UV-induced DSB-independent, and (e) associated with the recombination proteins BRCA1 and RAD51. As discussed above, this response to UV light is clearly different from the response to ionizing radiation. In eukaryotic cells, replication arrest can be overcome by different mechanisms (42): at a DNA lesion (e.g., a DNA photoproduct), the Y-branched structure of the replication fork can undergo a process called fork reversal, which leads to the formation of an X-shaped Holliday junction. By stabilizing the replication fork, fork reversal may allow bypass of the lesion or access for removal of the lesion (helicase-based replication restart). Alternatively, the Holliday junction might be resolved through a recombinogenic pathway. Our finding that UV-induced FANCD2 foci colocalize with recombination proteins points to recombination as the mechanism mediating the resolution of replication forks stalled at DNA photoproducts.

As repair recombination would only be the third line of defense against the mutagenic properties of DNA photoproducts, it cannot be expected that patients with FA would be as prone to UV-induced skin carcinogenesis as patients with xeroderma pigmentosum (deficient in nucleotide excision repair; first line of defense) or patients with a xeroderma pigmentosum variant (deficient in translesional DNA synthesis at DNA photoproducts; second line of defense; ref. 14). However, when patients with FA survive pancytopenia, and possibly myeloid leukemia, many of those patients later develop squamous cell carcinomas of the head and neck area, including cutaneous carcinomas (4–6). It is conceivable that the reduced ability to resolve stalled replication forks at environmentally induced DNA damage contributes to this cancer-prone phenotype. For this, genotoxic environmental agents may not be limited to UV light, but may also include other carcinogens. For example, tobacco and tobacco smoke are well recognized carcinogens and risk factors for head and neck cancers.

We suggest that the FA/BRCA pathway is part of a largely interconnected DNA damage response network that not only reacts to DNA damage introduced by ionizing radiation and DNA cross-linkers, but also reacts to DNA damage introduced by a larger variety of genotoxic agents, including those by UV light.


   Acknowledgments
 
Grant support: American Cancer Society grant RSG-03-006-01-CNE (T.M. Rünger).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/13/06. Revised 9/ 1/06. Accepted 9/15/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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