Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

Published OnlineFirst August 4, 2009; DOI: 10.1158/0008-5472.CAN-09-1208 Published Online First on August 4, 2009 as 10.1158/0008-5472.CAN-09-1208

Molecular Biology, Pathobiology, and Genetics

David Parra,1 Joan Manils,1 Ba`rbara Castellana,1 Arnau Vinã-Vilaseca,1 Eva Morań-Salvador,1 Nuria Va´zquez-Villoldo,1 Gemma Tarancoń,1 Miquel Borra`s,2 Sara Sancho,4 Carmen Benito,3 Sagrario Ortega,5 and Concepcio´Soler1

1Departament de Patologia i Terape`uticaExperimental, Facultat de Medicina, Campus de Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat; 2Unitat de Toxicologia Experimental, Parc Cientı´fic de Barcelona; and 3Proteccio´Radiolo`gica, Universitat de Barcelona, Barcelona, Spain; 4Promed, Fribourg, Switzerland; and 5Spanish National Cancer Research Center, Madrid, Spain

Abstract exonucleases TREX1 and TREX2 (1–5). Despite the numerous ¶ ¶ ¶ ¶ 3 -5 exonucleases present in the cell with putative overlapping TREX2 is a proofreading 3 -5 exonuclease that can be involved in vivo in genome maintenance; however, its biological role remains activities, functional studies have revealed specific and undefined.To better understand the function and physiologic nonredundant biological roles for many of these proteins (1, 6–11). relevance of TREX2, we generated mice deficient in TREX2 by Among these, the biological function of TREX2 is still largely targeted disruption of its unique coding exon.The knockout unknown. TREX2, which was identified in a database search by means mice are viable and do not show relevant differences in ¶ ¶ growth, survival, lymphocyte development, or spontaneous of its homology with TREX1, displays a robust nonprocessive 3 -5 exonuclease activity that can proofread the work of DNA tumor incidence compared with their wild-type counterparts polymerases and process 3¶ ends during DNA replication and over a period of up to 2 years.Also, we did not observe repair processes in vitro (12–14). In this regard, it has been chromosomal instability or defects in cell proliferation and reported that TREX2 may interact with Poly, and increase its cell cycle upon loss of TREX2.We have observed that TREX2 fidelity when deoxynucleotide triphosphates pools are imbalanced expression is not ubiquitous, being expressed preferentially (15). TREX2 biochemical and structural properties are similar to in tissues with stratified squamous epithelia, such as the TREX1, although they are not identical. The two proteins share a skin or esophagus, and specifically in keratinocytes.Interest- dimeric structure and can process ssDNA and dsDNA substrates ingly, TREX2-null mice are more susceptible to skin carcino- in vitro with almost identical k values. However, several features genesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) cat related to enzyme kinetics, structural domains, and subcellular compared with wild-type mice.This phenotype correlates distribution distinguish TREX2 from TREX1. TREX2 present a with a reduction of DMBA-induced apoptosis in both the 10-fold lower affinity for DNA substrates in vitro compared with epidermis and keratinocytes of TREX2-null mice.Altogether, TREX1. In contrast with TREX1, TREX2 lacks a COOH-terminal our results suggest a tumor suppressor role for TREX2 in domain that can mediate protein-protein interactions (12, 16–19). skin carcinogenesis through which it contributes to keratino- TREX2 is localized in both the cytoplasm and nucleus (20), whereas cyte apoptosis under conditions of genotoxic stress. [Cancer TREX1 is found in the endoplasmic reticulum, and is mobilized to Res 2009;69(16):6676–84] the nucleus during granzyme A–mediated cell death (21) or after DNA damage (22). Loss of TREX1 function does not trigger an increase in cancer development (11), but rather leads to cell- Introduction intrinsic immune activation by endogenous nondegraded ssDNA, 3¶-5¶ Exonucleases remove nucleotides from DNA 3¶ termini in resulting in the development of autoimmunity (23–26). Indeed, multiple processes of DNA metabolism, ranging from DNA TREX1 has been shown to be involved in the degradation of ssDNA synthesis to DNA degradation, and thereby can play a pivotal role generated from the processing of aberrant replication intermedi- in maintaining genome stability and preventing cancer, aging, ates (22) and endogenous retroelements (26). Otherwise, TREX2 immunologic abnormalities, and inherited diseases. The 3¶-5¶ seems to be important for chromosomal stability. As such, TREX2 exonuclease activity has been found in multidomain proteins, such deletion in embryonic stem (ES) causes high levels of Robertsonian as DNA polymerases (Poly,Polg, and Polq), base excision repair Translocations (27), but this effect is independent of its apurinic/apyrimidinic endonucleases (APE1 and APE2), double exonuclease activity and DNA binding domains (28). The strand break DNA repair proteins (MRE11), DNA repair nucleases biochemical activities and the mechanisms by which TREX2 (EXO1 and NM23-H1), helicases (WRN, Dna2), cell cycle checkpoint prevents chromosomal instability and the consequences of TREX2 proteins (RAD9 and RAD1), and the transcription factor p53. In deficiency in the organism remain unknown. addition, this activity has been described in the single-domain In this context, we aimed to asses the function and relevance of TREX2 in vivo. To that end, we generated and characterized the À/À TREX2 knockout mice, Trex2 . Furthermore, to evaluate putative Note: Supplementary data for this article are available at Cancer Research Online tissue-specific functions, we analyzed TREX2 tissue expression. (http://cancerres.aacrjournals.org/). Requests for reprints: Concepcio´Soler, Departament de Patologia i Terape`utica TREX2 knockout mice seem viable and fertile and do not show a Experimental, Facultat de Medicina, Campus de Bellvitge, Universitat de Barcelona, significant increase in spontaneous tumor incidence. Interestingly, Feixa Llarga s/n, E-08907, L’Hospitalet de Llobregat, Barcelona, Spain. Phone: 34- TREX2 deficiency increases susceptibility to carcinogen-induced 934039858; Fax: 34-934024249; E-mail: [email protected]. I2009 American Association for Cancer Research. skin tumorigenesis that correlates with impaired apoptosis. These doi:10.1158/0008-5472.CAN-09-1208 findings suggest a tumor suppressor role for TREX2 under

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TREX2 Prevents Skin Carcinogenesis

Figure 1. Targeted disruption of the Trex2 gene in mice. A, targeting strategy. Schematic representation of germ line Trex2 locus, targeting vector, mutated conditional (Trex2lox ), and knockout (Trex2À ) locus. The mouse Trex2 locus contains two exons (gray boxes). The full protein coding sequence is included in exon 2. LoxP sites (filled triangles) were placed flanking exon 2. The pgk-neo r cassette and pgk-tk cassette, flanked by frt sites (open boxes), used for selection of homologous recombinant ES clones are indicated. Flp, Flp recombinase; Cre, Cre recombinase. RV (EcoRV), RI (EcoRI). B, Southern blot (SB) and PCR analysis of recombinant ES clones and mice carrying the indicated alleles. The origin of the Southern blot probes is indicated in A. Sizes of the diagnostic DNA fragments are indicated in A and B. C, RT-PCR analysis of Trex2 mRNA expression in skin from wt, Trex2À/À, and Trex2lox/lox mice. D, Western blot (WB) analysis of skin extracts and immunoprecipitates from wt, Trex2À/À, and Trex2lox/lox mice with antibodies to mouse TREX2. Preimmune (PI) serum was used as a negative control for immunoprecipitation (IP). Bands corresponding to the 236 amino acid mouse TREX2 protein and to heavy immunoglobulin chain (IgH) are indicated. www.aacrjournals.org 6677 Cancer Res 2009; 69: (16). August 15, 2009

Cancer Research conditions of genotoxic stress in keratinocytes, cells where we have Fig. 1A. Genomic fragments corresponding to exon 2 (1 kb) and the found that this exonuclease is predominantly expressed. 5¶ (2.9 kb) and 3¶ (2.2 kb) arms were amplified using genomic DNA extracted from mouse R1 ES cells by PCR using the Expand High Fidelity PCR system (Roche). The targeting vector was electroporated into mouse R1 ES cells and recombinant clones were selected in the presence of G418 and Materials and Methods ganciclovir. Genomic DNA from resistant clones were digested with EcoRI, À/À Generation of Trex2 mice and genotyping. To conditionally and tested for correct targeting events via Southern blotting using disrupt the mouse Trex2 gene, a targeting strategy was designed to knock both 5¶ (probe a) and 3¶ (probe b) external probes. Targeted ES clones loxneo in two loxP sites flanking exon 2 via homologous recombination in ES cells. (Trex2 ) are hemizygous because the Trex2 gene is located on the The targeting construct was constructed by subcloning genomic DNA X chromosome and R1 ES cells are XY. Three ES clones, in which correct fragments encompassing exon 2 and the 5¶ and 3¶ flanking fragments into homologous recombination had occurred, were aggregated with eight- the loxP conditional vector pDELBOY-3Â, as schematically shown in cell-stage CD-1 embryos. Male chimeras transmitted the targeted allele

Figure 2. Tissue and cell expression of TREX2. A, TREX2 mRNA expression in various mouse (left) and human (right) tissues. The expression levels of the unique mouse TREX2 mRNA transcript and of the two human transcripts that code for the short (236 amino acid) and long (279 amino acid) isoforms were determined via real-time PCR in the indicated tissues. The results were normalized to the expression of HPRT and the mean values are displayed in relation to skin. B, TREX2 protein expression in various mouse tissues. Immunoprecipitation and Western blot analysis of extracts from various mouse tissues with antibodies to mouse TREX2. Bands corresponding to the 236 amino acid mouse TREX2 protein and to heavy immunoglobulin (IgH) chain are indicated. C, TREX2 immunostaining in mouse skin and esophagus sections and in cultured primary keratinocytes from wt and Trex2À/À mice. Nuclei were counterstained with 4¶,6-diamidino-2-phenylindole. Scale bars, 20 Am. D, DNA 3¶ exonuclease activity in extracts of wt and Trex2À/À mice cells and tissues. Top, extracts from epidermis were assayed for 3¶ exonuclease activity at the indicated times. Bottom, extracts from the indicated cells and tissues were assayed for 3¶ exonuclease activity after 10 min of incubation with the substrate.

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TREX2 Prevents Skin Carcinogenesis

Figure 3. Growth, survival, and lymphocyte cells in the Trex2À/À and wt mice. A, body weight of wt (n = 22) and Trex2À/À (n = 22) males (top) and of wt (n = 16) and Trex2À/À (n = 14) females (bottom) were followed for up to 2 y. Points, average; bars, SE. No significant differences were observed between wt and Trex2À/À genotypes. B, survival curves. Survival curves correspond to 36 Trex2À/À and 38 wt and mice. No significant difference in median survival was observed (log-rank test with 95% confidence interval). C, analysis of T-lymphocyte populations isolated from the thymus, spleen, and blood. D, analysis of B-lymphocyte populations isolated the bone marrow, spleen, and blood. Columns, average of six Trex2À/À and six wt are shown; bars, SE. No significant differences were observed between wt and Trex2À/À genotypes.

loxneo +/loxneo (Trex2 ) to their offspring. Female Trex2 mice were crossed with A-Sepharose beads for 3 h. Immunocomplexes were washed four times with transgenic Flp (pCAG-flpe/+) male mice to delete the neomycin resistance buffer containing 20 mmol/L HEPES (pH 7.5), 0.1% Nonidet P-40, 10% +/lox gene. Then, female Trex2 mice were bred with transgenic Cre (CMV-Cre, glycerol, and 150 mmol/L NaCl. Samples were boiled in Laemmli buffer and ubiquitous expression) male mice to delete exon 2 and simultaneously separated on 10% SDS-PAGE. They were then transferred to polyvinylidene +/ +/À segregate the Flp transgene. The resulting Trex2 , Trex2 females and difluoride membranes (Hybond-ECL; GE Healthware). Membranes were + À Trex2 , Trex2 males were subsequently intercrossed to eliminate the Cre blocked for 1 h at room temperature in 5% nonfat milk in TBST buffer [150 transgene. Crosses between these mice generated TREX2 knockout mice, mmol/L NaCl, 50 mmol/L Tris (pH7.4) and 0.05% Tween 20], incubated À/À Trex2 . Excisions were verified via Southern blotting and PCR analysis of with diluted antibody (anti-TREX2, 1/10,000) and followed by horseradish genomic DNA from tail biopsies of the mice (Fig. 1B). Targeting vector peroxidase–conjugated protein A/G (Bio-Rad) and detected with enhanced construction, primers, and PCR conditions are described in Supplementary chemiluminescence (ECL; GE Healthware). Antibodies against mouse Materials and Methods. TREX2 were generated using an MBP-fusion mouse TREX2 protein for Mice were maintained in a pathogen-free environment. All animal rabbit immunization. Antibodies were raised and affinity purified by procedures were approved by the Animal Care and Use Committee of standard procedures. This antibody does not cross-react with TREX1. The Spanish National Cancer Research Center and the Ethics Committee of the specificity of this antibody was unequivocally validated by the absence of University of Barcelona. immunodetection of TREX2 protein by immunoprecipitation, Western blot À/À RNA analysis. For reverse transcription-PCR (RT-PCR) analysis of TREX2 and immunofluorescence in samples from Trex2 mice compared with À/À lox +/+ mRNA expression, total RNA from tissues and cells of Trex2 , Trex2 , samples from Trex2 mice (Figs. 1D and 2C). +/+ and Trex2 mice was isolated using the NucleoSpin RNA extraction kit Exonuclease assay. Cells and tissues from mice were homogenized (Macherey-Nagel). Total RNA from human tissues was obtained from in an ice-cold lysis buffer containing 1% Triton X-100, 50 mmol/L Tris-HCl A Stratagene. cDNA synthesis was done with Transcriptor reverse transcrip- (pH7.5), 10 mmol/L MgCl 2, 1 mmol/L DTT, 100 mmol/L NaCl, 10 g/mL tase using oligo dT, according to the manufacturer’s instructions (Roche). aprotinin, 10 Ag/mL leupeptin, and 1 mmol/L PMSF, and 1 mmol/L sodium For a quantitative analysis, after cDNA was synthesized, real-time PCR was orthovanadate. Lysates were then assayed for DNA 3¶ exonuclease activity, performed using a LightCycler machine with LightCycler FastStart DNA as described elsewhere (11). MasterPLUS SYBR Green I (Roche). Values were normalized to hypoxanthine Flow cytometry analysis of B and T cells. Cells from the bone marrow, À À guanine phosphoribosyltransferase (HPRT1) mRNA expression. Primer thymus, spleen, and blood of 2-mo-old Trex2 / and Trex2+/+ mice were sequences, gene accession number, length of PCR amplicons, and PCR stained with the following antibodies to B- and T-cell surface markers: conditions are shown in Supplementary Table S2. FITC-anti-a/hTCR, PE-anti-CD4, Cy5-anti-CD8, FITC-anti-B220, and PE- Immunoprecipitation, Western blot, and antibodies. Cells and tissues anti-CD19 (Becton Dickinson) following standard protocols. Analysis was from mice were homogenized in ice-cold lysis buffer [1% Nonidet P-40, 1% performed with an Epics XL flow cytometer (Coulter Corporation). À/À deoxycolate, 0.1% SDS, 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl] Survival, growth, and spontaneous tumor development. Trex2 together with protease inhibitors [10 Ag/mL aprotinin, 10 Ag/mL leupeptin, mice and their Trex2+/+ littermates in the C57Bl/6/CD1/129 mixed genetic 86 Ag/mL iodoacetamide, and 1 mmol/L phenylmethylsulfonyl fluoride background were used in an untreated time course study, during which they (PMSF)]. For immunoprecipitation, 1 mg of protein was incubated with were observed weekly for up 2 y. Mice were monitored for body weight, 10 AL of rabbit polyclonal antibodies to mouse TREX2 coupled to protein fertility, survival, lymphocyte development, and spontaneous tumor www.aacrjournals.org 6679 Cancer Res 2009; 69: (16). August 15, 2009

Cancer Research development. Mice were euthanized when they seemed unhealthy, either colonies were stained with methylene blue following standard procedure, because of visible lesions, morbidity, or significant weight loss. Autopsies scored, and used to calculate the surviving fraction of cells in each condition. were performed on these animals and in all of the mice when they reached Apoptosis analyses. Apoptotic cells were evaluated in biopsies from the the age of 24 mo. Mouse tissues were fixed in 10% neutral-buffered formalin dorsal skin of mice that were exposed to single doses of DMBA (400 nmol solution and processed for paraffin embedding, cut into 5-Am sections, and for 24 h), DMBA+TPA (DMBA, 120 nmols for 4 d followed by TPA, 20 nmol mounted on slides. The sections were stained with H&E and examined by for 24 h), or acetone (control). Treated dorsal skin of mice was fixed in 10% two pathologists who were blinded to the tissue grouping. neutral-buffered formalin solution and processed for paraffin embedding. Skin tumor induction experiments. The strategy used for the Skin sections (5 Am thick) were stained with H&E, and the number of carcinogenicity study followed well-established mouse models of DMBA sunburn cells, defined as apoptotic cells within the epidermis exhibiting an and 7,12-dimethylbenz(a)anthracene (DMBA)+12-O-tetradecanoylphorbol- intensely eosinophilic cytoplasm and small and dense nuclei, were counted 13-acetate (TPA)–induced tumors in the skin. For the DMBA-induced throughout the epidermis. Sunburn cells for at least two sections of 1-cm tumor experiments, DMBA (120 nmols) was applied twice weekly for 14 wk. long epidermis of each skin sample were scored in a blinded fashion. To For the DMBA+TPA tumor initiation/promotion experiments, mice were quantify apoptotic DNA fragmentation in DMBA-treated keratinocytes, we treated with a single dose of the carcinogen DMBA (120 nmol in 200 AL used a known highly specific and sensitive sandwich ELISA kit (Roche) that of acetone; Sigma) followed by applications of the tumor promoter TPA quantifies cytoplasmic histone-associated DNA fragments in cells undergo- (20 nmol in 200 AL of acetone; Sigma) twice a week beginning 4 d after ing programmed cell death. initiation, and for a duration of 12 wk. Treatments were applied to the Spectral karyotyping analysis. Colcemid-treated cells were resus- À/À +/+ shaved backs of 8-wk-old Trex2 and Trex2 mice. Mice of the C57BL/6/ pended carefully in a KCl hypotonic solution (0.075 mol/l) for 30 min at CD1/129 mixed genetic background were backcrossed for six generations to 37jC, rinsed and fixed in 3:1 methanol/acetic acid, and dropped onto glass CD1 mice, which were then bred to each other to generate cohorts of slides. Slides were hybridized using the Spectral karyotyping (SKY) method À/À +/+ Trex2 and Trex2 mice that were used in the skin carcinogenesis according to the manufacturer’s protocol (Applied Spectral Imaging, Inc.). studies. Mice were visually inspected weekly, and tumor numbers and sizes Images were acquired with an SD300 Spectra Cube (Applied Spectral were quantified to calculate tumor incidence (percentage of tumor-bearing Imaging) mounted on a Zeiss Axioplan microscope using a custom- mice) and tumor multiplicity (average number of tumors per mouse). Mice designed optical filter (SKY-1; Chroma Technology). were sacrificed when they showed signs of poor health. Skin sections were fixed in 10% buffered formalin, embedded in paraffin, and subjected to histopathologic analysis. Results Cell cultures, proliferation, cell cycle, and survival assays. Primary À/À À/À Generation of Trex2 mice. To create the Trex2 mice, mouse embryonic fibroblasts (MEFs) from E14.5 embryos were obtained Trex2 according to standard protocols and cultured them in DMEM supple- gene was disrupted by deletion of exon 2, as detailed in A B mented with 10% fetal bovine serum (FBS). For proliferation assays, we Materials and Methods (Fig. 1 and ). Because exon 2 contains the plated 5 Â 104 cells on 24-well plates in triplicate and incubated the cells in full-length coding sequence of murine TREX2 (31), removal of this DMEM supplemented with 0.1% FBS for 60 h. We added DMEM sequence completely abrogates TREX2 expression. As described supplemented with 10% FBS, or with increasing FBS concentrations, and below, we did find that TREX2 is predominantly expressed in the measured 3H-thymidine incorporation at the indicated times, as described skin. Analysis of TREX2 mRNA (Fig. 1C) and protein expression (29). Primary keratinocytes were prepared from newborn mouse skin and (Fig. 1D) showed the presence of TREX2 expression in skin from +/+ cultured on collagen I precoated plates (BD Biosciences), as described (30). wild-type (wt; Trex2 ) and conditional gene–targeted mice Â 4 lox/lox À/À To assess keratinocyte proliferation, 1 10 cells were plated on 96-well (Trex2 ), and its absence in gene-targeted mice (Trex2 ), plates in triplicate and growth was monitored by the reduction of the confirming the null mutation. AlamarBlue reagent (Biosource) for up to 7 d. For cell cycle analysis, fixed cells were stained with propidium iodide and analyzed using an Epics XL Tissue- and cell-specific expression of TREX2. TREX2 mRNA flow cytometer (Coulter Corporation). To evaluate the effects of DMBA on transcripts had been previously detected in various human tissues, keratinocyte survival, we performed clonogenic assays. Keratinocytes (2.5 Â suggesting ubiquitous expression (20, 31). However, quantitative 105) were seeded on six-well plates in duplicated, and treated with the expression was not assessed in those studies. Although the human indicated doses of the genotoxic agent. Ten days after treatment, the and mouse TREX2 gene structure is highly homologue, mouse gene

À À Table 1. Incidence of common spontaneous tumors found in Trex2 / versus wt mice

Tumor Wt Trex2À/À

MFM&FMFM&F

Bronchioloalveolar adenoma 7/22 (32%) 2/16 (12.5%) 9/38 (24%) 8/22 (36%) 2/14 (14%) 10/36 (28%) Bronchioloalveolar carcinoma 2/22 (9%) 0/16 (0%) 2/38 (5%) 2/22 (9%) 2/14 (14%) 4/36 (11%) Hepatocellular adenoma 3/22 (14%) 0/16 (0%) 3/38 (8%) 4/22 (18%) 0/14 (0%) 4/36 (11%) Hepatic hemangioma 2/22 (9%) 2/16 (12.5%) 4/38 (10.5%) 1/22 (5%) 0/14 (0%) 1/36 (3%) Splenic lymphoma 0/22 (0%) 3/16 (19%) 3/38 (8%) 2/22 (9%) 7/14 (50%) 9/36 (25%) Mesenteric node lymphoma 0/22 (0%) 2/16 (12.5%) 2/38 (5%) 2/22 (9%) 4/14 (28%) 6/36 (17%) Thymic lymphoma 0/22 (0%) 4/16 (25%) 4/38 (10.5%) 1/22 (4.5%) 4/14 (28%) 5/36 (14%) Any lymphoma 0/22 (0%) 6/16 (37.5%) 6/38 (16%) 4/22 (18%) 7/14 (50%) 11/36 (30%)

NOTE: Number of mice with the indicated tumors followed by the total number of mice (the corresponding percentage is given in parentheses). Some À/À mice bore more than one tumor. Percentage of wt and Trex2 mice bearing at least one tumor was 42% and 63%, respectively. There were no À/À significant differences (Fisher’s exact test) in tumor incidence between wt and Trex2 mice.

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TREX2 Prevents Skin Carcinogenesis

Figure 4. DMBA-induced skin carcinogenesis in wt and Trex2À/À mice. Mice were untreated or treated with DMBA, or DMBA+TPA, as described in the Materials and Methods. A, average number of tumors per mouse in the skin of wt (n = 15) and Trex2À/À (n = 18) mice treated with DMBA. B, size distribution of skin tumors present in DMBA-treated wt and Trex2À/À mice at week 14. C, average number of tumors per mouse in the skin of wt (n = 25) and Trex2À/À (n = 25) mice following DMBA+TPA two-stage chemical carcinogenesis. D, tumor size distribution of skin tumors present in DMBA+TPA-treated wt and Trex2À/À mice at week 12. Tumor multiplicity data were statistically analyzed using the Mann-Whitney test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

codes for a single 26-kDa isoform, whereas human gene can gives in Fig. 2B, TREX2 protein is observed only in certain mouse tissues, rise to two additional 30-kDa isoforms that contain an extra such as the skin, tongue, esophagus, forestomach, or bladder, NH2-terminal region. The different human isoforms arise from which express relatively high levels of mRNA for this gene. In these mRNAs generated by alternative splicing, and by the use of mouse tissues, we only detected a single TREX2 polypeptide with alternative promoters. Analogous transcripts in human and mice an apparent molecular weight of f28 kDa, indicating the code for the 236 amino acid proteins (26 kDa; short isoform). Other expression of a single protein isoform in mice. This protein two human transcripts code for an extra 43 or 42 amino acids corresponds to the 236 amino acid isoform, as confirmed by Trex2 upstream the NH2 terminus of the 26 kDa isoform, and are heterologous expression in fibroblasts of the mouse cDNA translated into a protein of 279 and 278 amino acids, respectively coding for the 236 amino acid protein, which gives rise to an (30 kDa; long isoform). Interestingly, our results from the identical 28 kDa protein (data not shown). Collectively, mRNA quantitative RT-PCR analysis reveal a specific pattern of expression and protein data indicate that TREX2 shows a tissue-specific of TREX2 in both mice and human tissues. As seen in Fig. 2A, the expression. unique mouse TREX2 mRNAs transcript is highly expressed in To identify cell types expressing TREX2, we performed the skin, tongue, esophagus, and forestomach. Compared with skin, immunohistochemistry assays of major tissues expressing TREX2, 10-fold lower expression was detected in the bladder, prostate, and such as the skin and esophagus (Fig. 2C). As a negative control for thyroid, and a 100-fold lower levels were found in the trachea, lung, staining, we used corresponding knockout samples. We observed glandular stomach, small intestine, pancreas, and uterus. Expres- that TREX2 expression was confined to cells of the stratified sion was more than a 1,000-fold lower or almost undetectable in squamous epithelia of these tissues. Because keratinocytes are the the spleen, thymus, ganglia, kidney, colon, rectum, ovary, testis, most abundant cells that integrate the stratified squamous seminal vesicle, adrenal gland, salivary gland, brain, liver, skeletal epithelia, we next checked for TREX2 expression in primary muscle, or adipose tissue. Similarly, in human tissues, we observed keratinocyte cultures. Immunocytochemistry analyses illustrate major expression in skin; moderate expression in the tongue and that TREX2 is expressed in keratinocytes and localized mainly in cervix, low expression in the kidney, spleen, stomach, and thyroid; the nucleus, showing a punctate distribution with many foci per and it was undetectable in liver. An analogous pattern of cell (Fig. 2C). Quantitative mRNA analysis further confirmed that expression was observed for the two main human TREX2 TREX2 is preferentially and highly expressed in keratinocytes. Thus, transcripts, the predominant long isoform and the less abundant mouse TREX2 mRNA transcript levels were almost 105 times higher short isoform. Next, to evaluate protein expression, we performed in keratinocytes compared with MEFs and ES cells, whereas the immunoprecipitation followed by Western blot analyses. As shown transcript was undetectable in macrophages. www.aacrjournals.org 6681 Cancer Res 2009; 69: (16). August 15, 2009

Cancer Research

Finally, to evaluate the contribution of TREX2 activity to the Chromosomal stability, proliferation, and immunologic À À overall 3¶-5¶ exonuclease activity, we compared this activity in development are not altered in cells from Trex2 / mice. À/À +/+ several tissues from Trex2 and Trex2 mice (Fig. 2D). Another group has previously reported the reduced proliferation Consistent with the expression data, TREX2 does not contribute and presence of Robertsonian translocations, duplications, and to the 3¶-exonuclease activity of the dermis or liver, but does deletions in two clones of Trex2-deficient ES cells (27). Surprisingly, account for f50% of this activity in keratinocytes and the we did not detect any chromosomal abnormalities in more epidermis. than one hundred metaphases analyzed by SKY from MEFs Altogether, the expression and activity data indicate that TREX2 and keratinocytes isolated from at least four knockout and wt is predominantly expressed in keratinocytes and, consequently, in Trex2 mice (Supplementary Fig. S1A). Furthermore, we did not tissues with stratified squamous epithelia. find differences in the proliferation or cell cycle phases of several Trex2 loss does not lead to tumorigenic phenotype or cell types, such as keratinocytes (Supplementary Fig. S1B and D) reduced survival. The TREX2 knockout mice were viable, born and MEFs [from primary passage 2 (P2) to 6 (P6); Supplementary À À at the expected Mendelian frequency, fertile, and indistinguishable Fig. S1C and D), obtained from Trex2 / mice compared with +/+ by average weight and growth from their wt littermates (Fig. 3A). Trex2 cells. Mice lacking TREX2 seemed healthy and no apparent anatomic or Because defects in many genes involved in DNA repair and gross behavioral abnormalities were observed. No differences in life genome stability trigger impaired lymphocyte development (33), we span (Fig. 3B) or spontaneous tumor development (Table 1) were analyzed B- and T-cell populations in the thymus, bone marrow, À/À seen between the two genotypes for up to 2 years. Compared with spleen, and blood from Trex2 mice and their wt littermates. +/+ Trex2 mice, we did observe a slight increase in the percentage of Populations of lymphoid T cells bearing CD4, CD8, and T-cell À/À Trex2 mice that developed splenic lymphomas; however, the receptor ah (Fig. 3C) or lymphoid B cells expressing B220, CD19, or differences were not statistically significant. Although TREX2 is IgM (Fig. 3D) were not affected by loss of TREX2, revealing no highly expressed in the skin, its suppression does not lead to defects in B- and T-cell development. Together, these findings abnormalities in the skin or an increase in related diseases such as indicate that TREX2 is dispensable for chromosomal stability and skin hyperpigmentation, alopecia, dryness and dermatitis, or efficient cellular proliferation of several cell types. À/À +/+ spontaneous skin tumors. Both Trex2 and Trex2 mice Increased susceptibility to DMBA-induced skin tumorigen- À/À developed most common tumors in agreement with published rates esis in Trex2 mice. To further characterize TREX2 biological in aging male and female C57BL/6/129 mice (32). Collectively, these function, we tested whether TREX2 could play a relevant role in the results indicate that the loss of the Trex2 gene in mice does not DNA damage response to genotoxic stress. Specific expression of compromise survival or trigger a clearly cancer-prone phenotype. TREX2 in skin prompted us to examine DNA damage–induced skin

Figure 5. Apoptosis and clonogenic survival of DMBA-treated Trex2À/À and wt keratinocytes. A, apoptotic cells in the epidermis from carcinogen-treated Trex2À/À and wt mice. Mice were untreated (right), or treated with DMBA (middle)or DMBA+TPA (left), as described in Materials and Methods. Apoptotic cells (arrows) were analyzed 24 h after DMBA or TPA treatment. Scale bars, 50 Am. B, quantitative analysis of apoptotic cells in the epidermis. Columns, mean from seven mice of each genotype per group; bars, SE. Apoptotic cells are expressed as the number of cells per longitude of epidermis (cm). C, apoptosis in wt and Trex2À/À keratinocytes treated with DMBA (5 Ag/mL). When indicated, cells were pretreated with 20 Amol/L Z-VAD-FMK for 1 h, and then incubated for 18 h with or without DMBA. Apoptosis is displayed in relation to untreated cells. Columns, mean values of six independent experiments; bars, SE. The statistical significance of differences was analyzed with the Student’s t test for unpaired observations (*, P < 0.05; ***, P < 0.001). D, clonogenic survival of wt and Trex2À/À keratinocytes treated with the indicated doses of DMBA. Survival clones were counted 10 d after genotoxic treatment. Points, mean values of duplicates of a representative experiment of two independent experiments; bars, SE.

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TREX2 Prevents Skin Carcinogenesis

+/+ tumorigenesis. We compared the susceptibility of Trex2 and incidence of skin tumors, indicating that epithelial cells are À/À Trex2 age-matched mice to the development of skin tumors by especially susceptible to polymerase error-induced cancers (6). The À/À repeated DMBA application, or by the two-stage carcinogenesis absence of a cancer-prone phenotype in the Trex2 mice protocol, which involves tumor initiation by DMBA followed by indicates that the proofreading activity of TREX2 is not essential tumor promotion by TPA. Interestingly, the number of tumors was to maintain DNA replication fidelity or, alternatively, could be due À/À +/+ markedly increased in Trex2 mice compared with Trex2 mice to compensation by others proofreading exonucleases. Interest- À/À in both models (Fig. 4). As expected, in the DMBA model, the ingly, the increased susceptibility of the Trex2 mice to DNA period of latency was longer and tumor multiplicity was lower than damage–induced skin tumors suggests a relevant role of TREX2 in the DMBA+TPA model. Tumor size, which can be correlated under conditions of stress. In this regard, the Trex2 gene is similar with tumor growth rate, was similar between the two genotypes in to a few other tumor suppressor genes involved in genome À/À the DMBA+TPA group (Fig. 4D), and was slightly larger in Trex2 maintenance, DNA repair, and apoptosis pathways, such as XPA, +/+ mice compared with Trex2 in the DMBA group (Fig. 4B). (34), XPC (35), CHK2 (36, 37), or caspase-activated DNase (38). Furthermore, in the DMBA-treated group, the average number of Unlike p53 knockout mice, mice deficient in these genes do not À/À tumor-bearing mice was higher in the Trex2 mice than in the develop tumors spontaneously or are not tumor-prone at early +/+ Trex2 mice. By 7 weeks, f39% of the null mice had developed ages. However, these animals do show increased tumorigenic first tumors versus 6% of wt mice. By 9 weeks, 88% of null mice susceptibility when exposed to carcinogens. versus 60% of wt mice had developed tumors, and by 12 weeks, all Previous studies in ES cells lacking Trex2 have shown that À/À mice had developed tumors. Therefore, Trex2 mice show an TREX2 is required for efficient proliferation and chromosomal increased tumor susceptibility to DMBA-induced skin carcinogen- stability (20, 27). Severe chromosomal instability is usually esis compared with wt mice. This phenotype is independent of associated with lethality, growth retardation, premature aging, TPA-induced proliferation and tumor promotion, which suggests a immunologic abnormalities, or cancer (33). However, we have not suppressive role for TREX2 in the initiation phase of the tumors observed defects in cell proliferation or chromosomal abnormalities À/À À/À induced by DNA damage, rather than in the promotion stage. in cells from Trex2 mice. Also, Trex2 mice did not exhibit Reduced apoptosis in DMBA-treated skin and keratinocytes reduced survival, growth retardation, impaired lymphocyte devel- À/À of Trex2 mice. To determine the mechanisms by which opment, or a cancer-prone phenotype. Furthermore, the fact that the lack of TREX2 in the skin increases tumor development in Trex2-null mice showed increased susceptibility to carcinogen- response to genotoxic stress, we analyzed whether apoptosis was induced skin tumors and deficient apoptosis would be consistent altered in the untreated, DMBAÀ, and DMBA+TPA–treated skin of with a lack of chromosomal instability in this mouse. Thus, the À/À Trex2 mice. Interestingly, an approximately 2- to 3-fold greater DNA-PK knockout mouse, which is predisposed to chromosomal +/+ number of apoptotic cells was seen in the Trex2 compared instability, shows a decreased susceptibility to DMBA-induced À/À with the Trex2 epidermis (Fig. 5A and B). Next, we compared tumorigenesis due to the preexisting DNA damage in these cells DMBA-induced apoptosis in keratinocytes from wt and knockout (39). The chromosomal instability observed in the two TREX2- mice (Fig. 5C). In agreement with in vivo data, reduced levels of deficient ES clones (27) could be a consequence of replication stress DMBA-induced apoptosis, as measured by quantifying apoptotic generated during ES growth and selection. Therefore, our data À/À DNA fragmentation, were observed in Trex2 compared with wt indicate that TREX2 suppression does not lead to a general keratinocytes. When caspases were inhibited by the pan-caspase chromosomal instability, at least in the absence of genotoxic stress. inhibitor Z-VAD-FMK, no differences in DNA damage–induced The increased susceptibility to DMBA-induced skin carcinogen- À/À DNA fragmentation were observed between the two genotypes, esis in the Trex2 mice could be a consequence of deficient DNA indicating that TREX2 effects on apoptosis are dependent on prior editing or 3¶ end processing during nucleotide excision repair of caspase activation. Furthermore, data from survival colony assays DNA lesions generated by these genotoxics agents. However, unlike À/À indicated that Trex2 keratinocytes are more resistant than nucleotide excision repair–deficient cells (34, 35, 40), TREX2 +/+ Trex2 keratinocytes to DMBA-induced genotoxicity (Fig. 5D). deficiency in keratinocytes triggers a decrease in DNA damage– Thus, increased susceptibility to DMBA-induced skin tumorigen- induced apoptosis and an increase in survival. Therefore, a putative À/À esis in the Trex2 mice is associated with reduced carcinogen- role for TREX2 in nucleotide excision repair would probably not be induced keratinocyte apoptosis. Reduced apoptosis can contribute the main mechanism underlying TREX2 function in carcinogen- to survival of DNA-damaged keratinocytes and, therefore, to skin induced skin tumorigenesis. Our data support a proapoptotic role tumorigenesis. for TREX2 in which it facilitates the removal of keratinocytes that have suffered DNA damage. Apart from p53 (5, 41), other 3¶-5¶ exonucleases, such as WRN (42), NM23-H1, and TREX1 (21), can Discussion display a proapoptotic role through multiple mechanisms. In The present work shows that TREX2 is highly expressed in this regard, TREX1 and NM23-H1 have been implicated in DNA keratinocytes, and provides genetic evidence for a proapoptotic degradation during cell death mediated by caspase-independent tumor suppressor role for this exonuclease in carcinogen-induced granzyme A in cells targeted by cytotoxic T cells (21). Similarly, skin tumorigenesis. Cell requirements for 3¶-5¶ exonucleases can TREX2 could facilitate caspase-dependent DNA degradation once vary depending on homeostatic properties and environmental 3¶-hydroxyl DNA breaks are available, causing irreparable damage. exposure to exogenous stimuli. Specific TREX2 expression in In the absence of TREX2, a fraction of cells carrying mutagenic keratinocytes suggests that this exonuclease plays a relevant role DNA damage could indeed survive. Analogous to TREX2-null in tissues containing these cells. In fact, keratinocytes are the mice, caspase-activated DNase knockout mice, in which apoptotic first line of defense against exogenous genotoxic agents, such as DNA fragmentation is disrupted, do not show an increase in radiations, and carcinogens. In this context, inactivation of the spontaneous tumors, but are more susceptible to induced 3¶-5¶ exonucleolytic proofreading activity of Poly leads to a high carcinogenesis (38). www.aacrjournals.org 6683 Cancer Res 2009; 69: (16). August 15, 2009

Cancer Research

Skin is exposed to diverse and multiple exogenous genotoxic Disclosure of Potential Conflicts of Interest agents, and a large number of genes are required to maintain No potential conflicts of interest were disclosed. genome stability (43). Interestingly, TREX2 expression is largely reduced in the mice lacking IKKa, which is required to maintain Acknowledgments skin homeostasis and prevent skin cancer (44). Here, we found that Received 4/3/09; revised 5/14/09; accepted 5/21/09; published OnlineFirst 8/4/09. loss of TREX2 does not lead to a tumor-prone phenotype, but Grant support: Spanish Ministerio de Ciencia e Innovacio´n grants BFU2006-06076 rather increases susceptibility to induced skin carcinogenesis. and CSD2008-00005 (C. Soler), and BIO2006-3213 (S. Ortega), the Fondo de Investigaciones Thus, TREX2 can serve as a proapoptotic tumor suppressor that Sanitarias grant PI021192 (C. Soler), and the University of Barcelona (C. Soler). The costs of publication of this article were defrayed in part by the payment of page may contribute to genome maintenance in tissues with stratified charges. This article must therefore be hereby marked advertisement in accordance squamous epithelia under conditions of genotoxic stress. Further with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. D.J. Rossi (University of Helsinki) for providing the pDELBOY-3X work to search for TREX2 expression and mutations in squamous vector, Dr. J.C. Cigudosa and the Cytogenetic Unit of the CNIO for SKY analyses, and cell carcinomas is warranted. C. Go´mez and M. Riffo for ES clone aggregation and chimera crosses.

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Cancer Res 2009; 69: (16). August 15, 2009 6684 www.aacrjournals.org

Increased Susceptibility to Skin Carcinogenesis in TREX2 Knockout Mice

David Parra, Joan Manils, Bàrbara Castellana, et al.

Cancer Res Published OnlineFirst August 4, 2009.

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