Increased Susceptibility to Skin Carcinogenesis in
TREX2 Knockout Mice
David Parra,1Joan Manils,
1Barbara Castellana,
1Arnau Vina-Vilaseca,
1Eva Moran-Salvador,
1
Nuria Vazquez-Villoldo,1Gemma Tarancon,
1Miquel Borras,
2Sara Sancho,
4
Carmen Benito,3Sagrario Ortega,
5and Concepcio Soler
1
1Departament de Patologia i Terapeutica Experimental, Facultat de Medicina, Campus de Bellvitge, Universitat de Barcelona, L’Hospitaletde Llobregat; 2Unitat de Toxicologia Experimental, Parc Cientıfic de Barcelona; and 3Proteccio Radiologica, Universitat de Barcelona,Barcelona, Spain; 4Promed, Fribourg, Switzerland; and 5Spanish National Cancer Research Center, Madrid, Spain
Abstract
TREX2 is a proofreading 3¶-5¶ exonuclease that can be involvedin genome maintenance; however, its biological role remainsundefined. To better understand the function and physiologicrelevance of TREX2, we generated mice deficient in TREX2 bytargeted disruption of its unique coding exon. The knockoutmice are viable and do not show relevant differences ingrowth, survival, lymphocyte development, or spontaneoustumor incidence compared with their wild-type counterpartsover a period of up to 2 years. Also, we did not observechromosomal instability or defects in cell proliferation andcell cycle upon loss of TREX2. We have observed that TREX2expression is not ubiquitous, being expressed preferentiallyin tissues with stratified squamous epithelia, such as theskin or esophagus, and specifically in keratinocytes. Interest-ingly, TREX2-null mice are more susceptible to skin carcino-genesis induced by 7,12-dimethylbenz(a)anthracene (DMBA)compared with wild-type mice. This phenotype correlateswith a reduction of DMBA-induced apoptosis in both theepidermis and keratinocytes of TREX2-null mice. Altogether,our results suggest a tumor suppressor role for TREX2 inskin carcinogenesis through which it contributes to keratino-cyte apoptosis under conditions of genotoxic stress. [CancerRes 2009;69(16):6676–84]
Introduction
3¶-5¶ Exonucleases remove nucleotides from DNA 3¶ termini inmultiple processes of DNA metabolism, ranging from DNAsynthesis to DNA degradation, and thereby can play a pivotal rolein maintaining genome stability and preventing cancer, aging,immunologic abnormalities, and inherited diseases. The 3¶-5¶exonuclease activity has been found in multidomain proteins, suchas DNA polymerases (Poly, Polg, and Polq), base excision repairapurinic/apyrimidinic endonucleases (APE1 and APE2), doublestrand break DNA repair proteins (MRE11), DNA repair nucleases(EXO1 and NM23-H1), helicases (WRN, Dna2), cell cycle checkpointproteins (RAD9 and RAD1), and the transcription factor p53. Inaddition, this activity has been described in the single-domain
exonucleases TREX1 and TREX2 (1–5). Despite the numerous3¶-5¶ exonucleases present in the cell with putative overlappingactivities, in vivo functional studies have revealed specific andnonredundant biological roles for many of these proteins (1, 6–11).Among these, the biological function of TREX2 is still largelyunknown.TREX2, which was identified in a database search by means
of its homology with TREX1, displays a robust nonprocessive 3¶-5¶exonuclease activity that can proofread the work of DNApolymerases and process 3¶ ends during DNA replication andrepair processes in vitro (12–14). In this regard, it has beenreported that TREX2 may interact with Poly, and increase itsfidelity when deoxynucleotide triphosphates pools are imbalanced(15). TREX2 biochemical and structural properties are similar toTREX1, although they are not identical. The two proteins share adimeric structure and can process ssDNA and dsDNA substratesin vitro with almost identical kcat values. However, several featuresrelated to enzyme kinetics, structural domains, and subcellulardistribution distinguish TREX2 from TREX1. TREX2 present a10-fold lower affinity for DNA substrates in vitro compared withTREX1. In contrast with TREX1, TREX2 lacks a COOH-terminaldomain that can mediate protein-protein interactions (12, 16–19).TREX2 is localized in both the cytoplasm and nucleus (20), whereasTREX1 is found in the endoplasmic reticulum, and is mobilized tothe nucleus during granzyme A–mediated cell death (21) or afterDNA damage (22). Loss of TREX1 function does not trigger anincrease in cancer development (11), but rather leads to cell-intrinsic immune activation by endogenous nondegraded ssDNA,resulting in the development of autoimmunity (23–26). Indeed,TREX1 has been shown to be involved in the degradation of ssDNAgenerated from the processing of aberrant replication intermedi-ates (22) and endogenous retroelements (26). Otherwise, TREX2seems to be important for chromosomal stability. As such, TREX2deletion in embryonic stem (ES) causes high levels of RobertsonianTranslocations (27), but this effect is independent of itsexonuclease activity and DNA binding domains (28). Thebiochemical activities and the mechanisms by which TREX2prevents chromosomal instability and the consequences of TREX2deficiency in the organism remain unknown.In this context, we aimed to asses the function and relevance of
TREX2 in vivo . To that end, we generated and characterized theTREX2 knockout mice, Trex2�/� . Furthermore, to evaluate putativetissue-specific functions, we analyzed TREX2 tissue expression.TREX2 knockout mice seem viable and fertile and do not show asignificant increase in spontaneous tumor incidence. Interestingly,TREX2 deficiency increases susceptibility to carcinogen-inducedskin tumorigenesis that correlates with impaired apoptosis. Thesefindings suggest a tumor suppressor role for TREX2 under
Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).
Requests for reprints: Concepcio Soler, Departament de Patologia i TerapeuticaExperimental, Facultat de Medicina, Campus de Bellvitge, Universitat de Barcelona,Feixa Llarga s/n, E-08907, L’Hospitalet de Llobregat, Barcelona, Spain. Phone: 34-934039858; Fax: 34-934024249; E-mail: [email protected].
I2009 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-09-1208
Cancer Res 2009; 69: (16). August 15, 2009 6676 www.aacrjournals.org
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 (Trex2� ) locus. The mouse Trex2 locus contains two exons (gray boxes ). The full protein coding sequence is included in exon 2. LoxPsites (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 homologousrecombinant ES clones are indicated. Flp, Flp recombinase; Cre, Cre recombinase. RV (EcoRV), RI (EcoRI). B, Southern blot (SB ) and PCR analysis of recombinantES 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 inA and B. C, RT-PCR analysis of Trex2 mRNA expression in skin from wt, Trex2�/� , and Trex2lox/lox mice. D, Western blot (WB ) analysis of skin extracts andimmunoprecipitates from wt, Trex2�/� , and Trex2lox/lox mice with antibodies to mouse TREX2. Preimmune (PI ) serum was used as a negative control forimmunoprecipitation (IP ). Bands corresponding to the 236 amino acid mouse TREX2 protein and to heavy immunoglobulin chain (IgH) are indicated.
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conditions of genotoxic stress in keratinocytes, cells where we havefound that this exonuclease is predominantly expressed.
Materials and Methods
Generation of Trex2�/� mice and genotyping. To conditionally
disrupt the mouse Trex2 gene, a targeting strategy was designed to knock
in two loxP sites flanking exon 2 via homologous recombination in ES cells.
The targeting construct was constructed by subcloning genomic DNAfragments encompassing exon 2 and the 5¶ and 3¶ flanking fragments intothe loxP conditional vector pDELBOY-3�, as schematically shown in
Fig. 1A . Genomic fragments corresponding to exon 2 (1 kb) and the5¶ (2.9 kb) and 3¶ (2.2 kb) arms were amplified using genomic DNA extractedfrom 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 andganciclovir. Genomic DNA from resistant clones were digested with EcoRI ,
and tested for correct targeting events via Southern blotting using
both 5¶ (probe a) and 3¶ (probe b) external probes. Targeted ES clones(Trex2loxneo) are hemizygous because the Trex2 gene is located on theX chromosome and R1 ES cells are XY. Three ES clones, in which correct
homologous recombination had occurred, were aggregated with eight-
cell-stage CD-1 embryos. Male chimeras transmitted the targeted allele
Figure 2. Tissue and cell expression ofTREX2. A, TREX2 mRNA expression invarious mouse (left ) and human (right )tissues. The expression levels of theunique mouse TREX2 mRNA transcriptand of the two human transcripts that codefor the short (236 amino acid) and long(279 amino acid) isoforms were determinedvia real-time PCR in the indicated tissues.The results were normalized to theexpression of HPRT and the mean valuesare displayed in relation to skin. B, TREX2protein expression in various mousetissues. Immunoprecipitation and Westernblot analysis of extracts from variousmouse tissues with antibodies to mouseTREX2. Bands corresponding to the 236amino acid mouse TREX2 protein and toheavy immunoglobulin (IgH) chain areindicated. C, TREX2 immunostaining inmouse skin and esophagus sections and incultured primary keratinocytes from wt andTrex2�/� mice. Nuclei were counterstainedwith 4¶,6-diamidino-2-phenylindole. Scalebars, 20 Am. D, DNA 3¶ exonucleaseactivity in extracts of wt and Trex2�/� micecells and tissues. Top, extracts fromepidermis were assayed for 3¶ exonucleaseactivity at the indicated times. Bottom,extracts from the indicated cells andtissues were assayed for 3¶ exonucleaseactivity after 10 min of incubation with thesubstrate.
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glycerol, and 150 mmol/L NaCl. Samples were boiled in Laemmli buffer and
separated on 10% SDS-PAGE. They were then transferred to polyvinylidenedifluoride membranes (Hybond-ECL; GE Healthware). Membranes were
blocked for 1 h at room temperature in 5% nonfat milk in TBST buffer [150
mmol/L NaCl, 50 mmol/L Tris (pH 7.4) and 0.05% Tween 20], incubated
with diluted antibody (anti-TREX2, 1/10,000) and followed by horseradishperoxidase–conjugated protein A/G (Bio-Rad) and detected with enhanced
chemiluminescence (ECL; GE Healthware). Antibodies against mouse
TREX2 were generated using an MBP-fusion mouse TREX2 protein forrabbit immunization. Antibodies were raised and affinity purified by
standard procedures. This antibody does not cross-react with TREX1. The
specificity of this antibody was unequivocally validated by the absence of
immunodetection of TREX2 protein by immunoprecipitation, Western blotand immunofluorescence in samples from Trex2�/� mice compared with
samples from Trex2+/+ mice (Figs. 1D and 2C).
Exonuclease assay. Cells and tissues from mice were homogenized
in an ice-cold lysis buffer containing 1% Triton X-100, 50 mmol/L Tris-HCl(pH 7.5), 10 mmol/L MgCl2, 1 mmol/L DTT, 100 mmol/L NaCl, 10 Ag/mLaprotinin, 10 Ag/mL leupeptin, and 1 mmol/L PMSF, and 1 mmol/L sodiumorthovanadate. Lysates were then assayed for DNA 3¶ exonuclease activity,as described elsewhere (11).
Flow cytometry analysis of B and T cells. Cells from the bone marrow,
thymus, spleen, and blood of 2-mo-old Trex2�/� and Trex2+/+ mice were
stained with the following antibodies to B- and T-cell surface markers:FITC-anti-a/hTCR, PE-anti-CD4, Cy5-anti-CD8, FITC-anti-B220, and PE-
anti-CD19 (Becton Dickinson) following standard protocols. Analysis was
performed with an Epics XL flow cytometer (Coulter Corporation).
Survival, growth, and spontaneous tumor development. Trex2�/�
mice and their Trex2+/+ littermates in the C57Bl/6/CD1/129 mixed genetic
background were used in an untreated time course study, during which they
were observed weekly for up 2 y. Mice were monitored for body weight,
fertility, survival, lymphocyte development, and spontaneous tumor
Figure 3. Growth, survival, andlymphocyte cells in the Trex2�/� and wtmice. A, body weight of wt (n = 22) andTrex2�/� (n = 22) males (top ) and ofwt (n = 16) and Trex2�/� (n = 14) females(bottom ) were followed for up to 2 y.Points, average; bars, SE. No significantdifferences were observed between wt andTrex2�/� genotypes. B, survival curves.Survival curves correspond to 36Trex2�/� and 38 wt and mice. Nosignificant difference in median survivalwas observed (log-rank test with 95%confidence interval). C, analysis ofT-lymphocyte populations isolated from thethymus, spleen, and blood. D, analysis ofB-lymphocyte populations isolated thebone marrow, spleen, and blood. Columns,average of six Trex2�/� and six wt areshown; bars, SE. No significant differenceswere observed between wt and Trex2�/�
genotypes.
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development. Mice were euthanized when they seemed unhealthy, eitherbecause of visible lesions, morbidity, or significant weight loss. Autopsies
were performed on these animals and in all of the mice when they reached
the age of 24 mo. Mouse tissues were fixed in 10% neutral-buffered formalin
solution and processed for paraffin embedding, cut into 5-Am sections, andmounted on slides. The sections were stained with H&E and examined by
two pathologists who were blinded to the tissue grouping.
Skin tumor induction experiments. The strategy used for the
carcinogenicity study followed well-established mouse models of DMBAand 7,12-dimethylbenz(a)anthracene (DMBA)+12-O-tetradecanoylphorbol-
13-acetate (TPA)–induced tumors in the skin. For the DMBA-induced
tumor experiments, DMBA (120 nmols) was applied twice weekly for 14 wk.
For the DMBA+TPA tumor initiation/promotion experiments, mice weretreated with a single dose of the carcinogen DMBA (120 nmol in 200 ALof acetone; Sigma) followed by applications of the tumor promoter TPA
(20 nmol in 200 AL of acetone; Sigma) twice a week beginning 4 d afterinitiation, and for a duration of 12 wk. Treatments were applied to the
shaved backs of 8-wk-old Trex2�/� and Trex2+/+ mice. Mice of the C57BL/6/CD1/129 mixed genetic background were backcrossed for six generations to
CD1 mice, which were then bred to each other to generate cohorts ofTrex2�/� and Trex2+/+ mice that were used in the skin carcinogenesis
studies. Mice were visually inspected weekly, and tumor numbers and sizes
were quantified to calculate tumor incidence (percentage of tumor-bearing
mice) and tumor multiplicity (average number of tumors per mouse). Micewere sacrificed when they showed signs of poor health. Skin sections were
fixed in 10% buffered formalin, embedded in paraffin, and subjected to
histopathologic analysis.Cell cultures, proliferation, cell cycle, and survival assays. Primary
mouse embryonic fibroblasts (MEFs) from E14.5 embryos were obtained
according to standard protocols and cultured them in DMEM supple-
mented with 10% fetal bovine serum (FBS). For proliferation assays, weplated 5 � 104 cells on 24-well plates in triplicate and incubated the cells in
DMEM supplemented with 0.1% FBS for 60 h. We added DMEM
supplemented with 10% FBS, or with increasing FBS concentrations, and
measured 3H-thymidine incorporation at the indicated times, as described(29). Primary keratinocytes were prepared from newborn mouse skin and
cultured on collagen I precoated plates (BD Biosciences), as described (30).
To assess keratinocyte proliferation, 1 � 104 cells were plated on 96-wellplates in triplicate and growth was monitored by the reduction of the
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
flow cytometer (Coulter Corporation). To evaluate the effects of DMBA onkeratinocyte survival, we performed clonogenic assays. Keratinocytes (2.5 �105) were seeded on six-well plates in duplicated, and treated with the
indicated doses of the genotoxic agent. Ten days after treatment, the
colonies were stained with methylene blue following standard procedure,scored, and used to calculate the surviving fraction of cells in each condition.
Apoptosis analyses. Apoptotic cells were evaluated in biopsies from the
dorsal skin of mice that were exposed to single doses of DMBA (400 nmol
for 24 h), DMBA+TPA (DMBA, 120 nmols for 4 d followed by TPA, 20 nmolfor 24 h), or acetone (control). Treated dorsal skin of mice was fixed in 10%
neutral-buffered formalin solution and processed for paraffin embedding.
Skin sections (5 Am thick) were stained with H&E, and the number of
sunburn cells, defined as apoptotic cells within the epidermis exhibiting anintensely eosinophilic cytoplasm and small and dense nuclei, were counted
throughout the epidermis. Sunburn cells for at least two sections of 1-cm
long epidermis of each skin sample were scored in a blinded fashion. To
quantify apoptotic DNA fragmentation in DMBA-treated keratinocytes, weused a known highly specific and sensitive sandwich ELISA kit (Roche) that
quantifies cytoplasmic histone-associated DNA fragments in cells undergo-
ing programmed cell death.Spectral karyotyping analysis. Colcemid-treated cells were resus-
pended carefully in a KCl hypotonic solution (0.075 mol/l) for 30 min at
37jC, rinsed and fixed in 3:1 methanol/acetic acid, and dropped onto glassslides. Slides were hybridized using the Spectral karyotyping (SKY) methodaccording to the manufacturer’s protocol (Applied Spectral Imaging, Inc.).
Images were acquired with an SD300 Spectra Cube (Applied Spectral
Imaging) mounted on a Zeiss Axioplan microscope using a custom-
designed optical filter (SKY-1; Chroma Technology).
Results
Generation of Trex2�/� mice. To create the Trex2�/� mice,Trex2 gene was disrupted by deletion of exon 2, as detailed inMaterials and Methods (Fig. 1A and B). Because exon 2 contains thefull-length coding sequence of murine TREX2 (31), removal of thissequence completely abrogates TREX2 expression. As describedbelow, we did find that TREX2 is predominantly expressed in theskin. Analysis of TREX2 mRNA (Fig. 1C) and protein expression(Fig. 1D) showed the presence of TREX2 expression in skin fromwild-type (wt; Trex2+/+) and conditional gene–targeted mice(Trex2lox/lox), and its absence in gene-targeted mice (Trex2�/�),confirming the null mutation.
Tissue- and cell-specific expression of TREX2. TREX2 mRNAtranscripts had been previously detected in various human tissues,suggesting ubiquitous expression (20, 31). However, quantitativeexpression was not assessed in those studies. Although the humanand mouse TREX2 gene structure is highly homologue, mouse gene
Table 1. Incidence of common spontaneous tumors found in Trex2�/� versus wt mice
codes for a single 26-kDa isoform, whereas human gene can givesrise to two additional 30-kDa isoforms that contain an extraNH2-terminal region. The different human isoforms arise frommRNAs generated by alternative splicing, and by the use ofalternative promoters. Analogous transcripts in human and micecode for the 236 amino acid proteins (26 kDa; short isoform). Othertwo human transcripts code for an extra 43 or 42 amino acidsupstream the NH2 terminus of the 26 kDa isoform, and aretranslated into a protein of 279 and 278 amino acids, respectively(30 kDa; long isoform). Interestingly, our results from thequantitative RT-PCR analysis reveal a specific pattern of expressionof TREX2 in both mice and human tissues. As seen in Fig. 2A , theunique mouse TREX2 mRNAs transcript is highly expressed inthe skin, tongue, esophagus, and forestomach. Compared with skin,10-fold lower expression was detected in the bladder, prostate, andthyroid, and a 100-fold lower levels were found in the trachea, lung,glandular stomach, small intestine, pancreas, and uterus. Expres-sion was more than a 1,000-fold lower or almost undetectable inthe spleen, thymus, ganglia, kidney, colon, rectum, ovary, testis,seminal vesicle, adrenal gland, salivary gland, brain, liver, skeletalmuscle, or adipose tissue. Similarly, in human tissues, we observedmajor expression in skin; moderate expression in the tongue andcervix, low expression in the kidney, spleen, stomach, and thyroid;and it was undetectable in liver. An analogous pattern ofexpression was observed for the two main human TREX2transcripts, the predominant long isoform and the less abundantshort isoform. Next, to evaluate protein expression, we performedimmunoprecipitation followed by Western blot analyses. As shown
in Fig. 2B , TREX2 protein is observed only in certain mouse tissues,such as the skin, tongue, esophagus, forestomach, or bladder,which express relatively high levels of mRNA for this gene. In thesemouse tissues, we only detected a single TREX2 polypeptide withan apparent molecular weight of f28 kDa, indicating theexpression of a single protein isoform in mice. This proteincorresponds to the 236 amino acid isoform, as confirmed byheterologous expression in fibroblasts of the mouse Trex2 cDNAcoding for the 236 amino acid protein, which gives rise to anidentical 28 kDa protein (data not shown). Collectively, mRNAand protein data indicate that TREX2 shows a tissue-specificexpression.To identify cell types expressing TREX2, we performed
immunohistochemistry assays of major tissues expressing TREX2,such as the skin and esophagus (Fig. 2C). As a negative control forstaining, we used corresponding knockout samples. We observedthat TREX2 expression was confined to cells of the stratifiedsquamous epithelia of these tissues. Because keratinocytes are themost abundant cells that integrate the stratified squamousepithelia, we next checked for TREX2 expression in primarykeratinocyte cultures. Immunocytochemistry analyses illustratethat TREX2 is expressed in keratinocytes and localized mainly inthe nucleus, showing a punctate distribution with many foci percell (Fig. 2C). Quantitative mRNA analysis further confirmed thatTREX2 is preferentially and highly expressed in keratinocytes. Thus,mouse TREX2 mRNA transcript levels were almost 105 times higherin keratinocytes compared with MEFs and ES cells, whereas thetranscript was undetectable in macrophages.
Figure 4. DMBA-induced skin carcinogenesis in wtand Trex2�/� mice. Mice were untreated or treatedwith DMBA, or DMBA+TPA, as described in theMaterials and Methods. A, average number oftumors per mouse in the skin of wt (n = 15) andTrex2�/� (n = 18) mice treated with DMBA. B, sizedistribution of skin tumors present in DMBA-treatedwt and Trex2�/� mice at week 14. C, average numberof tumors per mouse in the skin of wt (n = 25) andTrex2�/� (n = 25) mice following DMBA+TPAtwo-stage chemical carcinogenesis. D, tumorsize distribution of skin tumors present inDMBA+TPA-treated wt and Trex2�/� mice at week 12.Tumor multiplicity data were statistically analyzedusing the Mann-Whitney test (*, P < 0.05; **, P < 0.01;***, P < 0.001).
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Finally, to evaluate the contribution of TREX2 activity to theoverall 3¶-5¶ exonuclease activity, we compared this activity inseveral tissues from Trex2�/� and Trex2+/+ mice (Fig. 2D).Consistent with the expression data, TREX2 does not contributeto the 3¶-exonuclease activity of the dermis or liver, but doesaccount for f50% of this activity in keratinocytes and theepidermis.Altogether, the expression and activity data indicate that TREX2
is predominantly expressed in keratinocytes and, consequently, intissues with stratified squamous epithelia.
Trex2 loss does not lead to tumorigenic phenotype orreduced survival. The TREX2 knockout mice were viable, bornat the expected Mendelian frequency, fertile, and indistinguishableby average weight and growth from their wt littermates (Fig. 3A).Mice lacking TREX2 seemed healthy and no apparent anatomic orgross behavioral abnormalities were observed. No differences in lifespan (Fig. 3B) or spontaneous tumor development (Table 1) wereseen between the two genotypes for up to 2 years. Compared withTrex2+/+ mice, we did observe a slight increase in the percentage ofTrex2�/� mice that developed splenic lymphomas; however, thedifferences were not statistically significant. Although TREX2 ishighly expressed in the skin, its suppression does not lead toabnormalities in the skin or an increase in related diseases such asskin hyperpigmentation, alopecia, dryness and dermatitis, orspontaneous skin tumors. Both Trex2�/� and Trex2+/+ micedeveloped most common tumors in agreement with published ratesin aging male and female C57BL/6/129 mice (32). Collectively, theseresults indicate that the loss of the Trex2 gene in mice does notcompromise survival or trigger a clearly cancer-prone phenotype.
Chromosomal stability, proliferation, and immunologicdevelopment are not altered in cells from Trex2�/� mice.Another group has previously reported the reduced proliferationand presence of Robertsonian translocations, duplications, anddeletions in two clones of Trex2-deficient ES cells (27). Surprisingly,we did not detect any chromosomal abnormalities in morethan one hundred metaphases analyzed by SKY from MEFsand keratinocytes isolated from at least four knockout and wtTrex2 mice (Supplementary Fig. S1A). Furthermore, we did notfind differences in the proliferation or cell cycle phases of severalcell types, such as keratinocytes (Supplementary Fig. S1B and D)and MEFs [from primary passage 2 (P2) to 6 (P6); SupplementaryFig. S1C and D), obtained from Trex2�/� mice compared withTrex2+/+ cells.Because defects in many genes involved in DNA repair and
genome stability trigger impaired lymphocyte development (33), weanalyzed B- and T-cell populations in the thymus, bone marrow,spleen, and blood from Trex2�/� mice and their wt littermates.Populations of lymphoid T cells bearing CD4, CD8, and T-cellreceptor ah (Fig. 3C) or lymphoid B cells expressing B220, CD19, orIgM (Fig. 3D) were not affected by loss of TREX2, revealing nodefects in B- and T-cell development. Together, these findingsindicate that TREX2 is dispensable for chromosomal stability andefficient cellular proliferation of several cell types.
Increased susceptibility to DMBA-induced skin tumorigen-esis in Trex2�/� mice. To further characterize TREX2 biologicalfunction, we tested whether TREX2 could play a relevant role in theDNA damage response to genotoxic stress. Specific expression ofTREX2 in skin prompted us to examine DNA damage–induced skin
Figure 5. Apoptosis and clonogenicsurvival of DMBA-treated Trex2�/� and wtkeratinocytes. A, apoptotic cells in theepidermis from carcinogen-treatedTrex2�/� and wt mice. Mice were untreated(right ), or treated with DMBA (middle ) orDMBA+TPA (left), as described inMaterials and Methods. Apoptotic cells(arrows ) were analyzed 24 h after DMBAor TPA treatment. Scale bars, 50 Am.B, quantitative analysis of apoptotic cells inthe epidermis. Columns, mean from sevenmice of each genotype per group;bars, SE. Apoptotic cells are expressed asthe number of cells per longitude ofepidermis (cm). C, apoptosis in wt andTrex2�/� keratinocytes treated with DMBA(5 Ag/mL). When indicated, cells werepretreated with 20 Amol/L Z-VAD-FMK for 1h, and then incubated for 18 h with orwithout DMBA. Apoptosis is displayed inrelation to untreated cells. Columns, meanvalues of six independent experiments;bars, SE. The statistical significance ofdifferences was analyzed with theStudent’s t test for unpaired observations(*, P < 0.05; ***, P < 0.001). D, clonogenicsurvival of wt and Trex2�/� keratinocytestreated with the indicated doses ofDMBA. Survival clones were counted10 d after genotoxic treatment. Points,mean values of duplicates of arepresentative experiment of twoindependent experiments; bars, SE.
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tumorigenesis. We compared the susceptibility of Trex2+/+ andTrex2�/� age-matched mice to the development of skin tumors byrepeated DMBA application, or by the two-stage carcinogenesisprotocol, which involves tumor initiation by DMBA followed bytumor promotion by TPA. Interestingly, the number of tumors wasmarkedly increased in Trex2�/� mice compared with Trex2+/+ micein both models (Fig. 4). As expected, in the DMBA model, theperiod of latency was longer and tumor multiplicity was lower thanin the DMBA+TPA model. Tumor size, which can be correlatedwith tumor growth rate, was similar between the two genotypes inthe DMBA+TPA group (Fig. 4D), and was slightly larger in Trex2�/�
mice compared with Trex2+/+ in the DMBA group (Fig. 4B).Furthermore, in the DMBA-treated group, the average number oftumor-bearing mice was higher in the Trex2�/� mice than in theTrex2+/+ mice. By 7 weeks, f39% of the null mice had developedfirst tumors versus 6% of wt mice. By 9 weeks, 88% of null miceversus 60% of wt mice had developed tumors, and by 12 weeks, allmice had developed tumors. Therefore, Trex2�/� mice show anincreased tumor susceptibility to DMBA-induced skin carcinogen-esis compared with wt mice. This phenotype is independent ofTPA-induced proliferation and tumor promotion, which suggests asuppressive role for TREX2 in the initiation phase of the tumorsinduced by DNA damage, rather than in the promotion stage.
Reduced apoptosis in DMBA-treated skin and keratinocytesof Trex2�/� mice. To determine the mechanisms by whichthe lack of TREX2 in the skin increases tumor development inresponse to genotoxic stress, we analyzed whether apoptosis wasaltered in the untreated, DMBA�, and DMBA+TPA–treated skin ofTrex2�/� mice. Interestingly, an approximately 2- to 3-fold greaternumber of apoptotic cells was seen in the Trex2+/+ comparedwith the Trex2�/� epidermis (Fig. 5A and B). Next, we comparedDMBA-induced apoptosis in keratinocytes from wt and knockoutmice (Fig. 5C). In agreement with in vivo data, reduced levels ofDMBA-induced apoptosis, as measured by quantifying apoptoticDNA fragmentation, were observed in Trex2�/� compared with wtkeratinocytes. When caspases were inhibited by the pan-caspaseinhibitor Z-VAD-FMK, no differences in DNA damage–inducedDNA fragmentation were observed between the two genotypes,indicating that TREX2 effects on apoptosis are dependent on priorcaspase activation. Furthermore, data from survival colony assaysindicated that Trex2�/� keratinocytes are more resistant thanTrex2+/+ keratinocytes to DMBA-induced genotoxicity (Fig. 5D).Thus, increased susceptibility to DMBA-induced skin tumorigen-esis in the Trex2�/� mice is associated with reduced carcinogen-induced keratinocyte apoptosis. Reduced apoptosis can contributeto survival of DNA-damaged keratinocytes and, therefore, to skintumorigenesis.
Discussion
The present work shows that TREX2 is highly expressed inkeratinocytes, and provides genetic evidence for a proapoptotictumor suppressor role for this exonuclease in carcinogen-inducedskin tumorigenesis. Cell requirements for 3¶-5¶ exonucleases canvary depending on homeostatic properties and environmentalexposure to exogenous stimuli. Specific TREX2 expression inkeratinocytes suggests that this exonuclease plays a relevant rolein tissues containing these cells. In fact, keratinocytes are thefirst line of defense against exogenous genotoxic agents, such asradiations, and carcinogens. In this context, inactivation of the3¶-5¶ exonucleolytic proofreading activity of Poly leads to a high
incidence of skin tumors, indicating that epithelial cells areespecially susceptible to polymerase error-induced cancers (6). Theabsence of a cancer-prone phenotype in the Trex2�/� miceindicates that the proofreading activity of TREX2 is not essentialto maintain DNA replication fidelity or, alternatively, could be dueto compensation by others proofreading exonucleases. Interest-ingly, the increased susceptibility of the Trex2�/� mice to DNAdamage–induced skin tumors suggests a relevant role of TREX2under conditions of stress. In this regard, the Trex2 gene is similarto a few other tumor suppressor genes involved in genomemaintenance, DNA repair, and apoptosis pathways, such as XPA,(34), XPC (35), CHK2 (36, 37), or caspase-activated DNase (38).Unlike p53 knockout mice, mice deficient in these genes do notdevelop tumors spontaneously or are not tumor-prone at earlyages. However, these animals do show increased tumorigenicsusceptibility when exposed to carcinogens.Previous studies in ES cells lacking Trex2 have shown that
TREX2 is required for efficient proliferation and chromosomalstability (20, 27). Severe chromosomal instability is usuallyassociated with lethality, growth retardation, premature aging,immunologic abnormalities, or cancer (33). However, we have notobserved defects in cell proliferation or chromosomal abnormalitiesin cells from Trex2�/� mice. Also, Trex2�/� mice did not exhibitreduced survival, growth retardation, impaired lymphocyte devel-opment, or a cancer-prone phenotype. Furthermore, the fact thatTrex2-null mice showed increased susceptibility to carcinogen-induced skin tumors and deficient apoptosis would be consistentwith a lack of chromosomal instability in this mouse. Thus, theDNA-PK knockout mouse, which is predisposed to chromosomalinstability, shows a decreased susceptibility to DMBA-inducedtumorigenesis due to the preexisting DNA damage in these cells(39). The chromosomal instability observed in the two TREX2-deficient ES clones (27) could be a consequence of replication stressgenerated during ES growth and selection. Therefore, our dataindicate that TREX2 suppression does not lead to a generalchromosomal instability, at least in the absence of genotoxic stress.The increased susceptibility to DMBA-induced skin carcinogen-
esis in the Trex2�/� mice could be a consequence of deficient DNAediting or 3¶ end processing during nucleotide excision repair ofDNA lesions generated by these genotoxics agents. However, unlikenucleotide excision repair–deficient cells (34, 35, 40), TREX2deficiency in keratinocytes triggers a decrease in DNA damage–induced apoptosis and an increase in survival. Therefore, a putativerole for TREX2 in nucleotide excision repair would probably not bethe main mechanism underlying TREX2 function in carcinogen-induced skin tumorigenesis. Our data support a proapoptotic rolefor TREX2 in which it facilitates the removal of keratinocytes thathave suffered DNA damage. Apart from p53 (5, 41), other 3¶-5¶exonucleases, such as WRN (42), NM23-H1, and TREX1 (21), candisplay a proapoptotic role through multiple mechanisms. Inthis regard, TREX1 and NM23-H1 have been implicated in DNAdegradation during cell death mediated by caspase-independentgranzyme A in cells targeted by cytotoxic T cells (21). Similarly,TREX2 could facilitate caspase-dependent DNA degradation once3¶-hydroxyl DNA breaks are available, causing irreparable damage.In the absence of TREX2, a fraction of cells carrying mutagenicDNA damage could indeed survive. Analogous to TREX2-nullmice, caspase-activated DNase knockout mice, in which apoptoticDNA fragmentation is disrupted, do not show an increase inspontaneous tumors, but are more susceptible to inducedcarcinogenesis (38).
TREX2 Prevents Skin Carcinogenesis
www.aacrjournals.org 6683 Cancer Res 2009; 69: (16). August 15, 2009
Skin is exposed to diverse and multiple exogenous genotoxicagents, and a large number of genes are required to maintaingenome stability (43). Interestingly, TREX2 expression is largelyreduced in the mice lacking IKKa, which is required to maintainskin homeostasis and prevent skin cancer (44). Here, we found thatloss of TREX2 does not lead to a tumor-prone phenotype, butrather increases susceptibility to induced skin carcinogenesis.Thus, TREX2 can serve as a proapoptotic tumor suppressor thatmay contribute to genome maintenance in tissues with stratifiedsquamous epithelia under conditions of genotoxic stress. Furtherwork to search for TREX2 expression and mutations in squamouscell carcinomas is warranted.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 4/3/09; revised 5/14/09; accepted 5/21/09; published OnlineFirst 8/4/09.Grant support: Spanish Ministerio de Ciencia e Innovacion grants BFU2006-06076
and CSD2008-00005 (C. Soler), and BIO2006-3213 (S. Ortega), the Fondo de InvestigacionesSanitarias 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
charges. This article must therefore be hereby marked advertisement in accordancewith 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
vector, Dr. J.C. Cigudosa and the Cytogenetic Unit of the CNIO for SKY analyses, andC. Gomez and M. Riffo for ES clone aggregation and chimera crosses.
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Cancer Research
Cancer Res 2009; 69: (16). August 15, 2009 6684 www.aacrjournals.org
2009;69:6676-6684. Published OnlineFirst August 4, 2009.Cancer Res David Parra, Joan Manils, Bàrbara Castellana, et al. Knockout MiceIncreased Susceptibility to Skin Carcinogenesis in TREX2
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