Tumors induce complex DNA damage in distantproliferative tissues in vivoChristophe E. Redona, Jennifer S. Dickeya, Asako J. Nakamuraa, Irina G. Karevaa, Dieter Nafb,1, Somaira Nowsheenc,Thomas B. Krystonc, William M. Bonnera, Alexandros G. Georgakilasc, and Olga A. Sedelnikovaa,2

aLaboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892; bLaboratory Animal Sciences Program,National Cancer Institute, Frederick, MD 21702; and cBiology Department, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville,NC 27858

Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved August 27, 2010 (received for review June 18, 2010)

That tumors cause changes in surrounding tissues is well docu-mented, but whether they also affect distant tissues is uncertain.Such knowledge may be important in understanding the relation-ship between cancer and overall patient health. To address thisquestion, we examined tissues distant to sites of implanted tumorsfor genomic damage using cohorts of C57BL/6 and BALB/cmicewithearly-stage subcutaneous syngeneic grafts, specifically, B16 mela-noma, MO5076 sarcoma, and COLON26 carcinoma. Here we reportthat levels of two serious types of DNA damage, double-strandbreaks (DSBs) measured by γ-H2AX focus formation and oxidativelyinduced non-DSB clustered DNA lesions (OCDLs), were elevated intissues distant from the tumor site in tumor-bearing mice comparedwith their age- and sex-matched controls.Most affectedwere cryptsin thegastrointestinal tractorgansandskin, bothhighlyproliferativetissues. Further investigation revealed that, comparedwith controls,tumor-bearingmice containedelevatedamountsofactivatedmacro-phages in the distant gastrointestinal tissues, as well as elevatedserum levels of several cytokines. One of these cytokines, CCL2/MCP-1, has been linked to several inflammation-related conditionsand macrophage recruitment, and strikingly, CCL2-deficient micelacked increased levels of DSBs and OCDLs in tissues distant fromimplanted tumors. Thus, this study is unique in being a direct dem-onstration that the presence of a tumormay induce a chronic inflam-matory response in vivo, leading to increased systemic levels of DNAdamage. Importantly, these findings suggest that tumors may havemore profound effects on their hosts than heretofore expected.

tumor-induced bystander effect | oxidative DNA damage | cytokines

The appearance of genome abnormalities and loss of viability incells other than those directly hit with ionizing radiation (IR) is

a well-documented process known as the radiation-induced by-stander effect (1). Other studies reported that senescent andcancerous cells that have not been subjected to IR or other stressesalso can affect normal cells sharing the same milieu (2, 3). Thesefindings suggest that bystander effects (BE) are widespread phe-nomena in which cells undergoing any of a variety of stresses re-lease substances that induce DNA damage in bystander cells. Thebystander cells have been found to contain elevated levels ofphosphorylated histone H2AX (γ-H2AX), indicating the presenceof DNA damage, leading to increased and consequent genomicinstability and decreased viability (4, 5).Possible mediators of BE may be various inflammation-related

cytokines that have been found at elevated levels in media con-ditioned on damaged, senescent, and cancerous cells. One, TGF-β,when added to cell cultures, induces elevated levels of DNAlesions similar to those induced by the conditioned media (3).Reactive oxygen species (ROS), including nitric oxide, have alsobeen implicated in the transmission of BE (4, 6).An important question is whether such effects demonstrated in

vitro also exist in vivo. In classic radiobiology there is the so-calledabscopal (out-of field or distant) effect, where irradiation of oneorgan results in a change in another, unirradiated organ (7). Al-though possibly caused by scatter from the main radiation source,

abscopal effects may also suggest the presence of bystander-likeprocesses in whole organisms.Similar bystander-like effects have been reported in vivo for

tissues neighboring injured, damaged, or stressed tissues. Elevatedlevels of inflammatory mediators, such as chemokines, cytokines,and prostaglandins (8), as well as elevated ROS levels (9–11), havebeen found in tumors and their immediate microenvironments.DNA damage has been reported in noncancerous cells neighbor-ing tumors (12, 13), for example, in normal liver tissue adjacent tohepatocellular carcinoma (14).In this study, we examined whether the presence of a tumor may

cause systemic effects (i.e., affect tissues other than those directlyproximal). We measured the levels of γ-H2AX, which generallymarks double-strand breaks (DSBs) and eroded telomeres but alsocan be induced in the absence of DNA damage (15, 16), as well asoxidatively induced clustered DNA lesions (OCDLs) in tissuesproximal and distant from nonmetastasizing implanted tumors inmice, and compared them to the levels in age- and sex-matchedcontrols. These DNA lesions were found to be elevated in sitesdistant from the tumors, particularly in gastrointestinal tract (GIT)tissues, skin, and hair follicles. As expected, tumor-associatedmacrophages (17) were found in these mouse tumors, but in ad-dition, increased amounts of activated macrophages were found inGIT tissues and skin of tumor-bearing mice compared with con-trols. To gain insight into possible mechanisms, we analyzed over50 cytokines in the serum from tumor-bearing and control mice.One cytokine elevated in tumor-bearing mice, CCL2 [chemokine(C-C) ligand 2], was first described as a factor necessary formonocyte recruitment to sites of injury and macrophage differ-entiation, and has since been implicated in the pathogenesis ofseveral inflammatory diseases (18–20). When implanted withtumors, CCL2-deficient mice failed to exhibit increased incidencesof DNA damage, indicating that CCL2 is essential for this process.These findings are unique in demonstrating that the presence of anearly stage nonmetastatic tumor may induce a level of systemicinflammation sufficient to induce potentially serious genomicdamage in tissues distant from the tumor site.

ResultsThree tumors were passaged through syngeneic donor mice, andtumor fragments were implanted s.c. into the experimental ani-mals, B16 melanoma and M5076 reticulum sarcoma, each into 6

Author contributions: C.E.R., D.N., W.M.B., A.G.G., and O.A.S. designed research; C.E.R.,J.S.D., A.J.N., I.G.K., S.N., T.B.K., and O.A.S. performed research; C.E.R., J.S.D., A.J.N., I.G.K.,W.M.B., A.G.G., and O.A.S. analyzed data; and C.E.R., W.M.B., A.G.G., and O.A.S. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1Present address: Ozgene Pty Ltd., Bentley DC, 6983 WA, Australia.2To whom correspondence should be addressed. E-mail: sedelnio@mail.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008260107/-/DCSupplemental.

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C57BL/6 (B6) mice, and COLON26 carcinoma into 6 BALB/cmice. All fragments formed subcutaneousmasses in the test cohort(TST). Each TST cohort was compared with two sex- and age-matched control cohorts in independent experiments, a negativecontrol of six PBS-injected mice (NC), and an acute inflammationcontrol of six mice injected with 0.05 mL complete Freund’s ad-juvant (IC) (see schematic in Fig. S1). Approximately 2 wk post-injection, when the tumor masses approached 200 mg (≈7-mmlength and 5-mm width), the animals were killed. Pathology anal-ysis showed that the zones of the Freund’s adjuvant injection werecompletely healed, with no signs of remaining skin inflammation.The tissues were then analyzed by immunostaining for γ-H2AX

foci and forOCDLs (comprised of at least one abasic or base lesionand a single-strand break in close vicinity) (21) in DNA. OCDLswere measured after digestion with enzymes that cleave the DNAat modified DNA residues, human APE1, which cleaves primarilyat abasic sites, Escherichia coli EndoIII at oxypyrimidine sites, andhuman OGG1 at oxypurine sites (21). The number of sites wasquantified by analysis of constant-field gel electrophoresis (22).

Affected Tissues. In the pilot experiment, mice bearing B16 mel-anomas subjected to this protocol exhibited elevated levels ofγ-H2AX foci in duodenum (2.30 ± 1.27 foci per cell; a 2.3-foldincrease over controls) and in skin adjacent to the tumor (2.66 ±0.04 foci per cell; a 3.5-fold increase over controls) (Tables S1 andS2). Other tissues examined including ovary, lung, liver, andkidney did not exhibit significant elevations in γ-H2AX levels.To examine if the tumor-induced bystander DNA damage is

caused by oxidative stress, we measured three types of OCDLs inthe collected mouse tissues and found significant increases in theduodenum (0.33–0.36 clusters per mega-base pairs for all threetypes; 1.9-, 2.1-, and 2.8-fold increases over controls for abasic,oxypyrimidine, and oxypurine clusters, respectively) and skin sur-rounding the tumor (0.32 ± 0.04, 0.36 ± 0.06, and 0.54 ± 0.08clusters per mega-base pair, 1.6-, 1.7-, and 1.6-fold increases overcontrols for abasic, oxypyrimidine, and oxypurine clusters, re-spectively) (Tables S1 and S3). Elevated levels ofOCDLs appearedto be more widespread than DSBs in TST animals. Of the othertissues examined, ovary and lung exhibited significant increases inall three or two of the three types of OCDLs, respectively.GIT and skin contain larger fractions of proliferating cells than

the other examined tissues (23, 24), suggesting that S-phase cellsmay be more sensitive to DSB formation as reported in vitro forbystander DSB formation induced by IR (25, 26). Because DSBsmay result if replication complexes encounter blocks resultingfrom non-DSB damage (15), greater numbers of OCDLs mayhave been converted to DSBs in duodenum and skin.The oxypurine and oxyprymidine residues in OCDLs are signa-

ture oxidative lesions induced by an oxidizing cellular environment.Certain types of OCDLs may be particularly challenging to repair:for example, attempted simultaneous repair of two nearby lesionson opposite strands by base excision repair. Simultaneous DNAglycosylase cleavage on opposite strands can result in the pro-duction of a DSB, enhancing the biological impact of oxidativelyinduced DNA lesions (21, 27). In addition, the GIT, containinghighly proliferative tissues, has been reported to be sensitive tooxidative stress (28, 29), a common source of non-DSB lesions.

Tumors Lead to the Induction of DNA Damage in GIT Organs and Skin.To determine how widespread this phenomenon may be, we ex-amined two other tumor models, M5076 reticulum cell sarcomaand COLON26 carcinoma. Because duodenum and skin of theB16 melanoma TST cohort exhibited elevated levels of bothγ-H2AX foci and OCDLs, we focused these experiments on theanalysis of GIT organs and regions of skin at various distancesfrom the tumor. Compared with controls, TST cohorts exhibitedelevated γ-H2AX focal frequencies in touch-print preparations ofthe four examined GIT tissues, duodenum, colon, rectum, and

stomach, 1.4- to 2.3-fold for sarcoma-bearing mice and 1.7- to 3.7-fold for carcinoma-bearingmice (Fig. 1A andE, andTables S1 andS2). A pronounced accumulation of abasic sites and oxidized baseswas found in all analyzed tissues of theGIT of the sarcoma- (1.8- to4.6-fold) and carcinoma- (1.6- to 4.1-fold) bearing mice comparedwith control cohorts (Fig. 2, and Tables S1 and S3).The GIT tissues consist of crypt structures (Fig. 1A, H&E

panel), with rates of cell turnover among the highest in the body.Intestinal crypts contain proliferating stem cells and transit-am-plifying cells in the basement layer, which then migrate to the wallsof the crypts and villi, becoming differentiated specialized celltypes (30). In touch-print preparations, γ-H2AX foci can be ac-curately counted in the cells (Fig. 1 A and E), but the origin of thecells within the tissue is unclear. In contrast, in frozen sections theorigin of the cells in the tissue is clear, but γ-H2AX foci oftencannot be accurately counted because a nucleus may be in-complete or may overlap one another (Fig. 1B). As an alternativeto counting foci, the total intensity of γ-H2AX staining was mea-sured in different areas of the colon and duodenum sections(duodenum images are presented in Fig. 1B). In the duodenum ofcarcinoma-bearing mice, the values of total γ-H2AX staining werethreefold higher at the base of the crypts, where proliferating cellsare located, than at the apexes; in the NC mice, the values wereonly 1.3-fold higher at the base compared with the top of thecrypts. The ratio of this value, 2.3, is somewhat greater than thevalue, 1.7, obtained by focus-counting in touch-prints because thenumbers of γ-H2AX foci in touch-prints are averaged over all ofthe cells, encompassing both cells with larger numbers of foci fromthe base of the crypts and cells with fewer foci from the top. Be-cause we did not use specific markers to identify stem cells, wecannot conclude if the elevated γ-H2AX staining at the base of thecrypts was stem cell-associated.In addition to the GIT cells, mouse dermal fibroblasts were

analyzed for γ-H2AX and OCDL levels. The low density of thesecells in frozen sections permitted counting of γ-H2AX foci in skinsamples proximal to the tumor and 0.5 cm distant from the tumor(in the sarcoma and carcinoma experiments), as well as 2 cm dis-tant from the tumor (in the carcinoma experiment) (Fig. 1C).γ-H2AX levels in all three locations exhibited statistically signifi-cant increases in TST mice compared with controls (1.5- to 2.5-fold) (Fig. 1 C and F and Tables S1 and S2). OCDL levels alsoincreased significantly (1.8- to 4-fold) in skin samples from all threelocations (Fig. 2 and Tables S1 and S3).Skin also contains hair follicles (Fig. S2 A and B), highly pro-

liferative miniorgans whose cells are profoundly influenced byenvironmental conditions. Elevated levels of γ-H2AX stainingwere notable in both transverse (Fig. 1D) and longitudinal (Fig.S2C) follicle sections in all three examined skin locations of TSTmice compared with controls. Measuring total intensity of γ-H2AX staining in various regions in longitudinal sections yieldedvalues 1.5-fold elevated in the base bulb region (example pre-sented in Fig. S2C), an area of high cell proliferation, in TSTmice;those of the NC mice showed no difference.Thus, this study is unique in demonstrating that the presence

of a tumor can induce DNA damage in tissues of the organismdistant from the tumor mass. In contrast to the two proliferativetissues, GIT and skin, no reproducible increases in γ-H2AX focalfrequencies were observed in splenocytes or whole-blood lym-phocytes (Table S2), both of which are composed primarily ofresting cells. This finding also supports the notion that the BE islinked to DNA replication (25, 26).

Tumor-Induced DNA Damage Is CCL2-Mediated. For damage to ap-pear in tissues distant from the site of a tumor, molecules, possiblyROS, cytokines, or cells, must migrate from the tumor to theseother tissues. We examined sections of colon, duodenum, and skinfrom the control and TST (M5076 sarcoma and COLON26 car-cinoma) cohorts of mice for the presence of several types of im-

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mune cells, F4/80+ mature macrophages, CD3+ T lymphocytes,and CD45+ B-lymphocytes. Of these three cell types, only F4/80+

macrophages were found to be elevated in the tissues from TSTanimals (Fig. 3 A and C). F4/80+ cells are present in the colon asmultifocal staining; in duodenum they are located in laminapropria; in skin they are often at the periphery of the neoplasm andin both epidermis and dermis (dermal layers are shown).We further measured the serum levels of 59 cytokines by

ELISA in the B16 melanoma and COLON26 carcinoma-bearingmice. Of these, four factors were substantially (>3.5-fold) ele-vated above control values in both cohorts, CCL2 (MCP-1,monocyte chemoattractant protein-1), CCL4 (MIP-1β, macro-phage inflammatory protein-1β), CCL7 (MCP-3), and CXCL10(IP-10, inducible protein-10) (Fig. 3D and Fig. S3).

We focused our attention on CCL2, which is associated withrheumatoid arthritis, atherosclerosis, asthma, ulcerative colitis, andmultiple sclerosis (31), and has been implicated in the progressionand prognosis of several cancers (32). It has been reported to besecreted by tumor cells, normal tissues, and immune cells (33), actsas an attractant for monocytes, lymphocytes, NK cells, and imma-ture dendritic cells to sites of injury, and is involved in macrophageactivation (20).CCL2 is amajor source of cyclooxygenase (COX)-2(18), and is involved in TGF-β up-regulation (34), both mainplayers in bystander signaling and carcinogenesis (6).Thus, we repeated our standard protocol using CCL2-deficient

mice implanted with WT M5076 reticulum cell sarcoma and B16melanoma. Strikingly, no elevation of γ-H2AX foci or OCDLs wasfound in GIT tissues in these mice, demonstrating that this process

Fig. 1. γ-H2AX foci are elevated in the GIT tissues and skin of COLON26 carcinoma-bearing mice. (A) (Left) GIT tissues consist of crypt structures (H&E-stainedparaffin sections of NC mice), which contain continually renewing cells. (Right) γ-H2AX immunostaining in touch-print preparations of the noted organs fromNC, acute IC, and TST cohorts. Images are representative maximum projections of optical sections with average numbers of γ-H2AX foci per cell. Higher-magnification images (Insets) more clearly show nuclei (red, counterstained with propidium iodide) containing γ-H2AX foci (green). (B) γ-H2AX staining infrozen duodenum sections. γ-H2AX levels are higher in the duodenum of TST mice compared with NC mice. (Insets) Crypt cells at higher magnification. (C)γ-H2AX staining in frozen sections of skin from control (NC and IC) mice compared with skin sections from tumor-bearing mice taken proximal (TST1) to thetumor mass (TST, tumor mass), about 0.5 cm (TST2) and 2 cm (TST3) from the tumor, with average numbers of γ-H2AX foci per cell. (D) Representative imagesof perpendicular hair follicle sections of NC and TST mice showing increased γ-H2AX staining in tumor-bearing mice. Graphs show the incidences of γ-H2AXfoci in the GIT (E) and skin (F). The numbers 1, 2, and 3 correspond to the skin-sample location. White bars, NC; black bars, IC; gray bars, TST cohorts. Error barsare SDs (n = 6 mice per group). *Statistically significant difference in TST mice separately compared with both NC and IC controls (P < 0.01 for GIT tissues andP < 0.05 for skin). Magnification, 400× for main panels; 200× for H&E panel.

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is dependent on the presence of the CCL2 gene in the host (Fig. 3E and F).When the CCL2 serum levels were assayed in the tumor-bearing KO mice, no measurable amounts were detected (Fig.3D), suggesting that immune cells of the host rather than tumorcells are the main CCL2 producers.

DiscussionThis report indicates that the presence of early stage nonmetastatictumormay havemorewidespread and serious consequences on thehost than previously documented. The findings demonstrate thattumors of different origin induce potentially dangerous oxidativeDNA lesions in tissues distant from the tumor site, substantiating

the existence of the tumor-induced BE in vivo. These findings alsosuggest that a source of persistent inflammation is required foraccumulation of DNA damage, because mice subjected to acuteinflammation (Freund’s adjuvant-injected) as well as PBS-injectedmice did not exhibit elevatedDNAdamage levels in distant tissues.Although the average elevation over the controls is approximatelytwofold for both OCDLs and γ-H2AX foci, some cells may harborconsiderably more damage than the average values if the processcreating the DNA damage is stochastic. Furthermore, the suscep-tibility of a cell population to genomic instability and oncogenictransformation may increase with the number of cells containingelevated DNA damage, resulting in long-term deleterious cellularand organismal outcomes.Although the elevation ofDNAdamage levels wasmost obvious

in the GIT and skin, significantly elevated levels of OCDLs werefound in several other tissue types that lacked significantly elevatedlevels of γ-H2AX foci. Thewider distribution ofOCDLs relative toγ-H2AX foci may be caused by different fractions of proliferatingcells in different tissues. DSB formation is possible in proliferatingcells where replication forks interacting with isolated oxidativelesions may form a DSB, but OCDLs form independently of thecellular replicative state. GIT and skin both contain large pro-liferative fractions, perhaps making them more sensitive for DSBformation. These results suggest that the elevated DNA damagelevels may be present in tissues throughout the organism, the levelsperhaps depending on the robustness of cellular defense processes,such as the efficiency of DNA repair, the antioxidant capacity ofthe tissues, or their extent of infiltration by immune cells. Of thetissues examined in this study, two (namely liver and kidney) didnot exhibit elevated OCDL levels in TST animals. We examinedthese two tissues and found that, in contrast to GIT and skin,kidney tissues in both control and TST animals were devoid of F4/80+ mature macrophages, a finding that strengthens the associa-tion between macrophages and OCDLs (Fig. S4A). On the otherhand, liver tissues exhibited substantial numbers of F4/80+ mac-rophages, even in control animals, and further elevated numbers intumor-bearing animals (Fig. S4A). Liver also exhibited manyfoldgreater antioxidant capacity than any of the other examined tissues(Fig. S4B), suggesting that the low levels of OCDLs in liver in thepresence of F4/80+ macrophages may be a result of ample anti-oxidant defense.There are several possible scenarios by which the presence of

a tumor may lead to elevated levels of DNA damage in distanttissues. The tumors were found to be infiltrated with maturedmacrophages and lymphocytes (Fig. 3B), which could be activatedby contact with the tumor cells to secrete a variety of factors. Inaddition, the tumor cells themselves are known to be capable ofsecreting a variety of cytokines and other factors (35), which mayalso activate resident or distant immune cells. However, the lack ofany increase in oxidative lesions in CCL2 KO tumor-bearing micesuggests that, although tumor cells are capable of generating DNAdamage-inducing signals, it is more likely that factors released bythe immune cells are responsible for the distant DNA damage.Conversely, immune cells themselves, activated in the tumor mass,may move throughout the body and secrete substances in varioustissues that induce DNA damage. Although some agents, such asROS, secreted by the tumor may be too unstable, macromoleculesand other signaling chemicals could be transported by the bloodand either induce processes in distant tissues to generate DNAdamage, or activate immune cells, perhaps already present indistant tissues for immune surveillance, which then secretechemicals inducing DNA damage. Skin and GIT tissues in thetumor-bearing mice contained elevated levels of activated mac-rophages, suggesting that these immune cells may be involved inthe local DNA damage induction in the tissues. Additionally,mucosa-associated lymphoid tissue is often highly amplified inchronically inflamed GIT (36), and may be another source of ac-tivated macrophages (Fig. 3B) and ROS.

Fig. 2. OCDLs in the GIT and skin from NC, IC, and TST cohorts of COLON26carcinoma-bearing mice. In TST mice, OCDL levels were measured in thetumor (diagonally marked), as well as in normal skin proximal to tumor(checkered), close to tumor (solid), and far from tumor (horizontallymarked). Clusters measured: (A) hAPE1 (abasic), (B) EndoIII (oxidized py-rimidine), and (C) hOGG1 (oxidized purine). Error bars are SD (n = 6) andthree to five independent measurements. *Statistically significant differencein TST mice compared with both NC and IC controls (P < 0.01).

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The skin areas examined were either adjacent to the tumor orat some distance away. In the adjacent skin samples, transport ofsignals through gap-junctions could also contribute to the in-duction of DNA damage, as suggested by the gradual decrease ofγ-H2AX foci in skin samples more distant from a tumor. Oxi-dative damage has been shown to increase in normal tissuesbordering tumors (14), and cell-cell transmission of ROS andother metabolites through gap-junctions has been suggested as

one possible signaling pathway for the radiation-induced by-stander effect in vivo (37).CCL2 up-regulation may be part of a general mechanism of

tumor response (38) and blocking CCL2 may enhance immu-notherapy (39). Our findings suggest that CCL2 may act withtumors to create a systemic inflammatory environment, whichinduces DNA damage in distant tissues throughout the organism.Because no obvious signs of acute inflammation were detected in

Fig. 3. (A) Elevated numbers of F4/80+ macrophages in colon, duodenum, and skin of M5076 sarcoma-bearing mice (TST) compared with NC mice as de-termined by immunoperoxidase staining of paraffin sections. (B) In tumor mass and mucosa-associated lymphoid tissue (MALT), F4/80 expression is oftensevere, and tumors are infiltrated with CD3+ and CD45+ cells. Nuclei are counterstained with propidium iodide. Magnification, 200×. (C) Mature/activatedmacrophage staining intensity determined by immunoperoxidase staining for F4/80 in colon, duodenum, and skin of M5076 sarcoma-bearing mice (TST, red)compared with control cohort (NC, black). F4/80 intensity grades were 1, minimal; 2, mild; 3, moderate. Each dot in each group represents data from oneanimal. F4/80+ cells in lymphoid aggregates were excluded from the analysis. Horizontal bars represent medians. (D) CCL2 levels are higher in serum of WT B6tumor-bearing mice over corresponding controls, but not in CCL2 KO mice. CCL2 levels are shown relative to the NC cohort (white). TST1, M5076 sarcoma-bearing mice (black); TST2, B16 melanoma-bearing mice (light gray). Error bars are SDs (n = 3–6). (E) The incidences of γ-H2AX foci increase in colon andduodenum of WT B6J tumor-bearing mice (TST1, TST2) compared with control mice (NC, IC), but not in CCL2 KO mice. Error bars are SDs (n = 5). *Statisticallysignificant difference in a TST mice compared with both NC and IC controls (P < 0.05). (F) OCDLs are increased in colon and duodenum of B6J WT but not ofCCL2 KO mice. Error bars are SDs (n = 5). *Statistically significant difference in TST mice compared with both NC and IC controls (P < 0.05).

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skin andGIT of tumor-bearingmice, the involvement of CCL2 andmacrophages in tumor-induced DNA damage suggests similaritieswith chronic tissue stress responses (40). In contrast to acute in-flammatory responses, the chronic response may involve a shift intissue homeostasis. Our findings link this type of chronic tissueresponse to the induction of oxidative DNA damage in normaltissues throughout the body, and provide a possible explanation forsome of the consequences of chronic inflammation, including el-evated cancer risk.

Materials and MethodsAdditional details are provided in SI Materials and Methods.

Mice and Tumors. Six-week-old C57BL/6NCr (B6) and BALB/cAnNCr (BALB/c)female mice were obtained from the Animal Production Area, NationalCancer Institute (NCI)–Frederick. Six-week-old Jax MCP-1/CCL2 KO mice onthe C57BL/6J background and WT female mice were purchased from theJackson Laboratory. Cryopreserved murine B16 melanoma (host strain: B6),M5076 reticulum cell sarcoma (host strain: B6), and COLON26 carcinoma(host strain: BALB/c) were obtained from the Division of Cancer Treatmentand Diagnosis tumor repository, NCI-Frederick, and were passaged throughsyngeneic “donor mice” before implanting into the experimental animals.Fragments of B16 melanoma and M5076 reticulum sarcoma were implanteds.c. into B6 mice, and COLON26 carcinoma was implanted into each BALB/cmice. The experimental scheme is presented in Fig. S1. In each of threeexperiments, three cohorts of mice were used: (i) TST: six animals wereimplanted with minced fragments of tumor harvested from donor mice; (ii)IC: six animals were subjected to a single s.c. injection of 0.05 mL completeFreund’s adjuvant to determine whether any response observed was be-cause of an acute inflammatory response rather than tumor growth; and (iii)NC: six animals were injected s.c. with sterile PBS. Mice were killed with CO2

when grafted tumors in the TST cohort reached a volume of ≈200 mg (early-stage nonmetastatic malignancies), and tissues were harvested for analysis.

DNA Damage Analysis. γ-H2AX immunostaining in tissue touch-print prepa-rations and frozen sections, as well as in cytospin samples of lymphocytesand splenocytes, was performed as previously described (41). The primaryrabbit polyclonal anti-γ-H2AX antibody (Novus Biologicals) and the second-ary goat anti-rabbit Alexa-488-conjugated IgG (Invitrogen) were used. Nu-clei were stained with propidium iodide. Laser scanning confocal microscopywas performed with a Nikon PCM 2000 (Nikon, Inc.).

ForOCDLdetection,DNA isolated from the tissueswas digestedwith humanrepair enzymes APE1 and OGG1, and E. coli EndoIII (New England Biolabs). Foreach enzyme-treated sample, a corresponding nonenzyme containing samplewas also run as a control following the same steps. An adaptation of constant-field gel electrophoresis was used (42). Electronic images for each gel lanewere processed using QuantiScan (BioSoft). DNA standards (λ-HindIII Digest)were used to obtain the corresponding dispersion curve with Origin 6.1 (Ori-ginLab). The numbers of average lengths (Ln) for each sample were calculatedusing the equations described in ref. 42. The frequencies of OCDLs weremeasured based on the Ln values of the enzyme-treated sample (+ lane) andthe accompanying control sample (− lane).

ACKNOWLEDGMENTS. We thank the Laboratory Animal Sciences Programand Pathology Histotechnology Laboratory staff (National Cancer Institute–Frederick) for help with animal maintenance and histological analysis andC. Richardson, P. Kalogerinis, B. Flood, and T. Santangelo (East CarolinaUniversity) for technical assistance. We thank Drs. M. Potter and G. Trinchieri(NCI) for critical reading of the manuscript. This work was funded by a Na-tional Cancer Institute Career Development Award (to C.E.R. and O.A.S.), theIntramural Research Program of the National Cancer Institute, the Center forCancer Research, the National Institutes of Health, and a Research/CreativeActivity grant and a College Research award from the Biology Departmentof East Carolina University (to A.G.G.).

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