Oncogenic bystander radiation effects in Patchedheterozygous mouse cerebellumMariateresa Mancuso*, Emanuela Pasquali†, Simona Leonardi*, Mirella Tanori*, Simonetta Rebessi*,Vincenzo Di Majo*, Simonetta Pazzaglia*, Maria Pia Toni‡, Maria Pimpinella‡, Vincenzo Covelli*, and Anna Saran*§

*Section of Toxicology and Biomedical Sciences and ‡Italian National Metrological Institute (INMRI), Biotechnologies, Agro-Industry and Health Protection,Ente per le Nuove Technologie, l’Energia e l’Ambiente (ENEA) Centro Ricerche Casaccia, 00123 Rome, Italy; and †Department of Experimental Oncology,Istituto Nazionale Tumori, 20133 Milan, Italy

Edited by Richard B. Setlow, Brookhaven National Laboratory, Upton, NY, and approved June 25, 2008 (received for review May 1, 2008)

The central dogma of radiation biology, that biological effects ofionizing radiation are a direct consequence of DNA damage occurringin irradiated cells, has been challenged by observations that genetic/epigenetic changes occur in unexposed ‘‘bystander cells’’ neighboringdirectly-hit cells, due to cell-to-cell communication or soluble factorsreleased by irradiated cells. To date, the vast majority of these effectsare described in cell-culture systems, while in vivo validation andassessment of biological consequences within an organism remainuncertain. Here, we describe the neonatal mouse cerebellum as anaccurate in vivo model to detect, quantify, and mechanistically dissectradiation-bystander responses. DNA double-strand breaks and apo-ptotic cell death were induced in bystander cerebellum in vivo.Accompanying these genetic events, we report bystander-relatedtumor induction in cerebellum of radiosensitive Patched-1 (Ptch1)heterozygous mice after x-ray exposure of the remainder of the body.We further show that genetic damage is a critical component of invivo oncogenic bystander responses, and provide evidence support-ing the role of gap-junctional intercellular communication (GJIC) intransmission of bystander signals in the central nervous system (CNS).These results represent the first proof-of-principle that bystandereffects are factual in vivo events with carcinogenic potential, andimplicate the need for re-evaluation of approaches currently used toestimate radiation-associated health risks.

cancer risk � DNA damage � in vivo � medulloblastoma � radiation

Ionizing radiation is a well known genotoxic agent and humancarcinogen that causes different short- and long-term effects (1,2). A longstanding paradigm for biological radiation effects hasbeen that radiation traversal through the cell nucleus is required forgenetic damage and biological responses. In the last two decadesthis view has been challenged by reports of radiation effects inunirradiated cells neighboring or cocultured with exposed cells(3–5). Bystander effects have been shown in single-cell systems andin more complex three-dimensional human tissue models (6, 7) forendpoints like sister chromatid exchanges (3), mutations (8), chro-mosome aberrations (9), cell transformation (10). In vivo, themouse spleen was a target of radiation-bystander DNAdamage (11).

Germ-line heterozygous inactivation of PTCH, the Sonic Hedge-hog (SHH) receptor and negative regulator of the pathway, pre-disposes to medulloblastoma, a childhood brain tumor originatingfrom neural granule cell progenitors (GCPs; ref. 12), and to tumorsin other tissues, including skin (13). Similarly, Ptch1 mutant micedevelop cerebellar tumors resembling human medulloblastoma(14). Neonatal irradiation greatly accelerates medulloblastoma, andpromotes basal cell carcinoma (BCC) precursor lesions to progressto large infiltrative BCC (15).

To prevent early mortality for medulloblastoma and improvecharacterization of the skin phenotype in irradiated Ptch1�/� mice,we have conducted experiments using expressly-designed leadshields for protecting mouse heads. Unpredictably, we observedmarked enhancement of medulloblastoma in mice irradiated withshielded brains. We therefore analyzed in shielded cerebella in-

duction of DNA double-strand breaks (DSBs) measured by�-H2AX focus formation in situ (16), and fractions of apoptoticcells. We found marked differences in magnitude and dynamics ofthese endpoints in shielded relative to exposed cerebellum, showingthat bystander-related damage is induced in remote unirradiatedtissues, and can initiate tumorigenesis in radiosensitive Ptch1�/�neural precursors. We explored the mechanisms of this phenom-enon and report significant modulation of initial GCP damage by12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein ki-nase C activator and cell-to-cell communication inhibitor (17),suggesting a mechanism mediated by gap-junctional intercellularcommunication (GJIC) for transmission of bystander damage toshielded cerebellum. These findings are the first demonstration thatbystander radiation responses can initiate tumorigenesis in unex-posed tissues in vivo.

ResultsHigh Medulloblastoma Incidence in Shielded Cerebellum of Ptch1�/�

Mice. Ptch1�/� mice (18) are highly responsive to radiation damagein neonatal cerebellum, and develop high medulloblastoma inci-dence (up to 80%) after irradiation in early postnatal age (14). Weirradiated progenies of Ptch1�/� and wild-type (WT) mice atpostnatal day 2 (P2) with 3 Gy of X rays. Mice were whole-body(WB) exposed, or irradiated with individual cylindrical lead shieldsproviding protection of heads (SH groups; Fig. 1 A and C). Cleardemarcation between exposed and shielded regions was evident atP10 due to hair-growth delay in exposed skin (Fig. 1B). Shieldingwas checked by dosimetry and Monte Carlo simulation. The dosedue to primary photons beneath the shields was �0.26%. Shieldedtissues, however, receive scattered radiation due to x-ray deflectionthrough irradiated tissues. A conservative value of the dose (atten-uated � scattered) of 1.2% of the total dose to shielded tissues wasestimated. Thus, another cohort of mice was WB exposed to 0.036Gy as internal control. Two additional cohorts were sham irradi-ated, or left untreated (CN).

Ptch1�/� mice were placed on a lifetime study and monitored fortumor development. Congruent with previous reports (14), WB-irradiated animals developed cerebellar tumors (Fig. 2 A–C). Ahigh percentage of mice (62%) died of aggressive disease by 23weeks, with median survival of 14 weeks (P � 0.0001 vs. CN, logrank test; Fig. 2D). Significantly, we also observed a remarkablyincreased medulloblastoma rate (39%) in SH-irradiated Ptch1�/�mice. Although this tumor response was lower than in WB-exposed

Author contributions: M.M. and A.S. designed research; M.M., E.P., S.L., M.T., S.P., M.P.T.,and M.P. performed research; V.C. contributed new reagents/analytic tools; M.M., S.R., andV.D.M. analyzed data; and A.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

§To whom correspondence should be addressed. E-mail: saran@casaccia.enea.it.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804186105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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animals (P � 0.0011), SH mice developed significantly more tumorswith highly reduced latency relative to CN mice (P � 0.0003; Fig.2D), showing that partial-body irradiation promotes Ptch1-driventumorigenesis in shielded cerebellum. Mice WB exposed to theestimated scatter dose (0.036 Gy) were, at the time this manuscriptwas written, well into the age (i.e., 31 weeks) when radiationtumorigenic effects become manifest in cerebellum. Yet, they hadnot shown signs of disease above background level (Fig. 2D).

The SH-irradiated Ptch1�/� mouse model is a highly reproduc-ible system, because the tumor results described here are consistentwith data from two independent experiments involving a total of 85mice, in which we used a simplified shielding method [supportinginformation (SI) Fig. S1].

Ptch1 Loss of Heterozygosity (LOH) in Tumors from WB or SH IrradiatedMice. Biallelic Ptch1 loss represents the major pathway to medul-loblastoma development in Ptch1�/� mice (14, 19). We analyzedtumors from irradiated and shielded mice for Ptch1 allelic imbal-ance by exploiting a T/C polymorphism at position 4016 of Ptch1,that discriminates the 129Sv-derived mutant from the CD1-derivedWT allele (14). We show that, similar to WB-irradiated Ptch1�/�mice, medulloblastomas from SH–exposed mice show loss of theWT-CD1 allele (Fig. 2 E and F).

To assess LOH mechanisms, panels of medulloblastomas fromSH or WB mice were analyzed using a minimum of 22 informativemicrosatellite markers spanning mouse chr 13 (Fig. 2G). None ofthe tumors had loss of the entire chr 13, because we never found

reduction to homozigosity of all markers analyzed. Instead, allmedulloblastomas had a chr-13 interstitial region in which severalcontiguous loci showed LOH. Overall, the pattern lacked highlystringent breakpoint clustering, supporting a genetic mechanismbased on loss of a critical gene within the chr-13 minimal deletedregion where we localized Ptch1 according to the Mouse Chromo-some 13 Linkage Map (http://www.informatics.jax.org/). Hence, ourresults define very similar LOH patterns in tumors from SH- andWB-exposed mice (Fig. 2G).

Induction of DSBs in Irradiated and Bystander EGL. Neural precursorcells are highly radiation sensitive, both in vitro and in vivo (20). Wecharacterized early responses to DNA damage in GCPs post-3Gyirradiation. DSBs were analyzed in irradiated and shielded cere-bellum by detection of �-H2AX foci that are formed at chromo-somal sites of DNA DSBs by phosphorylation of histone H2AX onser 139, and are evident in the nucleus soon after irradiation usingspecific antibodies (16). At P2, proliferating GCPs cluster over thesurface of the developing cerebellum to form the external granulelayer (EGL; see Fig. 4 A and I). Intense �-H2AX staining, involving�85% of GCPs, was present in the EGL at 0.5 h post-3Gy WBirradiation (Fig. 3A). In contrast, no �-H2AX staining was detectedin cerebellum of lead-shielded mice (Fig. 3B). �-H2AX stainingdeclined in irradiated EGL at later times (3, 6, and 18 h: 5.2, 2.9, and2.5%), while remaining very low or undetectable in shielded EGL(0.05, 0.15, and 0%).

Apoptosis in Irradiated and Bystander EGL. We examined apoptosislevels in irradiated and shielded EGL of the neonatal cerebellum bycounting apoptotic nuclei in the anterodorsal cardinal lobe (Fig. 4I)

Fig. 1. Irradiation set up for shielded irradiation. (A) Neonatal Ptch1�/� andPtch1�/� mice placed in polystyrene boxes were irradiated with heads and upperbody shielded by individual custom-built lead cylinders. (B) Demarcation be-tween exposed and shielded regions at P10 due to hair-growth delay in exposedskin. (C) Characteristics of the lead shields.

Fig. 2. Medulloblastoma development in Ptch1�/�

mice. (A) Normal brain. (B) Macroscopic aspect of cere-bellar tumors developing in the posterior fossa. (C) His-tology of medulloblastoma, composed of tightly packed,small round cells with minimal surrounding cytoplasm.(D) Kaplan–Meier kinetic analysis of medulloblastoma inwhole-body irradiated (WB), shield-irradiated (SH), andcontrol Ptch1�/� mice (CN). Included is the tumor-freesurvivalat31weeksofmicethatwereWBirradiatedwitha dose of 0.036 Gy (WB-0.036), equivalent to the scatterdose to SH cerebellum. (E and F) Representative electro-pherogram of genomic and tumor DNAs showing reten-tion of both Ptch1 alleles in normal tissue (E) and loss ofWT Ptch1 allele in medulloblastomas (F). (G) Analysis ofchr-13 LOH in tumors from WB and SH Ptch1�/� mice. Thedistanceofmicrosatellitemarkers (D13Mit) fromthecen-tromere is given in cM. Closed circles indicate no LOH;open circles denote LOH; gray circles indicate not infor-mative (NI) markers; mdr: minimum deleted region.

A B

Fig. 3. �-H2AXimmunohistochemical stainingoftheEGLofP2cerebellumafterirradiation. (A) A large fraction (� 85%) of GCPs exhibited numerous nuclear fociof�-H2AXat0.5hpost-3Gywhole-body(WB) irradiation. (B)Total lackof�-H2AXstaining in the EGL of shielded mice (SH) at 0.5 h postirradiation. (Scale bars, 20�m.)

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at different times postirradiation (Fig. 4 A–H and J). The extent andkinetics of apoptosis differed in exposed and bystander cerebellum,with substantial levels (20.3%) evident in the EGL of WB-exposedmice at 3 h postirradiation (Fig. 4 A and J), compared with onlyincidental apoptosis (0.25%) in SH mice (Fig. 4 B and J). At 6 h,however, maximal apoptosis was present in the EGL of WB- (40%)and SH-exposed mice (7.1%) (Fig. 4 C, D, and J). Analysis ofcaspase-3 activation was consistent with apoptotic cell counts (Fig.4 A–F). Increases in apoptosis were followed by marked decreasein cellularity and thinning of the EGL (Fig. 4 E and F). A sharpdecrease to 1% of apoptotic cells was observed in the EGL of SHmice at 18 h (Fig. 4 F and J), in contrast with markedly slower

kinetics of disappearance in WB-exposed mice, in which apoptoticneural progenitors decreased out at 48 h (0.26%) (Fig. 4 G and J).Consistently, cell repopulation and return to normal EGL thicknesswas slower and still incomplete in WB- vs. SH-irradiated mice at48 h (Fig. 4 G and H). No significant apoptosis was induced in theEGL post-0.036Gy (Table S1), or in sham-irradiated mice (data notshown) at all times.

Notably, although shielded cerebella received a scatter dose thatwas estimated to be as much as two orders of magnitude lowerrelative to unshielded cerebellum (0.036 vs. 3 Gy), only 5.5-foldlower apoptosis was detected at 6 h postirradiation in cerebella ofSH mice (Fig. 4 C, D, and J). This suggests a significant bystandercomponent of damage to shielded brains.

Radiation Damage by Expected Scatter Dose in Exposed vs. BystanderEGL. The obvious disparity, at tissue and whole-animal levels,between biological effects of the estimated scatter dose (i.e., 0.036Gy) from 3 Gy to the whole body and the effects of the same doseto shielded cerebellum, prompted us to confirm that the geneticdamage to cerebellum suffered by shielded mice did not result frominsufficient shielding or dose scatter. To test this hypothesis, and tomaximize downstream bystander responses, we compared themagnitude and kinetics of DNA DSBs and apoptosis in mice thathad been SH-irradiated with 8.3 Gy (SH-8.3Gy), or WB exposed tothe estimated 0.1 Gy scatter dose to cerebellum (WB-0.1Gy). Basedon the delayed apoptotic wave in SH cerebellum (Fig. 4 D and J),analyses were carried out starting from 3h postirradiation. Lackingadditional indirect effects, shielded EGL receiving 0.1 Gy throughdose scatter should theoretically suffer biological damage similar tothe EGL of mice receiving 0.1 Gy to the total body. �-H2AX focusformation was very low or undetectable in cerebellum directlyexposed to 0.1 Gy (Fig. 5 B and C). Instead, highly significantinduction of �-H2AX foci occurred in SH cerebellum at 4.5 (P �0.0015) and 6 h (P � 0.0139) after exposure to 8.3 Gy of theremainder of the body (Fig. 5 A and C), with decrease to controllevel at 18 h. Apoptosis was also significantly up-regulated incerebella of SH-8.3Gy mice (Fig. 5 D and F). A statisticallysignificant response in SH relative to 0.1-Gy exposed EGL wasalready evident at 4.5 h (P � 0.0001), with a 3.2-fold bystander-related enhancement. Apoptosis was further increased at 6 h in theEGL of SH-8.3Gy mice, reaching 7-fold over the scatter-dose group(P � 0.0001), and declined at 18 h postirradiation (Fig. 5F). Thus,significant bystander genotoxic responses occur in shielded cere-bellum in vivo after irradiation of the remainder of the body.

Significantly, short-term cellular responses were not specific ofradiosensitive Ptch1�/� mice, because WT siblings showed identicalbystander phenomena in neural precursors of P2 cerebellum (datanot shown). To ascertain that effects were not dependent on geneticbackground hosting the Ptch1 mutation, the SH-8.3Gy/WB-0.1Gyexperiment was replicated using the unrelated, carcinogen-resistantCar-R mouse strain (see SI Methods). Both DNA damage andapoptotic cell death in shielded EGL revealed similar magnitude ofbystander-related enhancements in cerebellum of SH-8.3Gy rela-tive to WB-0.1Gy Car-R mice (data not shown).

In all assays, we observed a non-random distribution of damagedcells in SH-exposed cerebellum, with �-H2AX-positive and apo-ptotic nuclei located preferentially in the highly-proliferative outercompartment of the EGL (Fig. 4 A, C, and D and Fig. 5 A and D),suggesting proliferation-dependent bystander effects. These resultsare consistent with published studies showing spatial dependence ofthe bystander effects (21).

Inhibiting Cell–Cell Communication via Gap Junctions Reverses By-stander Effects. If GJICs are essential in mediating bystanderresponses, it should be possible to reduce transmission of damagefrom directly hit to unexposed tissues by using inhibitors of GJIC.Thus, we examined whether modulation of GJICs can be elicited byTPA in neural precursors of P2 cerebellum, and whether this may

Fig. 4. Levels and kinetics of apoptosis in exposed and shielded P2 mousecerebellum. (A–J) Quantification of apoptosis in the anterodorsal cardinal lobe(boxed in I). (A–D Apoptosis at 3 and 6 h postirradiation in the EGL of WB- relativeto SH-exposed mice. (A–F, Insets) Immunohistochemical analysis of caspase-3activation. (E and G) Thinning of the EGL in WB-exposed and to lower degree inSH-exposed mice (F). Cell repopulation with mitotic figures in WB-exposed miceat 48 h (G). Compensatory hyperplasia at 48 h in SH-exposed EGL (F and H). Blackdashes in A and I delineate the EGL. (J) Percentage of apoptotic cells as a functionof time postirradiation in WB- and SH-exposed mice.

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prevent bystander responses in shielded cerebellum. Expression ofconnexin43 protein (Cx43), the most abundant gap junction proteinin the CNS (22), was evident in cerebellum post-8.3Gy SH irradi-ation (Fig. 6A). By contrast, Cx43 was almost undetectable in theEGL upon TPA treatment of SH-8.3Gy irradiated mice (Fig. 6B).For comparison, TPA influence on Cx43 expression is shown in thesubventricular zone (SVZ) of neonatal cerebellum (Fig. 6 C and D).In the EGL, protein blotting confirmed a 2.6-fold decrease of Cx43expression (P � 0.0025; Fig. 6 E and F). Remarkably, TPAtreatment abrogated DNA-DSB responses in the EGL post-8.3GySH irradiation. Moreover, apoptotic damage was reduced by 3.3-fold (Fig. 6G). This suggests that physiologic Cx43 expression isassociated with communication of damaging signals between ad-jacent cells in CNS. As a control of TPA effects on irradiatedtissues, we tested its influence on cerebellum post-3Gy-WB irradi-ation and found a TPA-related 1.2-fold reduction (39.6 vs. 31.1%)in apoptosis (Fig. 6H and Table S2). Thus, �8% of directly-hitGCPs were ‘‘protected’’ by TPA, a value surprisingly similar to the7.1% apoptosis in the SH-3Gy group (Fig. 6J). This may imply thatTPA is responsible for inhibition of the bystander component ofdamage in cerebellum of WB-irradiated mice, and that this com-ponent may account for �20% of total apoptotic damage.

Effect of Cyclooxygenase-2 (COX-2) Inhibitor on Bystander Damage.Non-irradiated bystander cells overexpress the COX-2 gene (23), adownstream target of mitogen-activated protein kinase (MAPK)pathways involved in radiation responses and linked to bystanderprocesses (24). We therefore assessed responses in shielded cere-bellum using a specific COX-2 inhibitor (25), nimesulide, combinedwith SH-8.3Gy. We show that nimesulide (1.5 �g/g body weight)caused a non-statistical reduction in DSBs, and had no effect onlevels of apoptotic damage in shielded cerebellum. Similarly, nosignificant modulation of apoptosis by nimesulide was detected incerebellum post-3Gy WB irradiation (Fig. 6H).

DiscussionIt is clear that the array of non-targeted radiation effects, e.g., sisterchromatid exchanges (3), DNA DSBs (10), micronucleus formation(9), mutations (8), chromosomal aberrations and instability (5, 7),reported to date mostly from in vitro systems, may have implicationsin cancer development. However, evidence that radiation-associated bystander responses are effectual in vivo has been limited(26), and there has been no proof that non-targeted radiationeffects contribute to carcinogenesis in bystander tissues. Here, weshow that genetic damage caused by bystander responses contrib-

Fig. 5. Radiation damage by expected scat-ter dose in exposed vs. bystander EGL. (A andB) �-H2AX positivity in the outer EGL of SH-8.3 Gy mice at 6h postirradiation comparedwith undetectable staining after exposure tothe scatter dose (0.1 Gy). (D and E) Increasedapoptosis in EGL of SH-8.3Gy mice at 6 hpostirradiationcomparedwithvery rareapo-ptosis after a 0.1 Gy dose. (C and F) Percent-age of �-H2AX-positive and apoptotic cells incerebellum at 3, 4.5, 6, and 18 h post-8.3-Gy(SH) and 0.1 Gy (WB) irradiation. *, P �0.0139; **, P � 0.0015; ***, P � 0.0001. (Scalebars, 20 �m.)

Fig. 6. Inhibition of GJICs reverses bystander effects incerebellum. (A) Immunohistochemical analysis showingwidespread expression of Cx43 protein in the EGL at 6hpost-8.3Gy SH irradiation. Arrows: Cx43 expression atcell–cell contacts. (B) LackofCx43positivity incerebellumofmicereceivingcombinedTPAandSH-8.3Gytreatment.(C) Strong Cx43 expression in the postnatal SVZ, and (D)reduced expression in the SVZ of SH-8.3Gy mice injectedwith TPA are shown for comparison. (E and F) Westernblot analysis showing decrease of Cx43 expression inTPA-treated relative to -untreated mice (P � 0.0025). (G)TPA (gray columns) dramatically reduced the levels of�-H2AX-positive (P � 0.0075) and apoptotic cells (P �0.0001) inshieldedcerebellum;notenumerousapoptoticfigures in A, compared with sporadic apoptosis (whitearrowhead) in B. No significant effect of nimesulide(Nms; white columns) administration on levels of short-term cellular responses. (H) TPA, but not Nms, signifi-cantly reduced apoptosis in cerebellum directly exposedto 3 Gy.

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utes to cancer risk in mouse CNS, with drastic acceleration ofmedulloblastoma in Ptch1�/� mice irradiated with shielded brains.

In the developing brain, radiosensitivity is highly dependent ondevelopmental stage, being higher in neural precursors comparedwith postmitotic neurons (20). In proliferating GCPs of P2 cere-bellum we have analyzed induction of �-H2AX foci. Whereasmassive DSB damage was present in directly-exposed EGL within0.5 h postirradiation, �-H2AX-positive cells were rare in shieldedcerebellum of mice exposed to 3 Gy. The finding of a highlyincreased tumor response in shielded tissue, despite low levels of�-H2AX induction, suggests that a persisting low level of DNAdamage in CNS after low radiation doses (�0.04 Gy) may facilitatechromosomal events relevant for carcinogenesis. This hypothesis isconsistent with findings that low levels of DNA damage (�10–20DSBs) fail to initiate the G2/M checkpoint in human fibroblasts(27), allowing genetically altered cells to proliferate. In addition,slower kinetics of disappearance of DSBs in neural progenitorsrelative to neurons (20) may contribute to a persistent low level ofunrepaired DSBs. As evidence of persisting damage, a steepincrease in apoptosis was detected at 6 h in the EGL of shieldedmice relative to internal controls (WB-0.036) or sham-irradiatedmice. Apoptotic cell death might be considered protective, becauseit selectively eliminates damaged cells that may contribute tocarcinogenic effects. However, cell death significantly stimulatedcompensatory hyperplasia in the EGL, which is potentially atumor-promoting factor in the presence of residual DNA damage.

Previous work (28, 29) has stressed the importance of DNA-DSBrepair in bystander phenomena, and the possibility that unrepairedor misrepaired DSBs underlie bystander induction of chromosomalaberrations and mutations involving large-scale genetic changes. Inthe Ptch1 model, time-course studies suggest a steadily increase ofPtch1-LOH rate in early preneoplastic cerebellar lesions, support-ing a critical role for biallelic Ptch1 loss in cerebellum tumorigenesis.Moreover, different Ptch1 LOH patterns were shown in radiation-induced and spontaneous medulloblastomas (14). Here, we reporta clear and consistent pattern of chr-13 interstitial deletions intumors from bystander cerebellum, resembling tumors from whole-body-exposed mice. Mechanistically, this suggests that bystanderradiation damage inherently has the potential to induce alterationand loss of genetic material and, in the CNS, promote tumorigenesisby facilitating LOH. Hence, genetic damage is a critical componentof in vivo oncogenic bystander responses. Although there are noknown reports of bystander-related tumorigenesis, long-term epi-genetic alterations have recently been described in mouse bystanderskin and spleen after partial-body irradiation (11, 30). Thus, thenature of long-term bystander responses may be tissue specific.

We observed spatial effects in the distribution of damaged cellsin bystander mouse cerebellum, with damage located preferentiallyin the outer compartment of the EGL. This could be explained byrapid GCP proliferation in that region. Interestingly, studies ofbystander effects in primary urothelial explants after 3He2� tra-versal of single cells revealed non-targeted damage at the prolif-erating periphery of explants, regardless of location of targeted cells(21). This suggested that only actively dividing cells were susceptibleto bystander effects. Our in vivo results in mouse CNS confirm thatthe proliferative or differentiation state is important for expressionof bystander damage.

We also observed a dose-response effect for bystander damage,because the magnitude of both endpoints analyzed increased aboutproportionally with the dose delivered to shielded animals. Previousin vitro studies involving different techniques and radiation typeshave shown that the bystander effect is an all-or-nothing processwith no apparent dose-effect relationship (31, 32). However, pro-duction of transmissible cell-to-cell effects can be substantiallydifferent in an in vivo context, in which physiologic cellular con-nections within a tissue, or cross talk among tissues, allow gateddiffusion of bystander signals. This type of intercellular communi-cation permits coordinated responses to cellular injury, a critical

feature for homeostasis during development and adult life ofmulticellular organisms.

One of the major challenges in the field is to understand themechanisms of non-targeted effects. In vitro studies have shownthat, in confluent cultures, physical contacts through GJICsbetween irradiated and non-irradiated cells are essential for theprocess (33, 34). However, in low-density cultures bystandereffects may rely on soluble factors released in the culturemedium by irradiated cells (35). In vivo, evidence that acuteradiation exposures result in the release of clastogenic solublefactors into the blood stream comes from several observations(36). For instance, clastogenic activity in human blood has beenfound after therapeutic or accidental exposure to high radiationdoses. The results described here strongly support the view thata Cx43-mediated gap-junction transfer of the bystander signal invivo mediates short-term cellular responses that may subse-quently trigger long-term carcinogenic effects in mouse CNS. Infact, by TPA-mediated inhibition of GJICs we have shownsuppression of bystander responses for both endpoints analyzed.As TPA, however, may have effects other than inhibition ofCx43-mediated intercellular communication, further studiestesting different GJIC inhibitors will be needed to assess thegenerality of the response. However, suppression of COX-2activity in bystander cerebellum by the chemical inhibitor nime-sulide did not influence bystander damage, suggesting thatinflammation/oxidative stress is not central in the effects ob-served here. GJICs and their constituent connexin proteins areknown to play a significant role in mediating radiation-inducedbystander effects (33). Because no structure is more intercon-nected than the mammalian CNS, it is conceivable that Cx43 gapjunctions are crucial in mediating bystander responses in mouseCNS in vivo. Although the nature of factor(s) transmittedthrough GJICs and connecting irradiated and bystander neuralcells remains unknown, only molecules �1 kDa (i.e., ions, smallsignaling molecules, some endogenous metabolites) could bepossible candidates (37).

The similarity of initial cellular responses in shielded cerebel-lum of different mouse strains demonstrates that bystandereffects occurring in CNS by exposure of distant tissues are notspecific of Ptch1-mutant mice, and represent a cross-strainphenomenon. Thus, our findings are potentially important toestimate bystander signals in other species, and to assess theirrelevance for human health.

Finally, whereas short-term bystander responses were reproduc-ible in different WT strains, longer-term carcinogenic effects incerebellum were specific of the Ptch1�/� genotype. The PTCHmutation underlies a recessive human syndrome characterized bycancer susceptibility and high radiosensitivity (13, 18). However,frequencies of PTCH mutant alleles within human populations arelow, and the contribution that such extreme sensitivity makes topopulation risk is very small. Greater contribution to radiation risksis probably due to genes that are less penetrant but more frequent,causing individual variability in biological responses to radiation,and increased risk in susceptible individuals. If ongoing and futurestudies confirm the tumorigenic role of bystander effects, this willhave significant impact on future assessment of cancer risks asso-ciated with occupational, diagnostic and environmental exposuresto radiation.

Materials and MethodsImmunohistochemistry, Statistics, and Mice. See SI Methods.

Animal Irradiation. Ptch1�/� mice and WT siblings were irradiated with X rays atP2. Full methods can be found in SI Methods.

For tumor induction, Ptch1�/� mice (n � 37) were whole-body (WB) irradiatedwith 3 Gy. Control mice (n � 51) were left untreated. For shielded irradiation (SH),Ptch1�/� mice (n � 46) were exposed to 3 Gy with heads protected by individualcustom-built lead cylinders that allowed exposing only the lower part of the

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body. A group of shielded mice (n � 34) was sham-irradiated at P2. To analyzeshort-term cellular responses, additional groups (3 per time point) were killed at0.5, 3, 6, 18, and 48 h postirradiation.

Protection of shielded parts was verified by dosimetry and Monte Carlosimulation(SIMethodsandFig.S2).Thedoseduetoprimaryphotonsbeneaththeshields was experimentally found to be �0.26%. In addition, shielded tissuesreceive scattered radiation due to x-ray deflection through the irradiated tissues.A conservative value of the total dose (attenuated � scattered) delivered to theshielded tissues of 1.2% of the total dose was estimated. Thus, another cohort of25 mice was WB- exposed to 0.036 Gy (i.e., the estimated scattered and attenu-ateddosetoshieldedcerebellumfromirradiationofthebodywith3Gy)atP2andused as internal control.

For characterization of DNA-damage responses in the WB and SH settings,Ptch1�/�, Ptch1�/�, and Car-R mice for comparative purposes (n � 3 for eachgenotype/strain), were exposed at P2 to 8.3 Gy of X rays with shielded heads.Corresponding groups were WB-exposed to 0.1 Gy (the approximate scatter doseto SH cerebellum resulting from exposure of the body to 8.3 Gy) and used asinternal controls. Mice were killed at 3, 4.5, 6, and 18 h after irradiation fordetection of �-H2AX foci and apoptosis. Mouse experimental procedures werecarried out according to the Italian legislation on animal experimentation, andreviewed by the internal Institutional Animal Care and Use Committee.

Histological Analysis and Tumor Quantification. The 3 Gy WB- and SH-exposedcohorts were observed daily for their lifetime. When moribund, mice were killedand autopsied. Brains were processed for histology by standard techniques.Macroscopic cerebellar tumors were partly frozen at �80°C.

LOH at the Ptch1 Locus. DNA was extracted from tumors and normal tissue usingWizard SV Genomic DNA Purification System (Promega). LOH analysis was per-formed as described in SI Methods.

Detection and Counting of �-H2AX. To study kinetics of �-H2AX formation inGCPs from WB- and SH-irradiated mice, we counted �-H2AX-positive nucleiin the entire EGL from midsagittal sections of P2 cerebellum (n � 3 per timepoint) at 3, 4.5 and 6 h after exposure to 3 or 8.3 Gy. Full methods can befound in SI Methods.

Apoptosis. Brains (n � 3 per time point) of Ptch1�/�, Ptch1�/�, or Car-R pupsirradiated with 3 or 8.3 Gy at P2 in the WB or SH settings were collected and fixedat 0.5, 3, 6, 18, 48 and 72 h postirradiation. Quantification of apoptosis wasperformed as described in SI Methods.

Modulation of GJICs by TPA. Mice (n � 6 per group) were injected i.p. with TPAdissolved in corn oil (200 ng/g body weight, 2-h intervals), or vehicle, at 0.5 hbefore, and up to 4.5 h post-8.3Gy SH irradiation (38). Mice showed no signs ofacute toxicity. Sacrifice was at 6 h postirradiation, and brains were fixed. Forimmunoblot analysis, cerebellum was dissected and snap frozen.

Suppression of COX-2 Activity by Nimesulide. Mice (n � 3 per group) weretreated with i.p. injections of nimesulide dissolved in water (1.5 mg/kg), or vehicle1 h before 8.3-Gy SH irradiation and killed at 6 h postirradiation.

Western Blot. Protein concentrations were determined by Bradford assay(Bio-Rad). Proteins (15 �g) from 3 cerebella were pooled for each lane andseparated on 12% SDS/PAGE. Antibodies included rabbit polyclonal antibodyagainst Cx43 (Cell Signaling Technology Inc.), 1:1000, detecting endogenouslevels of total Cx43, and monoclonal antibody against �-actin (Sigma–Aldrich),1:4000.

ACKNOWLEDGMENTS. We thank Orsio Allegrucci and Maurizio Quini for tech-nical assistance. This work was supported by European Union Contract FI6R-CT-2003-508842 RISC-RAD.

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12450 � www.pnas.org�cgi�doi�10.1073�pnas.0804186105 Mancuso et al.

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