Increased Artemis levels confer radioresistance to both high and low LET radiation exposures

12 Pages·2012·1.33 MB·English

Sridharanetal.RadiationOncology2012,7:96
http://www.ro-journal.com/content/7/1/96
RESEARCH Open Access
Increased Artemis levels confer radioresistance to
both high and low LET radiation exposures
Deepa M Sridharan1, Mary K Whalen1, Donna Almendrala1, Francis A Cucinotta2, Misako Kawahara1,
Steven M Yannone1 and Janice M Pluth1*
Abstract
Background: Artemis has a defined rolein V(D)J recombination and has been implicated in the repair ofradiation
induced double-strand breaks. However theexact function(s) ofArtemis in DNA repair and its preferred substrate(s)
in vivo remain undefined. Our previous work suggests that Artemis is importantfor the repair ofcomplex DNA
damage like that inflicted by high Linear Energy Transfer (LET) radiation. Toestablish the contribution of Artemis in
repairing DNA damage causedby various radiation qualities, weevaluatedthe effect ofover-expressing Artemis on
cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation.
Results: OurdatarevealthatArtemisover-expressionconfersmarkedradioprotectionagainstbothtypesofradiation,
althoughtheradioprotectiveeffectwasgreaterfollowinghighLETradiation.Inhibitorstudiesrevealthatthe
radioprotectionimpartedbyArtemisisprimarilydependentonDNA-PKactivity,andtoalesserextentonATMkinase
activity.Together,thesedatasuggestaDNA-PKdependentroleforArtemisintherepairofcomplexDNAdamage.
Conclusions:ThesefindingsindicatethatArtemislevelssignificantlyinfluenceradiationtoxicityinhumancellsand
suggestthatArtemisinhibitioncouldbeapracticaltargetforadjuvantcancertherapies.
Keywords:Artemis,Radioresistance,HighLETradiation
Background enzyme in V(D)J recombination, however its exact role in
Radiation therapy is one of the most broadly prescribed radioresistancehasbeenmoredifficulttodefine.Artemisis
treatments for malignancy. However, the reasons for the thought to function in non-homologous end joining
wide inter-individual variability in response to radiation (NHEJ), the primary repair pathway for double-strand
therapy are not apparent. Identifying factors that predict breaks (DSBs) in G1 phase of the cell cycle. Artemis has
resistance to therapy is an area of intense research that beenimplicatedasplayingaroleinthispathwaybecauseof
holds promise to facilitate personalized treatments and theradiosensitivityassociatedwithArtemisdefectsandthe
improve outcomes. Here we have identified elevated Ar- functional and physical interactions between Artemis and
temis levels asbeing a powerful determinant of radiation the DNA-dependent protein kinase (DNA-PK). DNA-PK
resistanceinhuman cells. plays a central role in NHEJ repair, phosphorylating itself
Defects in the Artemis gene were found to be causal in andotherproteinsduringtherepairprocessandactingasa
radiosensitive severe combined immunodeficiency disor- scaffold for repair protein assembly on DNA ends [3]. In
ders in a subset of Japanese, European, and Native Ameri- the presence of DNA-PK, Artemis acquires a DNA endo-
can SCID patients [1,2]. Individuals lacking functional nuclease activity which is important for its role in hairpin
Artemis are extremely sensitive to radiation and cannot processinginV(D)Jrecombinationandperhapskeyinpro-
generate mature B or Tcells due to incomplete V(D)J re- cessingspecificradiation-inducedsubstratesinNHEJ[4-7].
combination which results in immunodeficiency [1]. Arte- Studies have shown that DNA-PKcs recruits Artemis to
mis has been established as an essential hairpin-cleavage DSBs within the chromatin [8,9], aligns the ends of the
DSB to enable Artemis-mediated processing [4], and that
*Correspondence:[email protected] togethertheycancleaveblockedDNAtermini[10,11].
1LifeSciencesDivision,LawrenceBerkeleyNationalLaboratory,Berkeley,CA
A model has been proposed based on accumulated
94720,USA
Fulllistofauthorinformationisavailableattheendofthearticle data that Artemis phosphorylation is not required for its
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endonuclease repair function, but rather DNA-PKcs we used an extensively characterized human expression
autophosphorylation is, and that the latter modification embryonic kidney cell line. The wild type line, T-Rex-
allows conformational changes in the DNA that are ne- 293 (hereafter, HEK293), expresses native levels of Arte-
cessary for Artemis’ intra-stand incision capability [4]. mis and the derivative cell line carries a stably integrated
ATM, DNA-PKcs and ATR (ATM related) have been doxycycline-inducible Artemis cDNA (hereafter, 293Art)
shown to phosphorylate Artemis in vitro, evidence sug- [16]whichcanbeinducedtoexpresshighlevelsofArte-
gests ATM is the primary in vivo kinase phosphorylating mis. The T-Rex-293 cell line was chosen for this work
Artemis following ionizing radiation [12-17], however due to its ease of transfection, however our studies have
other studies reveal Artemis activity is entirely shown survival responses after radiation treatments
dependent on its interaction with DNA-PK [9]. Phos- comparable to primary fibroblasts when measured by a
phorylation of Artemis by DNA-PKcs or ATM is not standard clonal survival assay and the BrdU proliferation
required for the endonuclease activity of Artemis, and assay (unpublished data). Artemis protein levels were
hasbeenshowntobeuncoupledfromitsrepairfunction evaluated twenty-four hours after doxycycline treatment
[4], although it does influence cell cycle progression by Western blots of whole cell extracts. Artemis protein
[12]. Studies suggest that phosphorylation of Artemis by levels in HEK293 were very low, to non-detectable,
ATM is a negative regulator of G2/M checkpoint, and whereas 293Art extracts contained high levels of Arte-
that upon repair Artemis is de-phosphorylated [8,12,16]. mis protein, visualized as an intense band migrating at
DespitetheroleofArtemisphosphorylationoncellcycle ~100 kDa on Western blots (Figure 1A inset). Cells sta-
progression, there does not appear to be a clear indica- bly retain our inducible Artemis expression plasmid and
tion thatphosphorylationiskeytoitsrepair function. tolerate high levels of induced Artemis expression with
An increased sensitivity to agents that induce more no detectable changes in growth, unlike what has been
complex damage as compared to simple DSB inducing reported for constructs constitutively producing high
agents suggested an important role for Artemis in the re- Artemis levels [18]. Artemis protein levels were similarly
pair of complex double strand breaks [10,13]. Previously validatedforallsubsequent experiments.
we had shown Artemis defective cells exhibit elevated Using these HEK293 and 293Art cell lines we interro-
radiosensitivity, long-term unresolved damage demon- gated the role of Artemis in cellular survival after expos-
strated by persistent G1 and G2/M arrest, and a subtle ure to various radiation qualities. Cellular toxicity has
DSBrepairdefect,exhibiting~2foldmoreγH2AXfocias historically been measured with colony formation assays,
compared to controls [16]. These results are consistent where cells capable of continued proliferation form col-
with earlier findings showing that γH2AX levels can re- onies that are then stained and counted. To test the ef-
main elevated up to 14 days following alpha particle radi- fect of Artemis over-expression on radiation sensitivity
ation exposure in Artemis defective-cells [13]. Artemis in human cells we first measured how radiation affected
function in cellular response to radiation damage is evi- survival in HEK293 versus 293Art cell lines after X-ray
dentinthehypersensitivitytoradiationandpersistentcell exposureusing the clonogenic survivalassay. Clonal sur-
cyclearrestfollowingradiationdamageincellslackingAr- vival data indicate that cells having high levels of Arte-
temis[1,16].TogaininsightintothefunctionsofArtemis mis are significantly more resistant to X-rays than the
in cell survival after radiation exposure, we exposed parental cell line having low levels of Artemis
human cells over-expressingArtemis todifferent qualities (Figure 1A). To further investigate this result, the same
of radiation. Our data reveal that elevated Artemis levels celllineswereassayed forX-raytoxicityusingadifferent
provide a distinct survival advantage following exposure assay. This assay uses BrdU incorporation into the gen-
toionizingradiation(IR),andthatthiseffectismorepro- ome during replication to measure cellular proliferation
nounced following high LET exposure as compared to rather than colony formation. Cells are incubated with
lowLET,consistentwithanimportantroleforArtemisin BrdU 24 h after radiation and incubated for an add-
resolving complex DNA damage. We find that DNA-PK itional 24 h prior to harvesting and staining. Thus, cells
kinase activity influences the survival advantage imparted staining positive for BrdU have initiated DNA synthesis
by Artemis over-expression to a greater degree that ATM inthe24–48hafterradiationandtherefore‘survived’ra-
kinase activity and that Artemis phosphorylation is diation treatment. We have used this assay to reprodu-
uncoupledfromradioresistance. cibly and reliably measure toxicity and cell cycle arrest
following both radiation and genotoxic drug treatments
Results in normal and hypersensitive mouse and human lines
ElevatedArtemisproteinlevelsconferhighandlowLET [11,19,20]. Like the colony formation assay, the BrdU-
radioresistance proliferation assay also reveals increased radioresistance
To examine the influence of Artemis levels on cellular in cells with elevated Artemis protein levels (compare
survival after exposure to high and low LET radiation, Figure1AandB).
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Figure1ElevatedArtemislevelsconferaradioprotectiveeffectagainstHighandLowLETradiation.Westernblotsoflysatesfromhuman
HEK293cellscarryingintegratedvectorsharboringtetracycline-inducibleArtemisexpressionconstructs(293Art),orcontrolvector(HEK293),
probedwithantibodiesrecognizingArtemis(top)ortubulinforloadingcontrols(bottom)(1A-inset).Resultsfromaclonogenicsurvival
experimentusingHEK293(opencircles)and293ArtArtemisoverexpressingcells(solidtriangles),stainedandcoloniesscored14daysafter
exposuretovarieddosesofX-rays(A)comparedtoaBrdUproliferationexperimentsetupinparallelandharvested48hpostexposure(B).
ResultsfromBrdUproliferationexperimentsfollowinghighLETexposuresintheformofironnuclei(C)ortitaniumions(D)carriedoutusingthe
samedoserangeasX-rayexperimentsandprocessedatthesametimepoints.
The BrdU-proliferation assay was then used to evaluate with low LET X-ray exposure (1.7 fold) (Table 1). These
sensitivitytohighLETexposuresplusandminusArtemis data indicate that high levels of intracellular Artemis pro-
overexpressiontoassesswhetherasurvivaladvantagewas vide a distinct survivalbenefitfollowingradiation damage
observed with exposure to a radiation quality that pro- andthatthemagnitudeofthisradioprotectiveeffectisra-
ducedgreateramountsofcomplexdamage.Twodifferent diationqualitydependent.
types of high LET radiation, 1 GeV/u Titanium (LET
~100 keV/u), and 1 GeV/u Fe nuclei (LET~150 keV/u) HighlevelsofArtemisaccelerateDNAdouble-strand
wereused to investigate this point. As observed following breakrepair
X-ray exposure, Artemis over-expression significantly ThevarianthistoneH2AX,hasbeenshowntoberapidly
increased the survival of human cells following exposure phosphorylated at Ser139 (γH2AX) upon induction of
to high LET radiation (Figure 1C & D). As expected, a DNA DSBs and is a stoichiometric surrogate marker for
markedly higher toxicity was associated with high LETas this type of damage within cells [21,22]. The increase in
compared to low LET exposure (Figure 1, compare B, C phosphorylation and disappearance of phosphorylation
and D), however Artemis over-expression consistently at this site can be quantified by flow cytometry and
conferred a significant survival advantage with all radi- faithfully correlates with the kinetics of DSB repair
ationqualities.Notably,thedegreeofradioprotectioncon- [23,24]. To evaluate the effect of Artemis over-
ferred by Artemis over-expression varied with the expression on early DNA repair after high and low LET
radiation quality. The greatest radioprotection was exposures (Fe nuclei and X-ray), we quantified γH2AX
observedafterexposuretoFenuclei(~3fold),alesserde- phosphorylation in G1 cells by flow cytometry at thirty
greewas observed followingTi nuclei(2.2fold) exposure, minutes and two hours after exposure. Individual single-
and the lowest degree of radioprotection was observed cell flow cytometry data was analyzed and the
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Table1Calculationofsurvivalparametersbasedonproliferationassayusingtheequation
Survival=Exp(-αDose-βDose2)
A.Survivalparametersfor293ArtArtemisover-expressingCells
RadiationType α,Gy−1 β,Gy−2 D ,Gy Fold
37
293Art/HEK293c
X-rays – 0.057±0.002 4.21±0.087 1.7
Titaniuma 0.47±0.02 0.034±0.008 1.86±0.024 2.2
Ironb 0.22±0.002 0.065±0.007 2.59±0.074 3.0
B.Survivalparametersfor293WildtypeCells
RadiationType α,Gy−1 β,Gy−2 D ,Gy
37
X-rays 0.287±0.033 0.048±0.006 2.47±0.09
Titaniuma 0.025±0.044 – 0.85±0.03
Ironb 1.11±0.29 0.086±0.075 0.85+0.15
aTitaniumion1,000MeV/u.
bIronion1,000MeV/u.
cFoldadvantageinsurvival/proliferationfollowingradiationwithArtemisoverexpressionascomparedtowild-typecells.
distribution of γH2AX intensity was quantified and plot- SDS-PAGE,isdelayedfollowinghighLETexposurerela-
ted to visualize the DNA damage of cell populations at tive to low LET (Figure 3). More specifically, Artemis
these time points. As expected, γH2AX intensity protein mobility is markedly altered at early time points
increased after ionizing radiation exposure relative to (2 h) after X-rays while a parallel experiment with high
the untreated controls and the levels of damage were LET radiation shows no change in Artemis protein mo-
greater with Fe nuclei exposure as compared to X-ray bility at this same timepoint (Figure 3A). However, at
(Figure 2, compare A&B to C&D). At thirty minutes 48 h after high LET exposure a mobility shift in Artemis
after X-ray exposure, cells over-expressing Artemis show protein is observed, indicative of phosphorylation (com-
significantly less residual damage relative to the parental pare Figure 3A and B). These data point towards a delay
HEK293 (Figure 2A). Two hours after a 2 Gy X-ray ex- in Artemis phosphorylation following high LETas com-
posure the levels of residual γH2AX are nearly equiva- pared tolow LET exposure, consistent with ourprevious
lent between the two cell lines (Figure 2B). In contrast, studies showing ATM mediated signaling is delayed fol-
thirty minutes after exposure to 2 Gy of Fe nuclei, lowing high LET relative to low LET exposures in wild
HEK293 and 293Art show virtually no difference in type fibroblastcells [23].
γH2AX levels (Figure 2C). However, at two hours post
exposure to Fe nuclei the 293Art cells show markedly CellswithhighArtemislevelsshowdecreasedG2/M
decreasedγH2AXlevelsascompared totheparentalcell accumulationfollowingradiationexposure
line (Figure 2D). These data reveal that Artemis over- DNA damage initiates cell cycle checkpoints that arrest
expression diminishes γH2AX levels following both high cell cycle progression until the damage is repaired. To
and low LET exposures, but that the kinetics are differ- assess the effect of Artemis over-expression on progres-
ent between the radiation qualities. These results are sion through the G2/M checkpoint following radiation,
similar to previous results in wild type fibroblast cells we evaluated cell cycle distributions 48 h after radiation
[23] where high LET radiation results in markedly exposure using the DNA stain propidium iodide (PI).
slower repair kinetics as compared to low LET exposure. Cell cycle distribution was evaluated after exposure to
Furthermore,thesedata suggestthattherateofDNAre- various doses and qualities of ionizing radiation for the
pair is accelerated by Artemis overexpression for both 293ArtandHEK293cells (Figure 4).
high andlowLETradiation damage. High Artemis levels had no discernable effect on cell
cycle distributions in the absence of DNA damage. As
ArtemisphosphorylationisdelayedfollowinghighLET expected, both 293Art and HEK293 cells showed a
relativetolowLETradiation marked accumulation of cells at the G2/M boundary fol-
Artemis phosphorylation status was evaluated at 2 and lowing radiation exposure and this accumulation
48hfollowinglowandhighLETexposurestodetermine increased at higher doses, and was greater with high LET
if phosphorylation was temporally regulated with respect as compared to low LET exposure (Figure 4). In previous
to radiation quality. We find that Artemis phosphoryl- studieswithArtemisdefectivecellsweobservedpersistent
ation, which results in a mobility shift of the protein on cell cycle arrest and an absence of cycling 48 h post
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Figure2(Seelegendonnextpage.)
radiation exposure [16], in contrast, cells over-expressing HEK293 (Figure 4). Similar results are also observed at
Artemis traverse the G2/M checkpoint more readily and 72hpostradiationexposuresuggestingthattheprolifera-
move into G1 to a greater extent than control wild type tion defect in wild type cells is due to a damage-induced
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(Seefigureonpreviouspage.)
Figure2HighlevelsofArtemisincreasetherateofγH2AXresolutioninirradiatedcells.Flowcytometryanalysisofthedistributionsof
cellswithvariousγH2AXlevelsinHEK293and293Artcellsfollowingexposuretodifferentqualitiesofionizingradiation.Comparisonofthe
distributionofγH2AXsignalincontrolandirradiatedcellpopulationsatvarioustimespostexposure(leftpanels),includinganenlargedgraphof
theirradiatedsamples(rightpanels)comparingcontrolHEK293(dashedlines)and293Art(shaded).Measurementsweretaken30minafter2Gy
X-rays(A),2hafter2GyX-rays(B),30minafter2Gyironnuclei(C),and2hafter2Gyironnucleiexposure(D).
cellcycleblock,whichisnotrectifiedevenat72hafterir- by Artemis over-expression. We carried out cell prolifera-
radiation (not shown). Importantly, Artemis over- tion assays to quantify the survival advantage of Artemis
expression does not eliminate or bypass the G2/M cell over-expressionunderconditionswhereATMorDNA-PK
cycle checkpoint, but rather cells with elevated Artemis were catalytically inactivated with inhibitory drugs at a 10
levelshaveanattenuatedbiologicalresponsetoirradiation μM concentration, previously shown to totally block radi-
withcellcyclekineticssimilartotheparentallinealbeitat ation induced phosphorylation of down stream targets
a lower dose (i.e. cell cycle profile of HEK293 at 2 Gy is [25,26]. As expected, the parental HEK293 cells were sig-
comparable to 293Art at 3 Gy, Figure 4). This effect was nificantly more sensitive to X-ray treatments when either
observed at different doses and with different radiation ATMorDNA-PKwasinactivatedwithspecifickinaseinhi-
qualities (Fe and Ti), rendering the cell cycle distribution bitors (Ku55933, or Nu7026 respectively). Under these ex-
of 293Art cells at higher doses nearly identical to that of perimental conditions, sensitivity to radiation was greater
the parental HEK293 at lower doses (Figure 5, note pat- whenDNA-PKwasinhibitedrelativetoinhibitionofATM
ternshighlightedbydottedlines). (Figure6A).Thesurvivalof293Artcellswaslikewiseinflu-
Artemis over-expression consistently resulted in a larger enced by the inhibition of ATM and DNA-PK (Figure 6B),
percentage of cells cycling into G1 with fewer cells however the effect of ATM kinase inhibition was signifi-
remainingarrestedatG2/Mafterexposure,aphenomenon cantly less pronounced when Artemis levels were elevated
most consistent with an increased DNA repair capacity or (compare6A&B,shadedareas).Incontrast,DNA-PKkin-
repairrate. ase inhibition reduced survival dramatically in Artemis
over-expressing cells, approximately two orders of magni-
ATMandDNA-PKregulatetheradioresistanceconferred tude at the highest dose (Figure 6B), similar to what was
byArtemis observed in wild type cells with DNA-PK inhibition (com-
Both DNA-PK and ATM are reported to modify Artemis pare 6A & B). These data imply that the radioprotective ef-
and to be important mammalian DNA damage signaling fect conferred by elevated Artemis is significantly more
molecules.Wenextevaluatedthefunctionalsignificanceof dependentonDNA-PKthanATMactivity.Inaddition,these
thesekinaseactivitiesontheradioprotectiveeffectimparted data suggest that the mechanism of the radioprotection
A
B
Figure3ArtemisphosphorylationisdelayedfollowinghighLETdamage.WesternblotswereprobedfortotalArtemisproteinandmobility
shiftsoftheproteinindicatephosphorylationhasoccurred.Blotsareshown2hpostX-rayorironnucleiexposure(A,leftandrightpanel
respectively)and48hpostX-rayorironnucleiexposure(B,leftandrightpanelrespectively).Arrowsindicatelocationofbasally-phosphorylated
Artemis(lowerarrow)andhyper-phosphorylatedArtemis(upperarrow).
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Figure4Artemisover-expressionfacilitatesrepairandpassagethroughtheG2/Mcheckpoint.Flowcytometryofpropidiumiodine
stainedcellsrevealscellcyclekineticsfollowingvariousdosesofX-ray,Feion,orTiionexposure.CellcycleprofilesforwildtypeHEK293cellsare
showninwhiteandcellsover-expressingArtemis(293Art)areBlack.PeakscorrespondingtolocationofG1andG2cellsareindicatedatthebase
ofthefigure,withdoseandradiationqualitynotedontheYandXaxisrespectively.
resultingfromArtemisover-expressioninvolvesacoordinate therefore in order to assess DNA-PK and ATM activity
activity of Artemis and DNA-PK, most likely in enhancing on Artemis on phosphorylation levels Western blots of
DNArepair. 293Art cells two hours post X-ray exposure (0 Gy,
The kinase dependency of Artemis phosphorylation 0.5Gy,2Gyand4Gy) were run to assess the phosphor-
following radiation exposure was also examined. Mul- ylation status of Artemis in the presence and absence of
tiple kinases could potentially phosphorylate Artemis, inhibitors. In the absence of ATM inhibitor, Artemis
shows a dose dependent increase in phosphorylation,
visualized as a shift in mobility of the band specific for
Artemis protein (Figure 6C, control). Inhibiting ATM
kinase blocks the induction of Artemis phosphorylation
followingradiation asexhibitedbyalackofshiftedArte-
mis protein on Western blots (Figure 6C, ATMi). Treat-
ment of cells with the DNA-PKcs inhibitor (CSi) only
partially blocked the mobility shift of Artemis, with cells
showing less phosphorylation and lowered levels of
shifted protein (Figure 6C). The latter result suggests
that this phosphorylation is uncoupled from the survival
advantage imparted by Artemis over-expression, as inhib-
ition of DNA PKkinaseseverelyaffected the radioprotec-
tive effect but did not affect Artemis phosphorylation.
Furthermore, these data indicate that the mobility shift
Figure5QuantitationofcellcycledistributionofWTor associated with Artemis phosphorylation is primarily
Artemisover-expressingcellsfollowingexposuretovaried
ATMdependentwhereastheradioprotectiveeffectsofAr-
dosesofdifferentradiationqualities.Normalwild-type(HEK293)
temisarepredominantlyDNA-PKdependent.
orthesesamecellsover-expressingArtemis(293-Art)wereexposed
tovariousdosesandqualitiesofradiation.Followingradiationcells
wereincubatedfor24hours,thenlabeledfor24hourswithBrdU, Discussion
andfixedat48hourspostexposure.Flowcytometrywasusedto
Previous studies by our group and others have shown
quantifythepercentageofcellsinG1orG2.
that Artemis deficient cells exhibit elevated radiation
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A B
C
Figure6EffectofkinaseinhibitorsonthesurvivaladvantageimpartedbyArtemisover-expressionandArtemisphosphorylation
followingX-rayexposure.BrdUproliferationassayswereperformedonbothwildtypeHEK293(A)andcellsover-expressingArtemis(B)with
andwithouttwodifferentkinaseinhibitors,aATMkinaseinhibitor(ATMi)andaDNAPKcsinhibitor(CSi).Shadedregionsonthesegraphsarefor
visualcomparisonoftheeffectATMinhibitionontheabilityofwild-typeversusArtemisover-expressingcellstoproliferatepostX-rayexposure.
ArtemisphosphorylationisindicatedbyashiftofthetotalArtemisproteinonaWesterngel(arrowsindicatebasallyandhyper-phosphorylated
forms)followingvariousdosesofX-rayandwasalsomonitoredplusATMinhibition(C)andDNAPKcsinhibition(D).
sensitivity, yet have only a slight defect in DSB repair, that Artemis over-expression provides a survival advan-
implicating a role for Artemis in the repair of a specific tage post Iron exposure. Data from a representative ex-
class of DSBs [6,13,16]. Radiation-induced breaks consist periment with Titanium exposure shows a similar
of a combination of closely spaced single-strand breaks, pattern to Iron, again confirming that Artemis protein is
DSBs, sugar damage, base damage and breaks with more protective following high LET exposures and that
modified ends. Only specific subsets of these breaks are the level of protection is dependent on radiation quality.
thought to be processed by Artemis, with other proteins Western blot analysis (Figure 1A insert) verifies the dra-
apparently unable to substitute for Artemis in this func- matically increased Artemis levels in 293Art cells in
tion [10,11,13]. In G1 cells, the majority of radiation- comparison to wild type HEK293 cells and further con-
induced DSBs are rejoined quickly by NHEJ and this firms the correlation between the increased radioresis-
rapid repair appears not to require Artemis or ATM, tance and increased Artemis expression observed in
however a small subset of slow repairing damages have these cells. High Z particles like Fe and Ti are not com-
been shown to require ATM and Artemis for repair monly used in cancer therapeutic regimens as the LET
[13,27,28]. Interestingly, a recent report outlines a re- of these particles in normal tissue at the entrance and
quirement for ATM and Artemis in the repair of a frac- prior to the target site is very high. Typically, low charge
tion of breaks refractory to repair by homologous particles like carbon ions are used in particle therapy in
recombinationinG2cellsaswell [29]. centers offering high LET therapies (such as the Heidel-
Neither the precise role for Artemis in DSB repair nor berg Ion Therapy Centre (UKL-HD); the Heavy Ion
its substrate specificity in vivo has been completely Medical Accelerator in Chiba, Japan; the CNAO facility
defined. Some studies have suggested a specific role for near Milan in Pavia; and GSI in Darmstadt, Germany).
Artemis in the repair of complex DSBs, however it’s The suitability of carbon is mainly attributed to its low
exact role in repairing damage caused by high LET radi- LET (13 keV/micron) at the entrance of the tissue and
ation remains unclear [13,30]. The current study focuses substantial Bragg peak (several hundred keV/micron) at
on identifying the contribution of Artemis to the repair the site of the tumor. We have used Ti and Fe ions in
of various radiation qualities. In these studies we show these studies as these ion/energy combinations have a
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LET similar to that of the carbon ion beam near the addition,wefindthatArtemis-radioprotectionisprimarily
Bragg peak and therefore mimic the high LET deposited dependent on DNA-PK activity and not coupled to the
atthetumorsite duringtherapy. ATM-dependentphosphorylation(Figure5).Herewefind
Studies of γH2AX levels after damage reveal more that elevated Artemis levels alone can significantly alter
rapid resolution of phosphorylated H2AX in cells over- DNArepairandcellularsurvivalfollowingradiationexpo-
expressing Artemis. While elevated Artemis levels sures. Our data are most consistent with Artemis func-
reduced the γH2AX signal in cell populations after both tioning in the repair of complex DNA breaks as part of
high and low LET damages, the timing of these differ- theNHEJrepairpathwayand suggest thatthatArtemis is
ences is distinctly different. Artemis-mediated reduction probably rate limiting to this process within human cells.
in γH2AX levels (indirectly corresponding to reduced We have observed increased Artemis levels in a subset of
levels of DSBs) following high LET radiation exposures breasttumors,althoughwedidnotfindthistobethesin-
is only marginally detectable thirty minutes after expos- gular determinant of radiosensitivity (unpublished data).
ure, however a clear effect is observed at two hours post These findings suggest that Artemis levels in a subset of
exposure (compare Figure 2C & D). In contrast, low tumorcellsmaybeausefulindicatorofresponsivenessto
LET damage shows a dramatic difference in γH2AX radiation therapy. Moreover, Artemis inhibition may pro-
levels between Artemis over-expressing and wild type vide a functional adjuvant in radiation therapy in such
HEK293 cells at thirty minutes, whereas at two hours cases,apossibilitywearecurrentlyinvestigating.
post exposure the control and Artemis over-expressing
cells are nearly indistinguishable (compare Figure 2A & Methods
B). This likely reflects a slower repair of the more com- T-Rex-293 (Invitrogen) human kidney cells employed in
plex damage induced by high LET radiation as com- these studies have a rapid doubling time, high efficiency
pared to less complex low LET radiation damage as has of transfection and protein expression, accurate transla-
been previously observed [23,31]. Nonetheless, these tion and post translational folding and processing to
data show that Artemis levels significantly influence generate functional proteins and have been extensively
γH2AX resolution and likely the repair kinetics follow- used as an expression tool for recombinant proteins in
ingbothhigh andlowLETradiation. the last three decades. These cells expressing the tetra-
Inhibition of DNA-PK reveals that the radio-protective cycline repressor were cultured in DMEM supplemented
effect of Artemis over-expression was principally reliant with 10% tetracycline-free FBS and 5 mg/ml of blastici-
on DNA-PK activity and to a lesser degree on ATM ac- din. T-Rex-293 cellsweretransfected with pcDNA4/TO/
tivity. In contrast, we find that Artemis phosphorylation, Myc-HisA (Invitrogen) carrying wild type Artemis
indicated by a mobility shift of Artemis protein, was pri- cDNA, incubated for 48 h and then placed under zeocin
marily dependent on the activity of ATM, as has been selection (250 μg/ml). Clones were isolated, induced for
reported previously [14,17,32]. Furthermore, in cells Artemis expression with 1 μg/ml doxycycline 24 h prior
containing high levels of Artemis, cell proliferation fol- to radiation exposure, this induction is maintained dur-
lowing irradiation was more severely diminished by loss ingthe courseoftheexperimentandArtemis expression
of DNA-PK activity than by ATM inhibition (compare levels were evaluated by immunoblotting. For both
Figure 6A & B). Taken together, these data suggest that γH2AX flow cytometry and the BrdU proliferation assay,
Artemis phosphorylation as observed by mobility shifts T-25 flasks were plated for each dose and cells were
may not be relevant to Artemis’function in the repair of grownto85to95%confluence priortoexposure.
radiation damage,aswe hadpreviouslysuggested[16].
Irradiationconditions
Conclusions Cells were exposed to doses ranging from 1 to 6 Gy of
In summary, our data reveal that elevated Artemis levels 320 kV/X-rays, 1,000 MeV/nucleon Fe, or 1,000 MeV/
in human cells are able to confer a statistically significant nucleon Ti ions (LET of 150 keV/μm and 108.5 keV/μm
survival advantage following exposure to ionizing radi- respectively). X-ray exposures were performed at
W
ation.Moreover,thisArtemis-mediatedradioprotectiveef- Lawrence Berkeley National Laboratory using a Pantak
fectismorepronouncedfollowinghighLETexposuresas X-ray generator operating at 320 kV/10 mA with a
comparedtolowLETdamage(Figure1andTable1).We 0.5 mm copper filtration and using dose rates ranging
also find that Artemis over-expression accelerates the from 5 to 80 cGy/min depending on the dose. Fe and Ti
resolutionofγH2AX,andbyinferencetherateofdouble- ion exposures were performed at Brookhaven National
strandbreakrepairinvivo(Figure2).Thislatterfindingis Laboratory, NY using dose rates ranging from 10 cGy to
entirelyconsistentwithourobservationthathighlevelsof 1 Gy/min depending on the dose. Standard radiation
Artemis increase the rate at which cells are able to satisfy therapy regimens typically follow a fractionated regimen
theG2/Mcheckpointafterradiationdamage(Figure4).In of 1.5–2 Gy doses to a cumulative dose of 40–60 Gy,
Sridharanetal.RadiationOncology2012,7:96 Page10of12
http://www.ro-journal.com/content/7/1/96
depending on nature of the tumor. Radiation doses used said to arise from two or more tracks or miss-repair that
in this study are comparable to single fraction, or simu- increaseswithdose.Ifalphaiszero,orverysmall,thencells
lateanaccumulated dosefrom 2–3fractions. areresistantandonlybetacontributes.
FlowcytometryanalysisofγH2AXkinetics
Kinaseinhibitortreatments
Cells were exposed to an ATM kinase inhibitor (Calbio- Irradiated and mock-irradiated flasks were fixed at vari-
chem, EMD Biosciences, Gibbstown, NJ) and/or a DNA- ous times after exposure. Cells were harvested and fixed
with100%ice-coldmethanoladdeddrop-wisewhile vor-
Dependent Protein Kinase inhibitor (NU7026 SigmaAl-
drich, St. Louis, MO) in a subset of experiments to define texing. After fixation, cells were placed at −20°C until
the relative role of these kinases in imparting the survival stained. Cell suspensions were counted prior to staining
and diluted to 1.0×106/ml. For staining, an aliquot of
advantage in cells over-expressing Artemis (293Art). For
theseexperimentscellswerepre-incubatedwiththeseinhibi- the fixed cells containing 0.5×106 was removed and
torsusinga1μMconcentrationfor1hpriortoirradiation. washed in PBS. Cells were placed in a blocking solution
containing2%FBS/PBSandincubatedfor1hwithapri-
mary mouse monoclonal γH2AX antibody (1:800 dilu-
Clonogenicsurvivalassay
tion) from Molecular Probes (Invitrogen, Carlsbad, CA)
For colony formation assays, varying number of cells
on ice. After incubation, cells were pelleted, washed in
(1.5×102to1×104)wereplatedintriplicate60cm2plas-
PBS, and incubated in a secondary goat anti-mouse anti-
tic dishes and incubated for 12–15 h at 37°C, 5% CO
2 body(1:400dilution)fromMolecularProbes(Invitrogen,
priortoirradiation.CellswereirradiatedwithX-rayinthe
Carlsbad, CA) diluted in blocking solution. After 1 h
dishesandreturnedtotheincubatorfor7–8days.Result-
additional incubation on ice under foil, cells were pel-
ing colonies were stained with crystal violet, scored, and
leted and washedfor afinal timeinPBS. Flow cytometry
survivingfractioncalculatedandplotted.
was used to define cell cycle status by staining cellular
DNA with propidium iodide (10 μg/ml PI, Roche Ap-
BrdUproliferationassay plied Science, Indianapolis, IN) in a solution containing
Cells were irradiated with high or low LET radiation, RNase (100 μg/ml, Sigma-Aldrich, St. Louis, MO). Me-
incubated for 24 h then labeled with BrdU for 24 h, and
dian values of fluorescent signals corresponding to
finally fixed and stained 48 h after exposure using an γH2AX levels were obtained for each control and irra-
anti-BrdU antibody (20 μl antibody per 1×106 cells, BD
diated sample (X-ray or 1,000 MeV/u Fe ion). Cell data
Biosciences, San Jose, CA) following product directions.
was collected on a Beckman-Coulter EPICS XLMCL
Cell cycle distribution and BrdU incorporation were
flow cytometer using Cell quest Data Acquisition soft-
determined by data collected with a Beckman-Coulter
ware, and analyzed using Flowjo (Treestar, Inc. OR) soft-
EPICS XLMCL flow cytometer using Cellquest Data Ac-
ware. Individual cell data was exported for analysis and
quisition software, and analyzed using Flowjo (Treestar,
data is expressed as relative increase in phosphorylation
Inc. OR) software. Percentage of G1 cells incorporating
levelovercontrolsfor eachexperiment.
BrdU were determined for each dose and cell line, and
values graphedrelative tocontrols.
Westernblots
Cells were harvested and resuspended in cold high salt
Calculationofsurvivalparametersfollowingradiation lysis buffer (20 mM Tris, pH 8.0; 600 mM NaCl; 1 mM
exposure EDTA; 0.5% Igepal) containing protease inhibitor cock-
D gives the mean lethal dose that delivers an average of tail from Calbiochem (1 mM PMSF, 1 mM Na3VO4,
0
one lethal event/target. When D=D the surviving frac- and 1 mM NaF), incubated on ice for 30 min with occa-
0
tion is 37%. We calculated the D values for each cell sional vortexing, and then sonicated. Lysates were spun
37
line and radiation quality, and found them to be consist- at 14,000 rpm for 20 min at 4°C, and protein concentra-
ently lower in wild type HEK293 cells as compared to tion determined using the Biorad BCA protein assay.
the Artemis over-expressing cell line, 293Art. Alpha and Loading buffer (187.5 mM Tris, pH 6.8; 6% SDS; 30%
beta components were also evaluated for each cell line Glycerol, and 10% 1 M DTT) was added to samples,
followingeachradiationqualityonthe basis oftheequa- which were boiled and spun down. Samples were loaded
tion: S¼e∧ð(cid:2)αD(cid:2)βD2Þ, where S is the survival frac- ontoa7%SDS-PAGEgelandrunfor1hat150V(Run-
tion, D is the dose of exposure and α and β are two ning buffer: 25 mM Tris, 192 mM Glycine, 0.1% SDS).
numericalconstants.Thealphatermissaidtodominateat Proteins were transferred to 0.2um PVDF membrane at
lowdose[linearwithdoseforlog(survival)]andtobedue 4°C, 30 V, over night in Transfer Buffer (20% MeOH,
tosingleionsorelectrons.Thebetatermismoreprevalent 50 mM Tris, 400 mM Glycine, 0.1% SDS). After the
at high dose [quadratic with dose for log (survival)] and transfer, blots were rinsed with Tris buffered saline