Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair.

0.84 MB·English

by  Bulgar, A D

Citation:CellDeathandDisease(2012) 3,e252;doi:10.1038/cddis.2011.135
&2012MacmillanPublishersLimited Allrightsreserved2041-4889/12
www.nature.com/cddis
Removal of uracil by uracil DNA glycosylase limits
pemetrexed cytotoxicity: overriding the limit with
methoxyamine to inhibit base excision repair
ADBulgar1,LDWeeks1,YMiao1,SYang2,YXu2,3,CGuo1,SMarkowitz1,2,3,4,NOleinick2,5,SLGerson1,2andLLiu*,1,2
UracilDNAglycosylase(UDG)specificallyremovesuracilbasesfromDNA,anditsrepairactivitydeterminesthesensitivityofthe
celltoanticanceragentsthatarecapableofintroducinguracilintoDNA.Inthepresentstudy,theparticipationofUDGinthe
responsetopemetrexed-inducedincorporation ofuracilintoDNAwasstudiedusingisogenichumantumor celllineswithor
withoutUDG(UDGþ/þ/UDG(cid:2)/(cid:2)).UDG(cid:2)/(cid:2)cellswereverysensitivetopemetrexed.Cellkillingbypemetrexedwasassociated
with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDGþ/þ cells
were410timesmoreresistanttopemetrexedduetotherapidremovalofuracilfromDNAbyUDGandsubsequentrepairofthe
resultantAPsites(abasic sites)viathebaseexcisionrepair(BER). Theresistance topemetrexedinUDGþ/þ cells couldbe
reversedbytheadditionofmethoxyamine(MX),whichbindstoAPsitesandinterruptsBERpathway.Furthermore,MX-boundAP
sitesinducedcelldeathwasrelatedtotheircytotoxiceffectofdualinactivationofUDGandtopoisomeraseIIa,twogenesthatare
highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more
selectivecytotoxicityoncancercellsthroughasyntheticlethalmechanism.
CellDeathandDisease(2012)3,e252;doi:10.1038/cddis.2011.135;publishedonline12January2012
SubjectCategory:Cancer
Pemetrexed,anantifolate,inhibitsseveralkeyfolate-depen- chromosome segregation during mitosis, the association
dent enzymes in the thymidine and purine biosynthetic betweenUDGandCENP-AimpliesthatUDGmaybeinvolved
pathways, leading to the accumulation of dUMP and the incellproliferation.12
misincorporation of uracil into DNA.1–3 Incorporated uracil The base excision repair (BER) pathway is initiated
basesarerecognizedandexcisedbyuracilDNAglycosylase following the removal of a base lesion by a DNA glycosy-
(UDG).4–6 lase.13Glycosylaseexcisionofthedamagedbaseproceeds
UDG is a conserved DNA repair protein expressed in all viahydrolyticcleavageoftheN-glycosidicbond,leavingthe
typesofhumancells.7ItspecificallyremovesuracilfromDNA sugar–phosphate backbone intact and producing an abasic
andprotect cells fromcytotoxicity and mutagenicity. Human site(APsite).14,15TheresultantAPsiteisprocessedbyAP
UDG is encoded by the UNG gene. Alternative promoter endonuclease 1 (APE1), which generates a single-strand
usage and splicing of this gene produces two different break containing a 50-dRP residue and a 30-hydroxyl group.
isoforms: the mitochondrial UNG1 and the nuclear UNG2.8 The completion of BER occurs by short-patch (single
Nuclear UDG (UNG2) is the predominant form in cells and nucleotide)orlong-patch(2–13nucleotides)repairpathways
represents 490% of the total enzyme activity. Therefore, involvingDNApolymerasegapfillingandDNAligation.BERis
UDGusedinthisarticlereferstoUNG2.NuclearUDGactivity the most efficient mechanism for repairing a variety of base
is subject to cell cycle–dependent regulation and shows a lesions,includingthoseinducedbychemotherapeuticagents,
marked increase during the S-phase.9 During the S-phase, thereby rendering tumor cells resistant to DNA-damaging
UDG is localized in replication foci and interactswith PCNA chemotherapeuticagents.16–18ToovercomeBER-conferred
(proliferating cell nuclear antigen) and RPA (replication drugresistance,methoxyamine(MX)hasbeendevelopedas
protein A), two proteins that are required to form functional an active inhibitor of BER.19 MX reacts specifically with the
replication forks.9 This suggests that the UDG removal of aldehydegroupinthesugarmoietyoftheAPsite,formingan
incorporateduracilmaydirectlylinktotheprogressionofthe MX-bound AP-site. This structurally modified AP site is
replication fork.10,11 In addition, UDG has recently been refractory to the repair activity of APE1,20,21 leading to the
showntopromotetheassemblyofhumancentromereprotein interruption of the BER pathway. MX has been shown to
A(CENP-A).AsCENP-Aisanessentialproteinrequiredfor potentially enhance the therapeutic efficacy of various
1DepartmentofMedicine,DivisionofHematology/Oncology,CaseWesternReserveUniversity,Cleveland,OH,USA;2CaseComprehensiveCancerCenter,Case
WesternReserveUniversity,2103CornellRoad,Cleveland,OH44106,USA;3DepartmentofChemistry,ClevelandStateUniversity,Cleveland,OH,USA;4Department
ofGenetics,CaseWesternReserveUniversity,Cleveland,OH,USAand5DepartmentofRadiationOncology,CaseWesternReserveUniversity,Cleveland,OH,USA
*Correspondingauthor:LLiu,DepartmentofMedicine,DivisionofHematologyandOncology,CaseWesternReserveUniversity,Cleveland,10900EuclidAvenue,
OH44106,USA.Tel: þ12163685696;Fax: þ12163681166;E-mail:[email protected]
Keywords:UDG;baseexcisionrepair;APsites;methoxyamine
Abbreviations: UDG,uracilDNAglycosylase;BER,baseexcisionrepair;MX,methoxyamine;TopoIIa,topoisomeraseIIa;PCNA,proliferatingcellnuclearantigen;
RPA,replicationproteinA
Received30.8.11;revised09.11.11;accepted09.11.11;EditedbyRAKnight
TheimpactofUDGactivityonpemetrexedcytotocixity
ADBulgaretal
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DNA-damagingchemotherapeuticagents.22–25Itiscurrently in UDGþ/þ and UDG(cid:2)/(cid:2) cells. We first confirmed the UDG
being evaluated in multiple clinical trials (as TRC102, activity in these cells using an in vitro glycosylase cleavage
TRACON Pharmaceuticals, Inc., San Diego, CA, USA) assay,inwhicholigonucleotidesubstratescontaininguridine
and has completed phase-I testing in combination with residues were incubated with either purified UDG/APE1
pemetrexed. enzymes or cell extracts. As shown in Figure 1a, after the
Inthepresentstudy,weinvestigatedtheimpactofUDGand reaction with fluorescent probe-labelled oligonucleotide
BER on cell sensitivity to pemetrexed using isogenic UDG- substrates (40-mer) containing U:G mispairs, both purified
proficient and -deficient human cancer cells. Although peme- UDG/APE1 enzymes and cell extracts from UDGþ/þ cells
trexed has multiple targets, the different responses to peme- producedcleavedDNAfragmentsasan18-merband,which
trexedinUDGþ/þandUDG(cid:2)/(cid:2)cellswereinvestigatedonlywith resulted from the removal of uracil bases by UDG and
respect to the levels of uracil-DNA produced by pemetrexed. subsequent incision of the resultant AP sites by APE1. By
We also explored the inhibition of BER by MX as a novel contrast, no cleaved fragments were observed in UDG(cid:2)/(cid:2)
strategy to enhance the therapeutic efficacy of pemetrexed, cell extracts after incubation with an even higher
althoughMXisexpectedtopotentiateotheranticanceragents concentration of cell extracts. Dflag cells were capable of
capableofinducinguracilincorporationintoDNA. removing uracil bases, which were derived from UDG(cid:2)/(cid:2)
cellsbyrestoringUDGactivity.
We next determined the levels of uracil in the DNA of
Results UDGþ/þ and UDG(cid:2)/(cid:2) cells following pemetrexed exposure
using HPLC/MS/MS. There was an inverse relationship
UDG activity determines the level of uracil retained in between UDG activity and the level of uracil bases in the
DNA. To correlate UDG activity with the cytotoxicity DNA(Figure1b).Asignificantamountofuracilwasdetected
of pemetrexed, comparative studies were performed in UDG(cid:2)/(cid:2) cells, which correlated with the duration of
Cell extract (ug) - 5 5 5 10
UDG/APE + - - - -
40 mer
18 mer
UDG+/+ Dflag UDG -/-
120
ol2.5
Uracil (nfg/ml)16248000000 UUDDGG-+//-+ * ** ** sites relative to contr01..5152 UUDDGG-+//-+ * **
P
0 A 0
0 6 24 48 72 0 100 200 400
Time (hr) Pemetrexed (nM)
120
Uracil (nfg/ml)12468000000 UUDDGG-+//-+ * ** ** es relative to control 1234 RNeoa rcetaiocnti owni twh iUthD UGD*G **
sit
0 P
A 0
0 6 24 48 72
0 50 100
Time (hr) Pemetrexed (nM)
*P< 0.02, **P< 0.01
Figure1 UDGactivitydeterminesthelevelsofuracilandAPsitesinDNA.(a)UDGactivityassayinvitro.OligonucleotideduplexescontainingU:Gwereincubatedwith
cell extracts (5–10mg) from UDGþ/þ, DLD1flag, and UDG (cid:2)/(cid:2) cells at 371C for 1h. Reaction products were resolved by electrophoresis through denaturing 20%
polyacrylamidegels.(b)IncorporateduracildetectedinUDGþ/þandUDG(cid:2)/(cid:2)cellsbyHPLC/MS/MSanalysis.Cellsweretreatedwithpemetrexed(10mM)for6,24,48,and
72h.Cellswereharvestedand40mgofextractedDNAwereinvitroreactedwithpurifiedUDG(10U)for2h.(c)Cellsweretreatedwith5-FU(10mM)for6,24,48,and72h.
UracilwasquantifiedinthereactionproductbyLC-MSanalysis.(d)APsiteformedbypemetrexedinUDGþ/þ andUDG(cid:2)/(cid:2)cells.Cellsweretreatedwithpemetrexed
(0–400nM)for24h.DNAwasextractedandAPsitesmeasuredbyARPreagent.(e)APsitedetectedinDNAofUDG(cid:2)/(cid:2)cellsafterreactedwithpurifiedUDGinvitro.Cells
weretreatedwithpemetrexed(0–100nM)for24hand40mgDNAextractedfromcellswasinvitroreactedwithpurifiedUDG(10U)for2handAPsitesweremeasured
usingARP.Resultsarerepresentativeofthreeindependentexperiments
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TheimpactofUDGactivityonpemetrexedcytotocixity
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pemetrexedexposure.Bycontrast,thedetectableuracilinthe UDG(cid:2)/(cid:2) cells were more sensitive to 5-FU than either
DNA was very low in UDGþ/þ cells, suggesting rapid and UDGþ/þor Dflag cells (Figure 2b). By contrast, there was
efficientremovaloftheincorporateduracil.Similarly,agreater no significant difference in sensitivity to temozolomide, an
retention of uracil in UDG(cid:2)/(cid:2) cells than in UDGþ/þ cells alkylatingagent,orcisplatin,acrosslinkingagent(Figures2c
(Figure1c)wasdetectedfollowingexposureto5-fluorouracil and d), between UDGþ/þ and UDG(cid:2)/(cid:2) cells. Thus, results
(5-FU), a well-known thymidylate synthase inhibitor capable suggestthatUDGactivityspecificallyimpactsthecytotoxicity
ofintroducinguracilintoDNAthroughimbalancednucleotide of anticancer agents that are capable of inducing the
pools. incorporationofuracilbasesinDNA.
The AP sites formed by pemetrexed were measured in
cells, which are a surrogate marker for UDG activity in the The accumulation of incorporated uracil in DNA stalls
cells.AsshowninFigure1d,adose-dependentformationof DNA replication. To elucidate the underlying mechanisms
APsitesinDNAwasdetectedinUDGþ/þ butnotinUDG(cid:2)/(cid:2) responsible for pemetrexed’s cytotoxicity, we first examined
cells. The lack of detectable AP sites in UDG(cid:2)/(cid:2) cells is cellcycleprogressioninresponsetopemetrexed.Asshown
presumably due to the absence of UDG activity to remove inFigure3a,pemetrexed(25nM)causedthearrestofB25%
uracil,resultingintheaccumulationofuracilbasesintheDNA. and 36% of UDG(cid:2)/(cid:2) cells in the S-phase at 6 and 24h,
Toconfirmthis,pemetrexed-inducedAPsitesinUDG(cid:2)/(cid:2)DNA respectively (Figure 3a). The S-phase arrest lasted more
wereanalyzedafterincubationwithpurifiedUDGenzymesin than72h.Atthistimepoint,35%ofthecellswerestillinthe
vitro.AsshowninFigure1e,increasedAPsitesweredetected S-phase and B22% of the cells had undergone apoptosis
asafunctionofpemetrexeddoses. (subG1). By contrast, progression of the cell cycle in
UDGþ/þ cells was only slightly affected by this dose of
pemetrexed (Figure 3b). The effect of pemetrexed on cell
UDG activity determines cell sensitivity to
killing was further analyzed by annexin V staining at 24h
pemetrexed. To determine the correlation between the
after cells were treated with 50nM of pemetrexed. Results
expression of UDG and the sensitivity to pemetrexed, the
cytotoxiceffectofpemetrexedwasexaminedinUDGþ/þ and revealedthatpemetrexedatthesameconcentrationinduced
UDG(cid:2)/(cid:2)cellswithaclonogenicassay.UDG(cid:2)/(cid:2)cellswere10 30% of cell death in UDG(cid:2)/(cid:2) cells compared with 5% in
times more sensitive to pemetrexed than UDGþ/þ cells UDGþ/þ cells(Figures4aandb).
We next examined a network of proteins responsible for
(Figure 2a). The IC value for pemetrexed was 20nM in
50
UDG(cid:2)/(cid:2) cells, compared with 210nM in UDGþ/þ cells. The DNAdamagecheckpointsfollowingexposuretopemetrexed.
killing effect of pemetrexed in UDG(cid:2)/(cid:2) cells was reversed We found that phospho-Chk1 (Ser345) was significantly
when UDG activity was restored in Dflag cells. Similarly, increased in UDG(cid:2)/(cid:2) cells (Figure 3c) but not in UDGþ/þ
cells (Figure 3d). As a DNA damage checkpoint kinase, the
inductionofphosphor-Chk1suggeststhatpemetrexedstalls
100 100 replicationforksinUDG(cid:2)/(cid:2)cells.Wealsonotedthatinboth
cell lines, phospho-histone H3 (Ser 10), a mitotic marker,
increasedat6handdecreasedat24hfollowingexposureto
% Survival 10 UUDDGGf+la/+g % Survival 10 UUDDGGf+la/+g ppmpehhitmooorsteypictlahretocixo-ehenllids.cotoyfInnchelteeisrHtepo3srnoteigwnrgaHelsys3,sciwtoohnane.scopInnmeoriatitoadddndiirtcietiwocfltniulty,hcttauhthsaeestiooinicnndicauortceeftadiposhnewoisotihn-f
UDG-/- UDG-/- G1–S-phase–specificcyclinD1.Subsequently,theS-phase–
1 1 specific cyclin E was upregulated following the exposure to
0 100 200 300 400 0 5 10 15 20
pemetrexed (Figures 3c and d). It has been reported that
Pemetrexed (nM) 5-Fluorouracil (μM)
inhibition of cell proliferation can regulate histone H3
100 100 phosphorylation and that this modification enables the
transcription of genes that are activated as a consequence
ofavarietyofcell-signalingevents.26Therefore,ourdatamay
% Survival 10 UUDDGGf+la/+g % Survival 10 UUUDDDGGG-+fl/a/-+g srweuhggeugtlheaestortrtyhthaemt pephchohosaspnphihsoomrryylalaotitfoionngoeofnfehishttiosratnonensecHr3ipHti3isonai.sn Hilminopkweoedrvtaetnro,t
UDG-/- transcriptional activation of cyclin proteins in response to
DNA damage induced by pemetrexed needs to be further
1 1 investigated.AlthoughtheroleofcyclinD1andEremainsto
0 500 1000 1500 0 10 20 30 40 befullyelucidated,theyfunctionastheregulatorsoftheG1–S
TMZ (μM) Cisplatin (μM)
phase entry in response to inhibition of replication-stalled
Figure2 Comparisonofcellsensitivitywithchemotherapeuticagentsbetween S-phase. Furthermore, the upregulation of phosphor-cdc2
UDGþ/þ and UDG(cid:2)/(cid:2) cells. The cytotoxicity induced by different chemo-
and cyclin B1 also indicates that pemetrexed-induced DNA
therapeuticagentswasanalyzedbyclonogenicformationassayinUDG-proficient
damageactivatestheS-andM-phasecheckpoints.
or -deficient cells (a) pemetrexed treatment (0–400nM); (b) 5-FU treatment
(0–20mM); (c) temozolomide treatment (0–1500mM; P-value¼0.687); and In addition, pemetrexed induced an increase in the
(d)cisplatintreatment(0–40mM,P-value¼0.753).Resultsarerepresentativeof expression of topoisomerase IIa (topo IIa) that was
threeindependentexperiments much greater in UDG(cid:2)/(cid:2) cells than in UDGþ/þ cells
CellDeathandDisease
TheimpactofUDGactivityonpemetrexedcytotocixity
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UDG-/- cells UDG+/+ cells
6 hr 24 hr 72 hr 6 hr 24 hr 72 hr
G1: 62.3 G1: 62.2 G1: 36.2 G1: 29.3 G1: 67.1 G1: 69.8 G1: 57.6 G1: 51.2
S: 15.2 S: 25.3 S: 36.9 S: 35.0 S: 17.2 S: 16.4 S: 21.2 S: 16.5
G2/M: 20.9 G2/M: 11.4 G2/M: 14.2 G2/M: 13.7 G2/M: 15.7 G2/M: 12.9 G2/M: 16.1 G2/M: 22.3
SubG1: 1.6 SubG1: 1.2 SubG1: 12.4 SubG1: 22.4 SubG1: 0.8 SubG1: 1.0 SubG1: 7.3 SubG1: 6.6
Untreated Pemetrexed (25 nM) Untreated Pemetrexed (25 nM)
UDG-/- cells UDG+/+ cells
0 12.525 50 100 12.525 50100 Pemetrexed (nM) 0 12.525 50 100 12.525 50100
p-Chk1
Chk1
Cyclin D
Cyclin E
p-Histone H3
Histone H3
Cyclin B1
p-Cdc2
Cdc2
Tubulin
6 hr 24 hr
6 hr 24 hr
Figure3 Cellularresponsetouracil-DNAinducedbypemetrexed.Comparisonofcellcycleprogressionbeforeandaftertreatmentwithpemetrexedbetween(a)UDG(cid:2)/(cid:2)
and(b)UDGþ/þcells.ProteinalterationsintheresponsetoDNAdamage,cellcycleprogression,andcelldeathweredetectedin(c)UDG(cid:2)/(cid:2)cellsand(d)UDGþ/þcells
treatedwithpemetrexed.Cellswerecollectedat6and24hafterpemetrexedtreatment.Resultsarerepresentativeoftwoindependentexperiments
(Figures 4c and d). The induction of topo IIa may be We further investigated whether uracil-DNA lesions could
associated with either a global signal of DNA damage or a inhibit DNA replication. Cells were sequentially labeled with
more specific response to the S-phase arrest. As expected, the halogenated nucleotides chlorodeoxyuridine (CldU) and
gH2AX was markedly increased in UDG(cid:2)/(cid:2) cells, which is a iododeoxyuridine (IdU) to stain replication foci. Representa-
well-knownmarkerofDNAdouble-strandbreaks(DSBs)and tive cells are depicted in Figures 5b and c. Compared with
an indicator of stalled replication forks and replication fork replicationfocistainedwithCldUandIdUinuntreatedcells,a
collapse.27 In company with the induction of gH2AX, a significant reduction in IdU incorporation was observed in
significantcleavedPARP(ahallmarkofapoptoticcelldeath) UDG(cid:2)/(cid:2)cellsfollowinga6-hexposuretopemetrexedanda
wasdetectedinUDG(cid:2)/(cid:2)cells.Theactivationofcaspase3,8 further 18-h after incubation, suggesting that pemetrexed-
and 9 (Figures 4c and d) was then measured. Those are inducedaccumulationofuracilhasthepotentialtoinhibitDNA
considered to be the key effect of proteases on apoptosis replication. By contrast, there was no significant change in
through the degradation of numerous cellular substrates CldUandIdUincorporationinthereplicationfociofUDGþ/þ
including the cleavage of PARP. Surprisingly, the peme- cells before and after pemetrexed treatment (25nM). The
trexed-inducedPARPcleavageappearedtobeindependent fluorescencedensityofIdUorCldUwasquantifiedinUDG(cid:2)/(cid:2)
ofthecaspasepathway.Moreover,survivinwasnotedtobe andUDGþ/þ cellsbyusingtheNIHImageJsoftware(NIH,
upregulatedinresponsetopemetrexed.Asamemberofthe Bethesda,MD,USA;Figure5d).ThevaluesoftheratioofIdU
inhibitor of apoptosis protein family, survivin has been toCldUindicatethataccumulateduracilbasesinDNAblock
reported to bind to and inhibit effector caspase-3 and -7.28 DNAreplicationinUDG(cid:2)/(cid:2)cells.
Althoughthedirecteffectoncaspasesstillremaintobefurther
determined,theinductionofsurvivinmaylinktotheinhibition Blocking BER enhances pemetrexed cytotoxicity. We
of caspase 3. Taken together, our results suggest that have shown above that the lack of UDG activity sensitizes
pemetrexed-induced apoptotic cell death may not be tumor cells to antimetabolites. However, UDG deficiency
mediated through the caspase pathway or that other cell appears to be surprisingly rare in human tumors. In fact,
death regulators are more important in pemetrexed-treated human lung cancer express higher UDG than its
cells.29 corresponding normal tissues.30–32 Thus, the removal of
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TheimpactofUDGactivityonpemetrexedcytotocixity
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UDG -/- UDG +/+
3.3±0.7% 30.9±2.3 % 2.3±1.1% 5.0±0.3%
Untreated Pemetrexed (50 μM) Untreated Pemetrexed (50 μM)
UDG -/- UDG +/+
0 12.525 50100 12.525 50100 Pemetrexed (nM) 0 12.525 50100 12.525 50100
Topo II α
Topo I
γ-H2AX
Cleaved PARP
Bax
Bcl-2
Survivin
Caspase 3
Caspase 8
Caspase 9
Tubulin
6 hr 24 hr 6 hr 24 hr
Figure4 Pemetrexed-inducedapoptoticdeathisindependentofcaspasepathway.ThepercentageofcelldeathinducedbypemetrexedusingAnnexinVstainingin
UDG(cid:2)/(cid:2)(a)andUDGþ/þcells(b).Proteinalterationsinvolvedinregulationofapoptoticcelldeathweredetectedin(c)UDG(cid:2)/(cid:2)cellsand(d)UDGþ/þcellstreatedwith
pemetrexed.Cellswerecollectedat6and24hafterpemetrexedtreatment.Resultsarerepresentativeoftwoindependentexperiments
uracilbyUDGandsubsequentBERactivitylimitstheefficacy 110nM in H460 cells. MX was capable of enhancing
of pemetrexedin lung cancer treatment. We next studied a pemetrexed cytotoxicity in both cell lines four- to fivefold
therapeutic strategy to override the UDG-conferred (Figure6e).Thus,althoughmultiplemechanismsmayconfer
resistance to pemetrexed by interrupting the BER pathway resistance to pemetrexed, our results indicated that UDG
using MX. Cytotoxicity was measured in UDGþ/þ and activity in lung cancer cells is an important factor in
UDG(cid:2)/(cid:2) cells after treatment with pemetrexed alone or in pemetrexed-resistance. This was further confirmed by the
combinationwithMX.MXgreatlysensitizedUDGþ/þ cellsto studies,inwhichUDG,inH460cells,wasknockeddownby
pemetrexed and reduced the pemetrexed IC value from siRNA, resulting in the increase in cytotoxicity by threefold
50
220 to 80nM (Figure 6a). By contrast, no differential (data notshown).Importantly,MX reversesthisresistance.
sensitivity between pemetrexed alone and in combination AP sites were detected in H460 cells following treatment
with MX was observed in UDG(cid:2)/(cid:2) cells (Figure 6b). The withpemetrexed.AsshowninFigure7a,theformationofAP
failure of MX to potentiate pemetrexed toxicity in UDG(cid:2)/(cid:2) sites increased as the concentration of pemetrexed in-
cellscanbeexplainedbythefactthat,inUDG(cid:2)/(cid:2)cells,there creased. Co-treatment with MX formed MX-bound AP sites,
are no DNA-binding sites available for MX due to the resulting in the reduction of ARP (aldehyde-reactive probe)-
absence of AP sites in the DNA (Figure 1d). Similar detected AP sites. This is because ARP and MX react
experiments were performed in the human nonsmall cell competitivelywiththealdehydegroupinAPsitesandbinding
lungcancercelllinesH460andA549(Figures6c–e).These ofMXtotheAPsitesmakesthemunavailableforARPbinding
two cell lines retain wild-type p53 and harbor a mutation in (Figure 7a). Furthermore, the levels of UDG protein were
K-ras but express different levels of UDG. Western blotting significantly induced in cells treated with the combination of
revealedthatUDGproteinlevelsinA549wereapproximately pemetrexedandMX(Figure7b).Immunofluorescentstaining
9- and 17-fold higher than in H460 cells and normal lung revealed that the UDG protein was increased in both the
epithelial cells, respectively (Figure 6d). A549 cells were cytosolandthenucleusbutpredominantlyaccumulatedinthe
obviouslymoreresistanttopemetrexedthanH460cells.IC nucleus at 24h after treatment with the combination
50
valuesforpemetrexedwere1200nMinA549,comparedwith (Figure7c),suggestingthatMX-boundAPsiteswereableto
CellDeathandDisease
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CldU Peme (25 nM) ldU ldU
45’ 6 hr 45’ 18 hr 45’
Wash Harvest Harvest
UDG-/- sllec UDG+/+ cells
CIdU IdU Merge CIdU IdU Merge
Control
6 hr-exposure to
Pemetrexed
18 hr post
Pemetrexed
1.5 UDG+/+
Uy) UDG-/-
dsit
Cln
o de 1 **
U tce **
dn
o of Iesce 0.5
Rati(fluor 0
0 6 18
Time (hr)
Figure5 InhibitionofDNAreplicationinducedbypemetrexed.(a)SchematicdiagramoftheCldU,pemetrexedandIdUpulsetreatments.(b)UDG(cid:2)/(cid:2)and(c)UDGþ/þ
cellswerefixedat6and24haftertreatmentsandsubjectedtofluorescentimmunostaining.ReplicationfociwerelabeledwithCldU(green)andIdU(red).Resultsare
representativeoftwoindependentexperiments.(d)TheratioofIdUtoCldUwasmeasuredastheratioofthemeanfluorescentdensityofIdUversusCIdUforeachcell
(n¼40)
trap or stabilize UDG in DNA. In addition, concomitant 543±82mm3 (*P-valueo0.02) in mice treated with either
inductionsoftopoIIa,gH2AX,andcleavedPARPwereseen pemetrexedaloneorincombinationwith MX,respectively.
in cells treated with the combination of pemetrexed and MX Similarly, MX-potentiated antitumor effect of pemetrexed
(Figures7dande).Theseresultscouldbeexplainedbyour wasobservedinA549xenografts,whichwasconsistentwith
previous findings that MX-bound AP sites were capable of the in vitro data. In addition, at these doses, mice did not
poisoning topo IIa and inducing topo IIa-mediated DNA present evidence of systemic toxicity as evaluated by body
DSBs,33triggeringapoptosis. weightmeasurementsandcompletebloodcounttests(data
notshown).
MX potentiates the anti-tumour effects of pemetrexed
Discussion
in vivo. The MX-potentiated antitumour effect of
pemetrexed was further tested in vivo using human lung ThecurrentstudyprovidesevidencethatUDGhasaprofound
cancer xenografts. As shown in Figure 8, both H460 and impact on the cytotoxicity of uracil incorporation into DNA
A549 tumors were moderately sensitive to pemetrexed induced by pemetrexed. Because the expression of UDG in
alone. However, the antitumour activity of pemetrexed was lungcancertissueishighbutvariesbetweenindividuals,UDG
significantly enhanced by the combination of pemetrexed activity may be exploitable as a biomarker for predicting
(150mg/kg)andMX(4mg/kg)inthesetwoxenografttumors. susceptibility and response to pemetrexed-related treat-
At15days,H460xenograftstreatedwithPBS(controlgroup) ment. Moreover, our results demonstrated that the loss of
had a mean tumor volume of 2100±106mm3, compared UDG results in the accumulation of uracil in DNA, which
with a mean tumor volume of 1726±176mm3 or stalls replication forks and subsequently induces replication
CellDeathandDisease
TheimpactofUDGactivityonpemetrexedcytotocixity
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100 100
al al
v v
Survi 10 * Survi 10
% Peme % Peme
Peme + MX
Peme + MX
1 1
0 100 200 300 400 500 0 25 50 75 100 125
*P < 0.02Pemetrexed (nM) Pemetrexed (nM)
1500
100
Peme **
al 10 H460 A549 NC M)1000 Peme+ MX
v n
% Survi 1 *+MX UTuDbGlin IC50 ( 500
*
0.1 0
0 100 200 300 400
H460 A549
*P < 0.02Pemetrexed (nM) *P < 0.02 **P < 0.01
Figure6 ThepotentiationofpemetrexedcytotoxicitybyMX.(aandb)CytotoxicityofpemetrexedaloneandincombinationwithMXwasexaminedbyaclonogenic
survivalassayinUDGþ/þandUDG(cid:2)/(cid:2)cells.(c)MXpotentiatedthecytotoxicityofpemetrexedinH460cells.(d)ComparisonofUDGproteinlevelsbetweenH460andA549
bywesternblottingassay.(e)ComparisonofsensitivitytopemetrexedbetweenH460andA549cells.Resultsarerepresentativeofthreeindependentexperiments
35
elative to control 12235050 PPeemmeettrreexxeedd + MX MX-AP C P P+M P P+M P P+M M TUuDbGulin
P sites r 150 * * * 100 200 400(nM)
A 0
0 100 200 400
*P < 0.02 Pemetrexed (nM)
Untreated MX Peme Peme Peme + MX
C M P P + M
Peme + MX
Cleaved PARP
γ H2AX
Topo II α
6 hr 24 hr Tublin
Figure7 MX-boundAPsitesinducedbythecombinationofpemetrexedandMXarelethalDNAlesions.(a)Thedose-dependentrelationshipbetweenthelevelsofAP
sitesandtheconcentrationsofpemetrexed.TheAPsitesinDNAweremeasuredusingARPreagentafterH460cellsweretreatedwithpemetrexedalone(0–400nM)orin
combinationwithMX(6mM)for24h.MX-boundAPsitesweredeterminedbythedifferencesbetweenAPsitesinducedbypemetrexedaloneandthecombinationof
pemetrexedandMX.(b)UDGinductiondetectedincellstreatedwiththepemetrexedaloneandincombinationwithMX.CellswerecollectedandUDGproteinwasmeasured
bywesternblottinganalysis.(c)H460cellsgrownonthecoverslipweretreatedwithpemetrexedalone(100nM)andincombinationwithMX(6mM)for24handsubjectiveto
thefluorescentimmunostaining.ThesignalofUDGprotein(green)wassignificantlyenhancedandlocalizedinnucleus(blue)ofcellstreatedwiththecombinationof
pemetrexedandMX.(d)gH2AXfociformationdetectedinH460cellstreatedwithpemetrexed(100nM)aloneandincombinationwithMX(6mM)for24h;(e)Inductionofthe
cleavedPARPandgH2AXproteinswasdetectedbywesternblottingincellswiththesametreatments
collapse,leadingtoremarkableincreasesinthekillingeffect UDG removal of incorporated uracil links the DNA
of pemetrexed. These studies lead us to propose that, as a replication process. The progression of the DNA
therapeutic target, UDG may offer new avenues for the replication fork can be stopped by various factors, including
developmentofnovelanticanceragents. DNA damage, protein–DNA complexes, and depletion of
CellDeathandDisease
TheimpactofUDGactivityonpemetrexedcytotocixity
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H460 multiproteincomplexwithRPAandPCNA,bothofwhichare
2500 involved in various aspects of DNA replication.9,10
UDG-initiated BER operates at replication foci, suggesting
Control
2000 that UDG-mediated repair may directly and indirectly affect
3m) Pemetrexed the progression of DNA replication.35 Third, a significant
Pemetrexed+ MX
m
increase in the expression of topo IIa was observed in
me ( 1500 MX responsetopemetrexedexposure.Topoisomerasessustain
olu the progression of the replication machinery by removing
or v 1000 supercoilsaheadofthereplicationfork.Theinductionoftopo
m IIa may indicate that the progression of DNA replication is
Tu 500 * halted by pemetrexed-induced DNA damage. In UDG(cid:2)/(cid:2)
cells, gH2AX and PARP cleavage were concomitantly
increased with induction of topo IIa, suggesting that
0
replication fork collapse occurs when the replication
0 2 4 6 8 10 12 14 16 machinery arrives at the uracil-containing DNA template,
*P<0.02 Days leadingtocelldeath.Bycontrast,UDGþ/þ cellsweremore
resistanttotopemetrexed-inducedcelldeath.
2500 A549
UDG activity determines the levels of AP sites, key
Control
2000 targetsofMX. WhenuracilbasesarepresentinDNA,UDG
3m) PPeemmeettrreexxeedd+ MX makes almost all of its contacts with the uracil-containing
m DNA strand. The binding of UDG transiently forces DNA to
e ( 1500 MX undergo local distortions to flip out the uracil base.35 By
m
u contrast,normalbasesresistthedistortion,sothattheUDG
ol
or v 1000 * active site only allows uracil to bind in extra helical DNA,
m facilitatingtheUDGenzymaticexcisiontoproduceAPsites.
u
T 500 Interestingly, UDG remains bound to the AP site after
releasing the uracil base until the AP site is transferred to
APE1, thereby displacing UDG from an AP site. In fact,
0
bindingofUDGtoitsAPsiteresultintheinactivationofUDG
0 5 10 15 20 25 30
activitybecausethedissociationofUDGfromtheAPsiteby
*P<0.05 Days APE1isarate-limitingstep.WhenMXispresent,MXrapidly
bindstoAPsitesandmakesthemunrecognizablebyAPE1,
Figure8 MXsynergisticallyenhancesantitumoreffectofpemetrexed.Human
blockingthe crosstalkof APE1 with other BER proteins and
nonsmallcelllungcancerxenograftsweregrowninathymicnudemice.Whentumor
volumeofH460(a)orA549(b)reached100mm3,micereceivedthetreatmentwith interrupting BER activity. Importantly, the failure of
PBS(control),MX(4mg/kg),pemetrexed(150mg/kg),andpemetrexedplusMX,i.p recognition of MX-bound AP sites by APE1 would result in
injection/dailyfor5days.Tumorvolumewasmeasuredandusedtodeterminethe trapping/stabilising UDG in DNA. Although speculative, it is
therapeuticeffect possiblethattheexceptionallystrongbindingofUDGtoMX-
boundAPsitesformsprotein–DNAcomplexes,leadingtothe
obstructionofreplicationandcatastrophicDNADSBs.Thisis
nucleotide pools. It has been reported that the role of BER consistent with our observation that the combination of
becomes critical when damaged bases are produced or pemetrexed and MX induced UDG that was predominantly
persistatreplicationforks.34Onthebasisofourresults,we locatedinthenucleus,accompaniedbyasignificantincrease
suggestthattheaccumulationofuracilbasesinDNAwould in gH2AX. In addition to stopping the progression of DNA
stallDNAreplicationforks,whichimmediatelyactivatesDNA replication, MX-AP sites are also capable of poisoning topo
damage checkpoint pathways and robust mechanisms of IIaandinducingtopoIIa-mediatedDNADSBs.36–39Because
DNAdamagerepair.Thefollowinglinesofevidencesupport productionofthelethalMX-APsitedependsonUDGactivity,
thisconclusion.First,usingUDGþ/þ and UDG(cid:2)/(cid:2) cells,we UDGisanessentialfactorforMX-potentiationofpemetrexed
demonstratedthatcrosstalkbetweenuracilincorporationand cytotoxicity.
DNA damage checkpoints was mediated by UDG. When
uracil bases were present in DNA, particularly at replication Targeting UDG/BER enhances cytotoxicity of
forks,theDNAdamagecheckpointappearedtousetheATR/ pemetrexed through a synthetic lethal mechanism. A
chk1–cyclin/cdk network to stop damaged cells in the S- or synthetic lethal mechanism, in which tumor cells, but not
M-phase until repairs were made (such as through efficient normal cells, are specifically targeted for killing is highly
repairbyUDG)ortotriggercelldeathifrepairscouldnotbe attractive. The concept of synthetic lethality is based on the
made (as in UDG-deficient cells). Second, a fundamental reliance of cancer cells on DNA repair to maintain cell
level of coordination between BER and DNA replication is division.Becausecancercellsare‘addicted’toDNArepair,40
supported by evidence that BER proteins are active during inhibitingDNArepairimpedescancercellreplication,leading
DNA replication. In particular, nuclear UDG has been tocelldeath.Thepresentstudydemonstratesthattargeting
recently discovered to localize in replication foci to form a BERwithMXenhancespemetrexedcytotoxicitybythedual
CellDeathandDisease
TheimpactofUDGactivityonpemetrexedcytotocixity
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9
inactivationofUDGandtopoIIa,throughtrappinginMX-AP andgenomicDNAwasextractedbyphenol/chloroform.DNA(40mg)wasincubated
sites.AstheexpressionofUDGandtopoIIa´ ismuchhigher with10UofpurifiedUDG(NewEnglandBiolabs)in60mlofreactionbufferat371C
in tumor cells than in normal bone marrow cells,33,36 the for2h.Thereactionproductsweredriedat351CinaTurbovapunderastreamof
nitrogenandreconstitutedin150ml90%acetonitrile.Theanalytewasretainedbyan
MX-potentiated synergistic killing effect can be exploited to
Atlantis Hilic Silica analytical column (Waters Corporation, Milford, MA USA)
minimize hematopoietic toxicity. Therefore, BER-targeted,
(2.1(cid:3)100mm,3.5mM)andelutedisocraticallybyamixtureof90%acetonitrileand
synthetic lethal anticancer therapy has important clinical 10%2.0mMammoniumformateataflowrateof0.2ml/min.Thedetectionwascarried
implications. outbyanAPI3200MS/MSmassspectrometer(ABSCIEX,FosterCity,CA,USA).
Immunofluorescencemicroscopy. Cellsweregrownoncoverslipsand
MaterialsandMethods
weretreatedwithdrugsfor6–24h.Thencellswerefixedin2%paraformaldehyde
Cellsandreagents. StableandcompleteknockdownofUDGexpressionin
andpermeabilizedwith0.2%TritonX-100.CellswereincubatedwithprimaryUDG
DLD1 colon cancer cells (DLD1/UDG(cid:2)/(cid:2) cells) was achieved by homologous
(Abcam,Cambridge,MA,USA)orgH2AX(Bethyl,Montgomery,TX,USA)antibody
recombination,asdescribedbyZhangetal.41Briefly,homologousrecombination
for 1h at room temperature, followed by incubation with a secondary antibody
resultedininsertionofaDNAconstructcontainingaseriesofstopcodonswithin
conjugated with Alexa 488 (green) (Molecular Probes, Carlsbad, CA, USA).
Exon1oftheUNGgenetointerrupttranscriptionofboththemitochondrialand
ThenucleuswasstainedusingHoechst33258for15minatroomtemperature.
nuclear UNG isoforms. Recombinant virus was grown in 293T cells and
ImagesweredigitallycapturedusinganOlympusmicroscope(Olympus,Westmont,
subsequentlyusedtoinfectDLD1cells.Selectionofpositivelytransfectedclones
IL,USA)equippedwithadigitalcamera.
wasachievedbytheadditionofG418totheculturemedium.Asecondinfectionof
transfected clones with Cre-recombinase adenovirus caused excision of the
Westernblotanalysis. Cellularproteinwasquantifiedspectrophotometrically
neomycin-resistantcassette.DflagcelllinewasproducedbytransfectionofUDG
usingtheBio-Radassay.Equalamountsofproteins(30mg)wereseparatedby
expressionvectorinUDG(cid:2)/(cid:2)cellstorestoreUDGactivity.
SDS-PAGEandtransferredtopolyvinylidenefluoridemembrane(MilliporeCorp.,
Pemetrexed was obtained from LC Laboratories (Woburn, MA, USA),
Bedford,MA,USA).Themembranewasincubatedwithprimaryantibodyin1%
5-Fluorouracilandcisplatinwerepurchased fromSigma-Aldrich(St.Louis,MO,
nonfatdrymilksolutionovernightat41CandfollowedbytheincubationwithHRP-
USA)andtemozolomidewaspurchasedfromOchemInc.(DesPlaines,IL,USA).
conjugated secondary antibody at room temperature for 1h. Proteins were
UracilDNAglycosylasewaspurchasedfromNewEnglandBiolabs(Ipswich,MA,
visualized by ECL (Amersham Corp, Piscataway, NJ, USA) according to the
USA)andAPE1waspurchasedfromTrevigen(Gaitherburg,MD,USA).MX,uracil,
manufacturer’sinstructions.Sourcesofprimaryantibodywereasfollows:cleaved
and uracil-1,3-15N were purchased from Sigma-Aldrich. MX was dissolved in
2 PARP (BD Pharmingen, San Jose, CA, USA), gH2AX (Bethyl), pChk1, Chk1,
sterilizedwater(pH7.0)atastocksolutionof2.5Mandstoredat(cid:2)201C.Working
pcdc2,cyclinB1,topoIIa,topoisomeraseI,Bax,andBcl2(CellSignaling,Danvers,
solutionsweregeneratedbeforeexperimentaluse.Fluorescentdye-labeled40-mer
MA,USA),phospho-histoneH3(UpstateBiotechnologies,Billerica,MA,USA)and
oligonucleotidescontainingU:GmispairswerepurchasedfromOperonBiotechnol-
a-tubulin(Sigma-Aldrich).
ogies(Huntsville,AL,USA).
Cellcycleanalysis. Forcellcycleanalysis,106cells(DLD1/UDGþ/þ and
Colonysurvivalassay. Tumorcells(500–2000/dish)wereplatedandtreated DLD1/UDG(cid:2)/(cid:2)) were plated in 100-mm tissue culture dishes and exposed to
with pemetrexed (0–400nM), 5-FU (0–20mM), temozolomide (0–1500mM), or
pemetrexed(25nM).After6,24,and72hofculture,cellswerefixedwith80%
cisplatin(0–40mM).After7days,survivingcolonieswerestainedwithmethylene
methanolandwashedwithice-cold1%BSA/PBS.DNAwasstainedwith20mg/ml
bluefor30minatroomtemperatureandthecoloniescontainingmorethan50cells
propidiumiodide(Sigma-Aldrich)and2.5mg/ml of DNase-freeRNaseA(Roche,
werecountedtogeneratesurvivalcurves.
Branford,CT,USA).TheDNAfluorescenceofpropidiumiodide–stainedcellswas
measuredwithanEliteESPflowcytometer/cellsorter(Coulter,Miami,FL,USA).
Glycosylase activity assay. UDG activity (purified protein or whole-cell
extracts)wasmeasuredusinganoligodeoxynucleotidecontainingasingleuracil. Immunofluorescencemicroscopyofreplicationfocistainedwith
50-[HEX]GTAAAACGACGGCCAGTGUATTCGAGCTCGGTACCCGGGG CldU and IdU. DNA replication sites were visualized by incorporation of
30-CATTTTGCTGCCGGTCACGGAAGCTCGAGCCATGGGCCCC[Cy5]. CldU andIdU into DNA. UDG(cid:2)/(cid:2)andUDGþ/þcellswere labeled with 100mM
The fluorescent dye-labeled duplex oligonucleotides were incubated with purified CldU (ICN,Irvine,CA,USA)orIdU (Sigma-Aldrich) for45min atdifferenttime
UDGat371Cfor30min,followedby30minincubationwithAPE1orwithwhole-cell intervals.CellswerewashedwithPBS,fixedwithcold70%ethanolandstoredat
extract.Thereactionwasstoppedbyincubationat951Cfor5min.Reactionproducts, 41C.Forantibodystaining,theethanolwasremoved,and100%methanolwas
18-mer fragments, were resolved by electrophoresis on denaturing 20% addedfor5min.CellswerewashedtwicewithPBSandincubatedwith1.5MHClfor
polyacrylamidegels(7Murea,1(cid:3)Tris-borate-EDTA).Visualizationandquantitation 30mintodenaturetheDNA.CellswerewashedwithPBS,permeabilizedwith0.5%
wasachievedusingaTyphoon9200fluorescenceImager(AmershamBioScience, Tween-20inPBSfor5min,andthenincubatedin5%BSA(Sigma-Aldrich)andin
Piscataway,NJ,USA).UDGactivitywasdeterminedbasedonfluorescencedensity PBSfor20mintoreducenonspecificbinding.PrimaryantibodiesCldU(ratanti-
quantifiedusingtheImageQuantsoftware(AmershamBioScience). BrdU;AccurateChemicalandScienceCo.,Westbury,NY,USA)andIdU(mouse
anti-BrdU;BDBiosciences)weredilutedin1%BSAbuffer,addedtotheslides,and
APsiteassay. ThenumberofAPsiteswasmeasuredusingARPreagent.The incubatedinahumidenvironmentfor2h.SlideswerewashedwithPBS-Tween20
assay was performed as previously described.42 Briefly, cells (2(cid:3)106) were andtheninahigh-saltbuffer(200mMNaCl,0.2%Tween-20,and0.2%NP-40in
collectedafterdrugtreatmentandDNAwasextracted.DNA(10mg)wasincubated PBS)for15min.Thesampleswereincubatedwithsecondaryantibodiesfor1h.
with 15ml of 1mM ARP (Dojindo Laboratories, Kumamoto, Japan), and then Finally,slideswerewashedwithPBS-Tween20,mountedwithVectashieldantifade
precipitatedandwashedwithice-coldethanol.TheARP-labeledDNAwasthen mountingmedium(VectorLaboratories,Inc.,Burlingame,CA,USA),andstoredat
heat-denaturedat1001Cfor5min,quicklychilledoniceandmixedwithanequal 41C. Images were visualized on a Nikon EclipseTE-300 confocal microscope
volumeof2Mammoniumacetate.TheDNAwasthenimmobilizedonaBA-S85 (JenoptikLaserTechnologiesCorp.,Brighton,MI,USA).Fluorescencedensityof
nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) using a CldUorIdUwasquantifiedbytheNIHImageJsoftware.
minifold II vacuum filter device (Schleicher and Schuell). The membrane was
incubated with 0.25% BSA-PBS containing streptavidin-conjugated horseradish Xenografttumorsinnudemice. H460orA549tumorcells(5(cid:3)106)were
peroxidase(BioGenex,SanRamon,CA,USA)atroomtemperaturefor40minwith injectedintobilateralflanksoffemaleathymicNCr(nu/nu)mice(6weeksold).When
gentle shaking. ARP-labeled AP sites were visualized by chemiluminescence the tumor volumes reached 100–150mm3, mice were divided into control and
(Amersham)followedbyquantitativedensitometryusingtheNIHImageJsoftware. treatmentgroups(6–9mice/group).Nudemicecarryingtumorsweretreatedwith
pemetrexed(150mg/kg)alone,MX(4mg/kg)alone,orthecombinationofthetwo
Detection and quantification of uracil using HPLC/MS/MS agents, by daily intraperitonial injection (i.p.) for 5 consecutive days. Tumor
analysis. UDG(cid:2)/(cid:2)andUDGþ/þ cellswereexposedtopemetrexed(10mM) measurements were taken every 2 days. Tumor responses were quantified by
or5-FU(10mM)for6,24,48and72h.Atindicatedtimepoints,cellswereharvested tumorvolume.
CellDeathandDisease
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ADBulgaretal
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