Oxidation-Mediated DNA Cross-Linking Contributes to the Toxicity of 6-Thioguanine in Human Cells

Published OnlineFirst July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278

Cancer Therapeutics, Targets, and Chemical Biology Research

Reto Brem and Peter Karran

Abstract The thiopurines azathioprine and 6-mercaptopurine have been extensively prescribed as immunosuppressant and anticancer agents for several decades. A third member of the thiopurine family, 6-thioguanine (6-TG), has been used less widely. Although known to be partly dependent on DNA mismatch repair (MMR), the cytotoxicity of 6-TG remains incompletely understood. Here, we describe a novel MMR-independent pathway of 6-TG toxicity. Cell killing depended on two properties of 6-TG: its incorporation into DNA and its ability to act as a source of reactive oxygen species (ROS). ROS targeted DNA 6-TG to generate potentially lethal replication-arresting DNA lesions including interstrand cross-links. These triggered processing by the Fanconi anemia and homologous recombination DNA repair pathways. Allopurinol protected against 6-TG toxicity by acting as a ROS scavenger and preventing DNA damage. Together, our ﬁndings provide mechanistic evidence to support the proposed use of thiopurines to treat HR-defective tumors and for the coadministration of 6-TG and allopurinol as an immu- nomodulation strategy in inﬂammatory disorders. Cancer Res; 72(18); 1–9. 2012 AACR.

Introduction exports thiopurine mononucleotides from cells (5). TPMT The clinical effectiveness of the immunosuppressant, anti- expression is an important determinant of the clinical effec- inflammatory and anticancer thiopurines: azathioprine, 6- tiveness of thiopurines. Polymorphic TPMT variants with fi mercaptopurine (6-MP) and 6-thioguanine (6-TG) relies on signi cantly reduced activity are associated with high TGN their ability to selectively kill dividing immune effector or levels and extreme, potentially lethal, thiopurine toxicity. High cancer cells. Surprisingly, despite successful use for over 50 TPMT activity is associated with lower intracellular TGN fi years (1), the mechanisms by which they cause the death of concentrations and reduced clinical ef cacy (6). Prevention their target cells are still incompletely understood. Thiopurines of cell proliferation by limiting the supply of purine nucleotides are prodrugs. They are metabolized to the 6-TG nucleotides that are essential for DNA and RNA synthesis is another (TGN) that early studies identified as important determinants potential contributor to thiopurine toxicity. In this case, TPMT of toxicity. TGN are substrates for incorporation into DNA and methylates thioinosine monophosphate (TIMP), a metabolite the accumulation of DNA 6-TG is a major factor in thiopurine of azathioprine and 6-MP, to generate methyl-TIMP, a pow- fi de novo toxicity. Other contributors include the formation of toxic erful inhibitor of the rst enzyme in the pathway of thiopurine metabolites and oxidation of DNA 6-TG by exog- purine nucleotide biosynthesis. Purine nucleotide depletion enous chemicals or ultraviolet A (UVA) radiation (for review does not explain all thiopurine cytotoxicity, however, (7) and see ref. 2). the potently cytotoxic 6-TG is metabolized to TGN by a The most widely prescribed thiopurines, 6-MP and its different route that does not generate TIMP. prodrug azathioprine, are converted to TGN in several steps The DNA mismatch repair (MMR) system is also a major (for review see ref. 3). Conversion is counteracted by the contributor to thiopurine toxicity. DNA 6-TG deceives MMR activities of 3 enzymes: thiopurine methyltransferase (TPMT), into a potentially lethal intervention and, as a consequence fi which inactivates thiopurines by methylation (4), xanthine MMR-defects confer signi cant thiopurine resistance (for fi oxidase (XO), which catabolizes 6-MP and azathioprine to review see ref. 8). MMR-de cient cells do, however, retain a inactive thiouric acid, and the MRP4 transporter protein that susceptibility to killing by high thiopurine concentrations, indicating the existence of MMR-independent thiopurine cytotoxicity (9). Authors' Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, United Kingdom DNA 6-TG is also implicated in a cytotoxic pathway, which involves oxidative DNA damage. Cells containing DNA 6-TG Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). are hypersensitive to reactive oxygen species (ROS) generated chemically, biologically (10), or photochemically by UVA (11). Corresponding Author: Peter Karran, Cancer Research UK London fl Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, ROS in ict widespread DNA damage. In addition to the well- United Kingdom. Phone: 44-1707625870; Fax: 44-1707625812; E-mail: characterized DNA 8-oxoGuanine (12), they cause oxidation of [email protected] DNA 6-TG itself. Among these oxidation products, guanine-6 doi: 10.1158/0008-5472.CAN-12-1278 sulfonate (GSO3; ref. 11) and guanine-sulfinate (GSO2; ref. 13), 2012 American Association for Cancer Research. DNA interstrand cross-links (ICL; ref. 14), and DNA-protein

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cross-links (15) have been identified as replication-blocking RNA interference and potentially cytotoxic DNA lesions. The siRNA duplex smart pools were purchased from Dhar- Here we describe 6-TG-mediated cytotoxicity that requires macon. Cells were transfected using Lipofectamine RNAiMAX the incorporation of 6-TG into DNA but is independent of (Invitrogen) or Dharmafect (Dharmacon) according to the MMR, exogenous sources of ROS, and of UVA. It does require an manufacturers' instructions, with a final siRNA concentration oxidizing environment, however, and we show that 6-TG itself of 50 nmol/L. Cells were subcultured into normal medium 24 provides this by depleting endogenous antioxidant defenses hours after transfection. and thereby increasing steady-state ROS levels. Cell killing reflects the formation of potentially lethal DNA lesions that Cell survival, comet assay, 3H-thymidine incorporation inhibit replication. Its effects are particularly marked in cells To determine cell survival, treated cells were seeded into 96- with defects in the Fanconi anemia (FA) and homologous well plates (1,000 cells per well) in normal medium. Viability recombination (HR) pathways and cells with defects in either was assayed 5 days later using the MTT assay. For clonal pathway are extremely sensitive to 6-TG (9, 14, 16). Together survival, 500 cells per well were seeded into 6-well plates and the FA and HR pathway protect cells against DNA damage that colonies were counted 7 to 10 days later. Each analysis was arrests replication (reviewed in ref. 17). FA-deficient cells are carried out in triplicate. typically hypersensitive to killing, chromosome breakage and DNA interstrand cross-linking was determined by the comet particularly to radial chromosome induction by agents that assay. Cells were grown in the presence of 6-TG for 48 hours. cause DNA ICLs. They exhibit a similar pattern of sensitivity to They were then irradiated with 5 Gy IR and lysed. Following 2 6-TG (14), and we show that ICLs are among the DNA lesions hours digestion at 37C with 1 mg/mL Proteinase K (Roche) that efficiently block DNA replication in cells treated with 6-TG. they were analyzed by the alkaline comet assay as described (19). Materials and Methods DNA replication was assessed by measuring [3H]-thymidine Cell culture incorporation. Cells were treated with 6-TG and then pulsed The MMR-defective human leukemia cell line CCRF-CEM with [50 3H]thymidine (1 mCi/mL, 511 GBq/mmol) in normal was grown in RPMI, all other cells in Dulbecco's Modified medium for 30 minutes. Trichloroacetic acid insoluble radio- Eagle's Medium. Media were supplemented with 10% fetal calf activity in duplicate samples of 2 106 cells was determined by serum. The Fanconi anemia (FANCA / ) and wild-type scintillation counting. þ þ (FANCA / ) mouse embryonic fibroblasts (MEF) have been described (18). Their status was confirmed by isoenzyme Results analysis and DNA fingerprinting (January 2010). MSH2-defec- MMR-independent 6-TG sensitivity tive (HeLa-MSH2) and their control transfectant HeLa SilenciX Cells derived from FA patients are surprisingly sensitive to (HeLa-SX) cells (tebu-bio Cat Nr 01-00023) were cultured in the thiopurines (16). An early study (20) reported that FANCG- presence of hygromycin B. Nontransfected HeLa and MLH1- defective Chinese hamster ovary cells are even more sensitive deficient colon cancer cells HCT116 were obtained from to 6-TG than to acknowledged ICL-inducing agents such as Cancer Research UK Central Cell Services. Their identity was mitomycin C and diepoxybutane. This extreme 6-TG sensitivity confirmed by isoenzyme analysis and short tandem repeat was also apparent in FANCA / MEFs that were more than þ þ profiling (March 2011) and skin fibroblasts derived from a four-fold more sensitive to 6-TG than their FANCA / coun- Lesch-Nyhan syndrome patient (GM03467) (isoenzyme anal- terparts (Fig. 1A). FA-defective human cells were also hyper- ysis November 2010) were obtained from The Coriell Institute sensitive to 6-TG. siRNA-mediated depletion of FANCD2, a key (Camden, NJ). component of the FA pathway, significantly increased 6-TG sensitivity of HeLa cells (Fig. 1B). Importantly, FANCD2 silenc- ROS and glutathione detection ing in MMR-deficient HCT116 cells also resulted in 6-TG To measure ROS, trypsinized cells were washed once in PBS hypersensitivity (Fig. 1C), indicating that the FA pathway and incubated in 5 mmol/L CM-H2DCFDA (Invitrogen) in PBS provides protection against the cytotoxicity of 6-TG that is for 20 minutes at 37C. They were then washed twice in PBS partly independent of MMR. and green fluorescence was analyzed by flow cytometry. Total and oxidized levels of glutathione (GSH) were measured using MMR-independent activation of the FA pathway by 6-TG the Glutathione Assay (Trevigen) according to the manufac- Monoubiquitination of the FANCD2 protein is a sensitive turer's protocols. indicator of FA pathway activation by replication-arresting DNA damage. Western blotting revealed that 6-TG treatment Immunoblotting induced this FANCD2 modification in both MMR-proficient Whole cell extracts were prepared using radioimmunopre- and MMR-defective cells. FANCD2 monoubiquitination was cipitation assay buffer. Proteins (50 mg) were separated on 3% apparent in MMR-defective HeLa-MSH2 and HCT116 cells to 8% Tris-acetate polyacrylamide gels (Invitrogen). After following treatment with 6-TG concentrations of 0.8 mmol/L transfer, membranes were probed with antibodies against and above—approximately 2- to 3-fold higher than the con- FANCD2 (Novus Biologicals), MSH2 (BD Pharmingen), or XO centrations required to trigger FANCD2 activation in MMR- (Abcam). Antigen–antibody complexes were visualized using proficient HeLa-SX cells (Fig. 2). We conclude that 6-TG ECL blotting detection agent (GE Healthcare). treatment activates the FA pathway and that activation is

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catabolizing enzyme XO detoxiﬁes mercaptopurine in a reaction that generates ROS, although its role in 6-TG A 100 catabolism is less clear. We investigated whether XO was the source of the ROS produced by 6-TG. When HCT116 cells were treated with 6-TG in the presence of allopurinol, an acknowledged XO inhibitor (21), ROS levels declined to those 10 of untreated cells (Fig. 3B). The lower ROS levels in allopu- ﬁ

Survival (%) rinol-treated HCT116 cells were associated with a signi - FancA+/+ FancA–/– cantly reduced 6-TG sensitivity. Whereas treatment with 0.8 mmol/L 6-TG for 48 hours reduced cell viability to <50%, 1 0 0.06 0.12 0.18 0.24 inclusion of allopurinol reversed this toxicity and cell sur- 6-TG (μmol/L) vival was comparable to that following treatment with B allopurinol alone (Fig. 3C). Allopurinol also signiﬁcantly 100 increased the concentration of 6-TG required to trigger MMR-independent FANCD2 ubiquitination. In the absence of allopurinol, monoubiquitinated FANCD2 was detectable in extracts of HCT116 cells treated with 0.4 mmol/L 6-TG. When 6-TG treatment was carried out in the presence of 10 allopurinol, FANCD2 was not detectably ubiquitinated even after treatment with 1.2 mmol/L 6-TG (Fig. 3D). Allopurinol

Survival (%) HeLa sicontrol mocktransf. siControl siFancD2 also protected MSH2-deficient CCRF-CEM cells against HeLa siFancD2 FANCD2 β-Actin FANCD2 activation (data not shown). Allopurinol is a powerful XO inhibitor at micromoles per 1 liter concentrations. In our experiments, however, protection 0 0.5 1 1.5 2 against 6-TG toxicity and FANCD2 modification required its 6-TG (μmol/L) C inclusion at millimoles per liter levels. This suggested that 100 its effect was not a consequence of XO inhibition. To examine this possibility, cells were treated with 6-TG in the presence or absence of an alternative XO inhibitor, febuxostat (22). Febuxo- stat concentrations up to 200 mmol/L had no detectable effect on ROS levels, FANCD2 ubiquitination or HCT116 cell survival 10 (data not shown), confirming that XO was not the source of ROS. In addition, Western blotting and direct assays of XO Survival (%) HCT116 sicontrol activity indicated that, with the exception of HeLa cells, XO was HCT116 siFancD2 siControl siFancD2 undetectable in cultured cells including HCT116, CCRF-CEM FANCD2 and several lymphoblastoid cell lines (Supplementary Fig. S1). 1 β-Actin 0 0.5 1 1.5 2 These findings indicate that allopurinol protection against 6-TG (μmol/L) 6-TG is independent of its ability to inhibit XO. It therefore most likely reflects the ability of allopurinol to act as a ROS scavenger. þ þ Figure 1. 6-TG cytotoxicity. A, MEFs. FANCA / and FANCA / MEFs Because 6-TG metabolism by XO was excluded as a possible were grown in the presence of 6-TG at the concentrations as indicated source of ROS, we examined whether 6-TG increased ROS for 16 hours. They were then returned to normal medium and survival was assessed by clonal assay 7 days later. B and C, human cells. levels by depleting antioxidant protection. GSH is one of the FANCD2 was depleted by siRNA transfection of HeLa (B) and HCT116 (C) most important cellular antioxidants. Figure 4A shows that cells. Three days after transfection, cells were seeded into 96-well plates 6-TG induces a dose-dependent reduction of GSH levels in both (1,000 per well) in medium containing the indicated doses of 6-TG. HCT116 and HeLa-MSH2 cells. The depletion was exacerbated Two days later, the medium was replaced with normal medium and growth continued for a further 3 days. Survival was determined by MTT by cotreatment with buthionine sulfoxide (BSO), a GSH assay. Data are means of at least 3 experiments. FANCD2 levels were synthesis inhibitor that prevents GSH replenishment. BSO and analyzed by Western blotting (inserts) 4 days after transfection. allopurinol had opposite effects. In HeLa-MSH2 cells, BSO potentiated 6-TG mediated ROS formation (Fig. 4B). It was associated with FANCD2 monoubiquitination at lower 6- partly independent of MMR-mediated processing of DNA TG concentrations (Fig. 4C) and an enhanced sensitivity to 6-TG. killing by 6-TG (Fig. 4D). Similar results were obtained in HCT116 cells (data not shown). We conclude that 6-TG treat- 6-TG sensitivity and FA activation: the role of ROS ment increases steady-state ROS levels by depleting the levels FACS analysis of cells stained with CM-H2DCFD, a report- of protective GSH. The ensuing oxidative stress results in the er for ROS, revealed that 6-TG treatment induced a dose- formation of potentially lethal DNA lesions that activate the dependent increase in intracellular ROS (Fig. 3A). The purine FA pathway.

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HeLa-SX HeLa-MSH2 HCT116 6-TG (μmol/L) 0 0.4 0.8 1.2 0 0.4 0.81.2 0 0.40.20.8 FANCD2-ubiq FANCD2 β-Actin

MSH2

Figure 2. Mismatch repair-independent activation of FANCD2. Mismatch repair-proficient (HeLa-SX) or repair-defective (HeLa-MSH2 and HCT116) cells were grown in the presence of the indicated concentrations of 6-TG for 48 hours. Western blots of extracts were probed with antibodies against FANCD2. The positions of unmodified (FANCD2) and activated FANCD2 (FANCD2-ubiq) are arrowed. The MSH2 deficiency of HeLa-MSH2 cells was confirmed by reprobing the blots with anti-MSH2. b-Actin served as a loading control.

6-TG sensitivity and FA activation: the role of DNA 6-TG DNA was also a requirement, we examined the effects of The observations described above firmly implicate 6-TG on Lesch-Nyhan GM03467 fibroblasts. These HPRT- increased ROS levels in MMR-independent 6-TG cytotoxic- negative cells are extremely resistant to 6-TG and do not ity. To address whether the ROS generated from 6-TG are incorporate 6-TG into DNA. CM-H2DCFD staining and sufficient for toxicity or whether incorporation of 6-TG into FACS analysis indicated that 6-TG treatment of GM03467

0.4 μmol/L 6-TG Untreated 0.8 μmol/L 6-TG Untreated +6-TG μ 1.2 mol/L 6-TG + allop. +6-TG Number of cells Number of cells

1 1 ROS fluorescence ROS fluorescence (arbitrary units) (arbitrary units)

CD100 0 0.4 0.8 1.2 6-TG (μmol/L) 80 –+–+ –+ –+allop. FANCD2-ubiq. 60 FANCD2 β-Actin 40 % survival

0 6-TG – – + + allop. – + – +

Figure 3. ROS generation by 6-TG and the protective effects of allopurinol in HCT116 cells. A, 6-TG dose dependence of ROS production. Cells were harvested after treatment for 48 hours with 0, 0.4 mmol/L, 0.8 mmol/L, or 1.2 mmol/L 6-TG as indicated. ROS levels were determined by FACS. The ﬂuorescence intensity is expressed relative to that of untreated cells. B, protection by allopurinol against ROS. Cells were treated with 1.2 mmol/L 6-TG for 48 h in the presence or absence of 500 mmol/L allopurinol. ROS production was analyzed as described in A. C, allopurinol protection against 6-TG toxicity. Cells were grown in the presence of 0.8 mmol/L 6-TG 500 mmol/L allopurinol for 48 hours and seeded into 96-well plates. Survival was determined 6 days later by MTT assay. The data are means of 3 independent experiments. D, prevention of FANCD2 activation by allopurinol. Cells were treated for 48 hours with the concentrations of 6-TG as indicated in the presence or absence of 500 mmol/L allopurinol. Western blots of extracts were probed for FANCD2. Unmodiﬁed FANCD2 and the activated form (FANCD2-ubiq) are arrowed. b-Actin was included as a loading control.

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A C 100

80 0 0.4 0.8 1.6 6-TG (μmol/L) –+– +–+ –+BSO 60 FANCD2-ubiq. FANCD2 β 40 -Actin % glutathione 20 D 0 100 6-TG (μmol/L) 000.8 1.2 2.4 0.80.4 1.6 HCT116 HeLa-MSH2 B 6-TG

6-TG 6-TG/BSO Untreated BSO

6-TG/BSO Survival (%) Number of cells 10 1 0 0.8 1.6 2.4 ROS fluorescence 6-TG (μmol/L) (arbitrary units)

Figure 4. 6-TG and GSH. GSH depletion and potentiation of 6-TG toxicity by BSO. A, GSH depletion. HCT116 or HeLa-MSH2 cells were treated with 6-TG at the concentrations indicated for 48 hours. Extracts were prepared and GSH levels were measured. Values are expressed as percentage of those from untreated cells. Values are the mean of 2 independent determinations. B, the effects of BSO on 6-TG induced ROS. HeLa-MSH2 cells were treated for 48 hours with 0.8 mmol/L 6-TG in the presence or absence of 200 mmol/L BSO. ROS production was analyzed by FACS. C, potentiation of FANCD2 activation by BSO. Extracts of HeLa-MSH2 cells treated for 48 hours with the concentrations of 6-TG shown were analyzed by Western blotting. The positions of unmodified (FANCD2) and activated (FANCD2-ubiq) forms of FANCD2 are arrowed. b-Actin was included as a loading control. D, 6-TG cytotoxicity enhancement by BSO. HeLa-MSH2 cells were treated for 48 hours with the concentrations of 6-TG shown in the presence or absence of 200 mmol/L BSO. Survival was determined by MTT assay. Data are the means of 3 experiments. cells also induced ROS (Fig. 5A). The survival of GM03467 MMR-independent manner and was observed in HCT116 and cells was completely unaffected by 6-TG concentrations HeLa-MSH2 cells. Replication arrest was significantly allevi- that induced ROS at levels that caused significant lethality ated by treatment with allopurinol indicating that it was in HCT116 cells indicating that 6-TG-induced oxidative dependent on ROS and not simply a consequence of 6-TG stress is insufficient in itself to cause cell death. Impor- incorporation into DNA. tantly, 6-TG treatment did not induce detectable activation Defects in the FA pathway are particularly associated with of FANCD2 in GM03467 cells—even at extremely high sensitivity to DNA interstrand crosslinking agents. In a concentrations. The FA pathway was functional in these previous publication (14) we reported that the ROS pro- cells, however, and FANCD2 ubiquitination was detectable duced when DNA 6-TG is activated by UVA induce ICLs and following mitomycin C treatment (Fig. 5B). Confirmation the chromosome aberrations that are typically associated that DNA damage derived from incorporated 6-TG is with these DNA lesions. To investigate whether 6-TG required to trigger the FA pathway and is responsible for induced ICLs independently of UVA activation, cells were 6-TG induced cytotoxicity, was provided by GM03467 cells treated with 6-TG and the introduction of ICLs was analyzed in which FANCD2 was down-regulated by RNA interference. by the Comet assay. By comparing ICL formation in cells Abrogation of the FA pathway did not detectably alter their treated with 6-TG in the presence or absence of allopurinol, 6-TG sensitivity. It did, however, significantly sensitize them the contribution of ROS was also assessed. Cell lysates were to mitomycin C (Fig. 5C). Taken together, these findings extensively digested with proteinase K before electrophore- indicate that both DNA 6-TG and ROS are required to sis to remove any DNA–protein cross-links. Figure 6B shows generate potentially lethal DNA lesions that activate the that 6-TG treatment reduced the comet tail moment pro- FA pathway. duced by IR consistent with the formation of ICLs. ICL induction was 6-TG dose dependent and was largely abol- The lethal DNA 6-TG lesions ished when allopurinol was present during incubation with The FA pathway is activated by replication-arresting DNA 6-TG. We conclude from these data that 6-TG incorporated lesions. 6-TG treatment induced a dose-dependent inhibition into DNA is a target for damage by ROS induced by the 6-TG of DNA replication as assessed by [3H]-thymidine incorpo- treatment itself and that oxidation of DNA 6-TG results in ration into nascent DNA (Fig. 6A). Inhibition occurred in a the formation of ICLs.

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effects of agents that cause replication stalling and FA-defec- A tive cells are very sensitive indicators of the presence of a variety of DNA lesions that block advancing replication forks. – 6-TG Some time ago, FANCG-defective hamster cells were reported to be unusually sensitive to 6-TG (20). We conﬁrmed this hypersensitivity in FANCA-deﬁcient MEFs and showed that + 6-TG abrogation of the FA pathway also confers 6-TG sensitivity in

Number of cells human cells. Importantly, this sensitivity is independent of MMR indicating that it represents a novel mechanism of 6-TG 1 toxicity. Taken together with a previously noted hypersensi- ROS fluorescence tivity of HR-defective xrcc2 cells (14) and the recently reported (arbitrary units) fi B 6-TG sensitivity of cells de cient in the BRCA2 (also known as FANCD1) or BRCA1 proteins (9), these findings firmly impli- 6-TG (μmol/L) 0 4 MMC8 cate the FA and HR pathways in preventing some of the FANCD2-ubiq. potentially lethal effects of 6-TG. FANCD2 The observations reported here define 2 properties of 6-TG C that are necessary and sufficient for MMR-independent cyto- 100 toxicity. The first is its ability to increase intracellular ROS 50 by depleting antioxidant levels. This is probably a general property of thiopurines. Consistent with our observations, Survival (%) Mocktransf. sicontrol siFancD2 FANCD2 5 β-Actin A 100 – 6-TG MMC – 6-TG MMC sicontrol siFANCD2 80

60 Figure 5. The effects of 6-TG in Lesch-Nyhan cells. A, ROS levels: GM03467 cells were grown in the presence of 4 mmol/L 6-TG for 72 hours. ROS were analyzed by FACS. B, FANCD2 monoubiquitination. GM03467 40 cells were treated for 72 hours with the indicated doses of 6-TG or with 30 ng/mL mitomycin C. FANCD2 activation was analyzed by Western 20 blotting. C, the effects of FANCD2 depletion in Lesch-Nyhan cells. Forty- H-thymidine incorporation (%) H-thymidine eight hours after transfection of GM03467 with FANCD2 or control RNAi, 3 0 cells were seeded into 96-well plates in the presence or absence of 6-TG (μmol/L) 0 0.4 0.8 1.6 0 0.4 0.8 1.6 4 mmol/L 6-TG or 15 ng/mL MMC. Three days later the medium was – Allopurinol + Allopurinol replaced by drug-free medium and survival was determined by MTT assay after a further 6 days. FANCD2 silencing was confirmed by Western B blotting (insert). 40 35 Discussion 30 Our findings define a novel mechanism of 6-TG toxicity that 25 does not rely on MMR. Both pathways require incorporation of 20 6-TG into DNA and its post-incorporation modification. One 15 important difference is that MMR intervention is triggered by moment Tail 10 S-methylation of DNA 6-TG rather than the oxidation that is 5 implicated in MMR-independent cell killing. In both cases, 0 Allopurinol – + – + – + – + toxicity is counteracted by HR. The 2 pathways contribute to 6- 6-TG (μmol/L) 0 0.25 0.5 1 TG toxicity in MMR-proficient cells. They are illustrated sche- matically in Fig. 7. The FA pathway comprises 14 known FANC proteins that Figure 6. DNA synthesis and interstrand cross-linking. A, HeLa-MSH2 coordinate the sensing and repair of DNA lesions, including cells treated with the indicated concentrations of 6-TG for 48 hours in the presence or absence of 500 mmol/L allopurinol were pulse labeled for 20 ICLs, that arrest replication (for review see ref. 17). Current minutes with [3H]-thymidine and incorporation of radioactivity into TCA- models of lesion processing invoke damage recognition and insoluble material was determined. Values are expressed as percentage recruitment of a ubiquitinated FANCD2:FANCI heterodimer to of the incorporation of untreated cells. B, FANCAþ/þ MEFs were grown in sites of DNA damage by the FA "core" complex. Subsequent the presence of 6-TG at the concentrations shown in the presence or absence of allopurinol (500 mmol/L) for 48 hours. Cells were irradiated nuclease-mediated incisions generate DNA double-strand with 10 Gy ionizing radiation. DNA damage was analyzed by alkaline breaks that are then directed towards HR for resolution. The comet assay after digestion with proteinase K and is expressed as comet FA pathway provides significant protection against the lethal tail moment. The data shown are representative of 2 experiments.

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FA cells to crosslinking drugs reﬂects their inability to process S ICLs correctly (17). UVA activation of DNA 6-TG produces ROS HN N that contribute to the formation of ICLs (14). The ﬁnding that

H N ROS-dependent DNA cross-linking occurs even in unirradiated 2 N N H cells treated with 6-TG is surprising but is, however, consistent 6-Thioguanine (6-TG) with our previous observations of 6-TG-induced chromosome

HPRT (1) Incorporation aberrations. FA cells are hypersensitive to the induction of chromosomal aberrations, breaks and radials, by UVA activat- DNA 6-TG ed DNA 6-TG (14). Importantly, that study also revealed that 6- TG induced the same spectrum of aberrations in unirradiated ROS (2) S-Adenosylmethionine (2) DNA damage cells. This pattern of chromosomal damage—generally associated with ICLs—is consistent with ICL formation by UVA- dependent and UVA-independent mechanisms. In our experiments, 6-TG-induced ICLs were detected by the comet assay DNA ox6-TG DNA me6-TG DNA ICL (3) and inferred from FANCD2 activation. These are both extreme- DNA damage Replication Replication ly sensitive indicators of DNA damage and probably reflect processing MMR (5) relatively rare DNA lesions. It is also important to note that the FA/HR FA pathway is activated as a general response to stalled (4) HR (6) replication and its function seems to be to direct processing Survival Cell death Survival Cell death Outcome of replication-related DNA breaks away from the alternative MMR independent MMR dependent nonhomologous end joining pathway (29). It is likely that other DNA 6-TG oxidation products such as GSO2,GSO3, or DNA- protein crosslinks also engage the FA pathway in 6-TG treated Figure 7. Mismatch repair-independent and repair-dependent pathways of 6-TG cytotoxicity. Purine salvage by HPRT (1) leads to the cells. incorporation of 6-TG into DNA. DNA 6-TG is a substrate for damage (2) Our experimental findings have implications for the clinical by methylation or by ROS generated from 6-TG–mediated depletion of use of thiopurines. Azathioprine and 6-MP are widely used to cellular antioxidants. DNA 6-TG oxidation causes DNA replication- treat leukemia and, increasingly, inflammatory bowel disease. blocking lesions, including ICLs (3). These are lethal unless correctly 6-TG has been less frequently prescribed. Its limited use seems processed by the FA and HR DNA repair pathways (4). Base pairs containing methylated DNA 6-TG are recognized by MMR, which to stem from its liver toxicity (30) although this seems some- processes them into potentially lethal DNA lesions (5). Survival may be what controversial (discussed in ref. 31). 6-TG is now consid- increased by HR processing of DNA breaks resulting from the ered a viable alternative for inflammatory bowel disease intervention of MMR. patients who fail to respond to azathioprine or 6-MP (32), however. Indeed, in view of its more direct metabolism to TGN, both 6-MP and azathioprine have been shown to deplete appropriate doses of 6-TG may offer a more predictable and reduced GSH in cultured human cells (23). ROS scavenging generally better treatment option than 6-MP or azathioprine. abrogated both 6-TG-mediated replication inhibition and FA The effectiveness of thiopurines is thought to reflect the ratio pathway activation. The second essential property for toxicity between 2 principal metabolites, the TGNs and methylmer- is the provision, in the form of DNA 6-TG, of a DNA target for captopurine (MMP) ribonucleotide. TGNs, the precursors of damage by ROS. Our findings indicate that the FA and HR DNA 6-TG, are regarded as the pharmacologically active pathways process the potentially lethal oxidized DNA 6-TG metabolites whereas the therapeutically inactive TPMT- lesions that are generated by these reactions. derived MMP contributes to the dose-limiting hepatotoxicity HR is also implicated in reversing MMR-dependent 6-TG (30). Direct conversion of 6-TG to TGN avoids the generation of toxicity (9). In that case, by analogy to MMR involvement in 6-MMP and would therefore seem to be therapeutically advan- methylating agent toxicity (24), HR processing lies down- tageous. Indeed, 6-TG is particularly efficacious in a subgroup stream of MMR-induced DNA breakage and replication fork of patients for whom 6-MP or azathioprine treatment is disruption most likely after incision at me6-TG:T mismatches associated with subtherapeutic TGN, high MMP levels and (8, 25). TGN are good substrates for incorporation into DNA severe hepatotoxicity. (26) and unmodified DNA 6-TG is not a replication block in The effectiveness of azathioprine or 6-MP is improved by vitro (27). The strict requirement for both 6-TG and ROS coadministration of the XO inhibitor allopurinol (33). By indicates that replication disruption in the MMR-independent preventing the catabolism of a significant fraction of these pathway is caused by oxidized DNA 6-TG lesions. drugs to inactive thiouric acid, XO inhibition increases the The complete absence of detectable toxicity in L-N cells is availability of thiopurines for conversion to TGN. When com- eloquent testimony to the requirement for DNA substitution by bined with an appropriately reduced thiopurine dose, allopu- 6-TG in both MMR-dependent and MMR-independent killing. rinol permits the attainment of therapeutic TGN levels. This is The oxidation potential of 6-TG is lower than that of canonical accompanied by a surprising and dramatic reduction in the DNA bases (28) and the well-documented oxidation of DNA 6- levels of methylated intermediates (33), which may be a reason TG to lesions that inhibit replication is consistent with involve- for the less severe adverse side effects of combined treatment. ment of the FA and HR pathways. The extreme vulnerability of Coadministration of allopurinol also alleviates some of the side

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Brem and Karran

effects of 6-TG treatment. Unlike 6-MP, 6-TG is not a substrate possible clinical effectiveness of combined 6-TG and allopu- for XO and it can be administered with allopurinol without rinol in certain patient groups. dose reduction. In our experiments, allopurinol protected cells against 6-TG toxicity by acting as a ROS scavenger and pre- Disclosure of Potential Conflicts of Interest fl venting DNA 6-TG oxidation. It is possible that these scaveng- No potential con icts of interest were disclosed. ing properties contribute to ameliorating toxic side effects in fi Authors' Contributions patients and that some of the bene cial effects of allopurinol in Conception and design: P. Karran, R. Brem combination with azathioprine and 6-MP may also stem from Development of methodology: R. Brem its antioxidant properties. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Brem In summary, we show that the cytotoxicity of 6-TG partly Analysis and interpretation of data (e.g., statistical analysis, biostatistics, reflects its accumulation in DNA where it serves as a target for computational analysis): P. Karran, R. Brem Writing, review, and/or revision of the manuscript: P. Karran, R. Brem the intracellular ROS that are present at increased levels Study supervision: P. Karran because 6-TG also depletes antioxidant defences. The cytotoxic effects of oxidized DNA 6-TG are independent of MMR and Grant Support reflect the formation of DNA lesions, including ICLs that This work was supported by Cancer Research UK. require processing by the FA and HR DNA repair pathways. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in These properties of 6-TG suggest that it may be particularly accordance with 18 U.S.C. Section 1734 solely to indicate this fact. efficient in the treatment of tumors with inactive BRCA1 or fi BRCA2 (9) that are defective in these pathways. Our ndings Received April 11, 2012; revised June 27, 2012; accepted July 11, 2012; also provide support, and a mechanistic rationale, for the published OnlineFirst July 20, 2012.

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Oxidation-Mediated DNA Cross-Linking Contributes to the Toxicity of 6-Thioguanine in Human Cells

Reto Brem and Peter Karran

Cancer Res Published OnlineFirst July 20, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-1278

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