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Oxidation-Mediated Dna Crosslinking Contributes to the Toxicity of 6-Thioguanine in Human Cells

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Oxidation-Mediated Dna Crosslinking Contributes to the Toxicity of 6-Thioguanine in Human Cells Author Manuscript Published OnlineFirst on July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. OXIDATION-MEDIATED DNA CROSSLINKING CONTRIBUTES TO THE TOXICITY OF 6-THIOGUANINE IN HUMAN CELLS Reto Brem & Peter Karran1 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK 1Corresponding author: Tel. + 44 1707625870 Fax + 44 1707625812 email [email protected] Running head: Thioguanine toxicity in human cells Keywords: thioguanine, reactive oxygen species, DNA damage, DNA crosslinks, Fanconi anemia proteins This work was supported by Cancer Research UK The authors state there is no actual or potential conflict of interest Word count: 3824 Figures 7 Tables 0 1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 utilized 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 crosslinks. 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 immunomodulation strategy in inflammatory disorders. 2 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION The clinical effectiveness of the immunosuppressant, anti-inflammatory and anticancer thiopurines: azathioprine, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) relies on their ability to selectively kill dividing immune effector or cancer cells. Surprisingly, despite successful use for over 50 years (1), the mechanisms by which they cause the death of their target cells are still incompletely understood. Thiopurines are prodrugs. They are metabolized to the 6- TG nucleotides (TGN) that early studies identified as important determinants of toxicity. TGN are substrates for incorporation into DNA and the accumulation of DNA 6-TG is a major factor in thiopurine toxicity. Other contributors include the formation of toxic thiopurine metabolites and oxidation of DNA 6-TG by exogenous chemicals or ultraviolet A (UVA) radiation (for review see (2)). The most widely prescribed thiopurines, 6-MP and its prodrug azathioprine, are converted to TGN in several steps (for review see (3)). Conversion is counteracted by the activities of three enzymes: thiopurine methyltransferase (TPMT) which inactivates thiopurines by methylation (4), xanthine oxidase (XO) which catabolises 6-MP and azathioprine to inactive thiouric acid, and the MRP4 transporter protein that exports thiopurine mononucleotides from cells (5). TPMT expression is an important determinant of the clinical effectiveness of thiopurines. Polymorphic TPMT variants with significantly reduced activity are associated with high TGN levels and extreme, potentially lethal, thiopurine toxicity. High TPMT activity is associated with lower intracellular TGN concentrations and reduced clinical efficacy (6). Prevention of cell proliferation by limiting the supply of purine nucleotides that are essential for DNA and RNA synthesis is another potential contributor to thiopurine toxicity. In this case, TPMT methylates thioinosine monophosphate (TIMP), a metabolite of azathioprine and 6-MP, to generate methylTIMP, a powerful inhibitor of the first enzyme in the de novo pathway of purine nucleotide biosynthesis. Purine nucleotide depletion does not explain all thiopurine cytotoxicity, however, (7) and the potently cytotoxic 6-TG is metabolized to TGN by a different route that does not generate TIMP. The DNA mismatch repair (MMR) system is also a major contributor to thiopurine toxicity. DNA 6-TG deceives MMR into a potentially lethal intervention and, as a consequence MMR-defects confer significant thiopurine resistance (for review see (8)). MMR-deficient cells 3 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. do, however, retain a susceptibility to killing by high thiopurine concentrations, indicating the existence of MMR-independent thiopurine cytotoxicity (9). DNA 6-TG is also implicated in a cytotoxic pathway which involves oxidative DNA damage. Cells containing DNA 6-TG are hypersensitive to reactive oxygen species (ROS) generated chemically, biologically (10), or photochemically by UVA (11). ROS inflict widespread DNA damage. In addition to the well-characterized DNA 8-oxoGuanine (12), they cause oxidation of DNA 6-TG itself. Among these oxidation products, guanine-6 sulfonate (GSO3) (11) and -sulfinate (GSO2) (13), DNA interstrand crosslinks (ICLs) (14), and DNA-protein crosslinks (15) have been identified as replication-blocking and potentially cytotoxic DNA lesions. Here we describe 6-TG-mediated cytotoxicity that requires the incorporation of 6-TG into DNA but is independent of MMR, exogenous sources of ROS, and of UVA. It does require an oxidizing environment, however, and we show that 6-TG itself provides this by depleting endogenous antioxidant defences and thereby increasing steady-state ROS levels. Cell killing reflects the formation of potentially lethal DNA lesions that inhibit replication. Its effects are particularly marked in cells with defects in the Fanconi anemia (FA) and homologous recombination (HR) pathways and cells with defects in either pathway are extremely sensitive to 6-TG (9, 14, 16). Together the FA and HR pathway protect cells against DNA damage that arrests replication (reviewed in (17)). FA-deficient cells are typically hypersensitive to killing, chromosome breakage and particularly to radial chromosome induction by agents that cause DNA interstrand crosslinks (ICLs). They exhibit a similar pattern of sensitivity to 6-TG (14), and we show that ICLs are among the DNA lesions that efficiently block DNA replication in cells treated with 6-TG. 4 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 20, 2012; DOI: 10.1158/0008-5472.CAN-12-1278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS Cell culture The mismatch-repair defective human leukaemia cell line CCRF-CEM was grown in RPMI, all other cells in DMEM. Media were supplemented with 10% FCS. The Fanconi anemia (FANCA-/-) and wildtype (FANCA+/+) MEFs have been described (18). Their status was confirmed by isoenzyme analysis and DNA fingerprinting (January 2010). MSH2-defective (HeLa-MSH2) and their control transfectant HeLa SilenciX (HeLa-SX) cells (tebu-bio Cat Nr 01- 00023) were cultured in the presence of hygromycin B. Non-transfected HeLa, MLH1-deficient colon cancer cells HCT116 were obtained from Cancer Research UK Central Cell Services. Their identity was confirmed by isoenzyme analysis and STR profiling (March 2011) and skin fibroblasts derived from a Lesch-Nyhan syndrome patient (GM03467) (isoenzyme analysis November 2010) were obtained from The Coriell Institute. ROS and glutathione detection To measure ROS, trypsinized cells were washed once in PBS and incubated in 5 µM CM- H2DCFDA (Invitrogen) in PBS for 20 min at 37°C. They were then washed twice in PBS and green fluorescence was analyzed by flow cytometry. Total and oxidized levels of glutathione were measured using the Glutathione Assay (Trevigen) according to the manufacturer’s protocols. Immunoblotting Whole cell extracts were prepared using RIPA buffer. Proteins (50 µg) were separated on 3-8% Tris-Acetate polyacrylamide gels (Invitrogen). After transfer, membranes were probed with antibodies against FANCD2 (Novus Biologicals), MSH2 (BD Pharmingen) or Xanthine Oxidase (abcam). Antigen-antibody complexes were visualized using ECL blotting detection agent (GE Healthcare). RNA interference siRNA duplex smart pools were purchased from Dharmacon. Cells were transfected using Lipofectamine RNAiMAX (Invitrogen) or Dharmafect (Dharmacon) according to the manufacturers’ instructions, with a final siRNA concentration of 50 nM. Cells were subcultured into normal medium
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