Immune effector cells produce lethal DNA damage in cells treated with a thiopurine

Abstract

Azathioprine, a widely used immunosuppressant, is also used in the control of inflammatory disorders. These are characterized by the local accumulation of immune effector cells that produce reactive oxygen species (ROS). The DNA of azathioprine-treated patients contains 6-thioguanine (6-TG), a base analogue that is particularly susceptible to oxidation. Here, we show that 6-TG is vulnerable to ROS produced by chemical oxidants and that cells containing DNA 6-TG are hypersensitive to these oxidants. We also show that 6-TG incorporated into the DNA of macrophages sensitizes them to killing by endogenously produced ROS. ROS generated by macrophages are also a hazard for cocultured nonmacrophage cells containing DNA 6-TG. This bystander vulnerability of cells containing DNA 6-TG to oxidation by ROS generated by immune effector cells has implications for the long-term use of azathioprine in the management of inflammatory disorders.

Figure 1. H₂O₂ oxidises 6-TGdR to G^SO3dR

An aqueous solution of 6-TGdR (0.1mM) was treated with 1mM H₂O₂ for 60 min and the reaction products were analyzed by reverse-phase HPLC. Column eluates were monitored by A₃₄₂ and fluorescence (excitation 324 nm and emission at 410 nm). a) Untreated (Black trace). 6-TGdR elutes at 20 minutes. b) H₂O₂ treated (Grey trace). G^SO3dR elutes at 10 minutes. c) The amounts of 6-TGdR (grey line) and G^SO3dR (black line) after treatment with the H₂O₂ concentrations shown were quantified by HPLC.

Figure 2. DNA 6-TG sensitizes cells to killing by chemical oxidants that induce ROS

HCT116 cells that had been grown in the presence of 6-TG (1μM) DNA for 24h were treated with (a) H₂O₂ (1h) or (b) KBrO₃ (3h) or (c) γ-irradiated at the doses indicated. Survival of HCT116 in absence of 6-TG (black line) or containing 6-TG in their DNA (grey line) was determined by clonogenic assay (a - c). The data represent the mean ± SD of 4 independent experiments. In parallel, the same HCT116 cells were loaded with the ROS reactive dye CM-H₂DCFDA prior to exposure to H₂O₂, KBrO₃ or IR. Changes in fluorescence intensity were analysed by FACS (insert a - c). The solid histogram represents untreated cells. d) DNA replication. Control (black line) or 6-TG treated HCT116 cells (grey line) were treated with H₂O₂ for 1h and [³H]-TdR incorporation into TCA-insoluble material was measured. Incorporation is expressed as a percentage of that in untreated cells. Data are the mean ± SD from 2 separate experiments.

Figure 3. Lethal DNA lesions induced by H₂O₂ oxidation of DNA 6-TG are not excised by NER

SV40 transformed GM04429f (circles) or MRC5-VA (squares) cells were cultured for 48h in 0.5μM 6-TG then treated with H₂O₂ for 1h. Clonal survival was determined. Solid lines: no 6-TG treatment. Dashed lines: + 6-TG treatment. Data are the mean ± SD of 4 independent experiments. Insert. Confirmation of the XP phenotype. Cells not treated with 6-TG were irradiated with UVC at the doses indicated and survival determined as above.

Figure 4. ROS production by activated J774a.1 cells

J774a.1 macrophage precursor cells were incubated for 48h in medium containing LPS at the indicated concentrations. (a) H₂O₂ accumulated in 60 min was determined by microassay based on the peroxidase dependent oxidation of phenol red. (b) Superoxide (O₂⁻) accumulated in 60 mins was determined by O₂⁻ microassay based on the reduction of cytochrome c. (c) Cells were loaded with the ROS reactive dye CM-H₂DCFDA prior to activation with LPS (100μg/ml) and analysed by fluorescent microscopy 24h later. Following LPS activation, macrophages differentiated to produce a varied cell morphology which included a number of large and very granular cells (arrow). Fluorescent staining for ROS reflected this variation in morphology and cells displaying punctated cytoplasmic staining as well as whole cell staining were observed. Scale bar is 50μm. The fraction of ROS positive cells (d) includes all these predominant staining patterns.

Figure 5. Oxidation of J774a.1 DNA 6-TG by endogenous ROS and sensitization to killing

a) HCT116 or J774a.1 cells were grown for 48h in the presence or absence of LPS (100μg/ml) and 6-TG (1μM, HCT116; 0.5μM, J774a.1). DNA was extracted, digested to deoxynucleosides and separated by HPLC. 6-TGdR and G^SO3dR were quantified by A₃₄₂ and by fluorescence. In HCT116 cells, 6-TG replaced 0.42% DNA G. For J774a.1 cells, this value was 0.7%. Data are expressed as the percentage conversion from 6-TG to G^SO3 deoxynucleoside. Insert: ROS levels of HCT116 (light grey) or J774a.1 cells (dark grey) containing 6-TG used for the measurement of 6-TG:G^SO3 described above were assessed by CM-H₂DCFDA staining and FACS, b) 6-TG treated CCRF-CEM cells (48h, 1μM) were co-incubated for further 48h with LPS-activated (100μg/ml) J774a.1 cells. DNA from non-adherent cells was extracted, digested to deoxynucleosides, separated by HPLC, and quantified as above. c) HCT116 and J774a.1 cells were grown in the presence of 6-TG (as shown) for 48h and either left untreated (black bars) or treated with LPS (100μg/ml; grey bars) for a further 48h. Cell viability was determined by Annexin V/PI staining and FACS analysis. The data represent the mean ± SD of 3 independent experiments.

Figure 6. Cells containing 6-TG are susceptible to killing in co-culture with J774a.1

a) Representative dot plot of 6-TG-treated HCT116 cells. Cells in the lower left quadrant (Annexin V negative/PI negative) were considered viable. b) Co-culture. 6-TG-treated or untreated HCT116 cells were plated together with non-activated or LPS-activated J774a.1 cells. 48h later, HCT116 viability was determined by Annexin V/ PI staining and FACS. Insert: Forward (size) and side (granularity) FACS scatter plot from a mixture of HCT116 target cells and J774a. These data were used to exclude J774a.1 cells (R1) from subsequent analyses. c) The percentage of viable 6-TG-treated HCT116 cells (grey bars) or CCRF-CEM leukemia cells (black bars) either untreated or pre-incubated with NAC (10μM, 3h) after 48h co-culture with non-activated or LPS activated J774a.1 cells. Data are from 4 independent experiments ± SD for HCT116 and 2 experiments for CCRF-CEM. d) 6-TG treated CCRF-CEM leukemia cells were left untreated or incubated with NAC (10μM) for 3h before co-culture with LPS-activated J774a.1 cells for 48h. DNA from non-adherent cells was extracted, digested to deoxynucleosides and separated by HPLC. DNA 6-TGdR was quantified by A₃₄₂ and G^SO3dR by fluorescence. The data shown are the mean conversion to G^SO3dR from 4 independent experiments ± SD.