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. 2015 May;172(9):2246-57.
doi: 10.1111/bph.13046. Epub 2015 Feb 27.

The catalytic topoisomerase II inhibitor dexrazoxane induces DNA breaks, ATF3 and the DNA damage response in cancer cells

Shiwei Deng  1 , Tiandong Yan, Teodora Nikolova, Dominik Fuhrmann, Andrea Nemecek, Ute Gödtel-Armbrust, Bernd Kaina, Leszek Wojnowski
Affiliations

The catalytic topoisomerase II inhibitor dexrazoxane induces DNA breaks, ATF3 and the DNA damage response in cancer cells

Shiwei Deng et al. Br J Pharmacol. 2015 May.

Abstract

Background and purpose: The catalytic topoisomerase II inhibitor dexrazoxane has been associated not only with improved cancer patient survival but also with secondary malignancies and reduced tumour response.

Experimental approach: We investigated the DNA damage response and the role of the activating transcription factor 3 (ATF3) accumulation in tumour cells exposed to dexrazoxane.

Key results: Dexrazoxane exposure induced topoisomerase IIα (TOP2A)-dependent cell death, γ-H2AX accumulation and increased tail moment in neutral comet assays. Dexrazoxane induced DNA damage responses, shown by enhanced levels of γ-H2AX/53BP1 foci, ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), Chk1 and Chk2 phosphorylation, and by p53 accumulation. Dexrazoxane-induced γ-H2AX accumulation was dependent on ATM. ATF3 protein was induced by dexrazoxane in a concentration- and time-dependent manner, which was abolished in TOP2A-depleted cells and in cells pre-incubated with ATM inhibitor. Knockdown of ATF3 gene expression by siRNA triggered apoptosis in control cells and diminished the p53 protein level in both control and dexrazoxane -treated cells. This was accompanied by increased γ-H2AX accumulation. ATF3 knockdown also delayed the repair of dexrazoxane -induced DNA double-strand breaks.

Conclusions and implications: As with other TOP2A poisons, dexrazoxane induced DNA double-strand breaks followed by activation of the DNA damage response. The DNA damage-triggered ATF3 controlled p53 accumulation and generation of double-strand breaks and is proposed to serve as a switch between DNA damage and cell death following dexrazoxane treatment. These findings suggest a mechanistic explanation for the diverse clinical observations associated with dexrazoxane.

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Figures

Figure 1
Figure 1
Dexrazoxane induces DSB in tumour cells. (A) HTETOP cells expressing (+TOP2A) or depleted of TOP2A (−TOP2A) by tetracycline (TET) pre-treatment (1 μg·mL–1 for 24 h) were treated with dexrazoxane (DRZ) at the indicated concentrations. Cell viability was determined 24 h following dexrazoxane exposure. *P < 0.05, **P < 0.01, significantly different from +TOP2A. (B) TOP2A-expressing HTETOP cells were treated with 100 μM dexrazoxane or 1 μM doxorubicin (DOX) for specified time periods. γ-H2AX foci were detected by immunofluorescent staining. (C) γ-H2AX foci following 24 h treatment with 100 μM dexrazoxane in HT1080, NYH and DLD-1 cells were determined by immunofluorescent staining. *P < 0.05, **P < 0.01, significantly different as indicated. (D) Neutral comet assay of TOP2A-expressing HTETOP cells performed after 24 h of treatment with 100 μM dexrazoxane. Con: untreated controls, dexrazoxane : dexrazoxane -treated cells. n = 3. *P < 0.05, significantly different as indicated. (E) Immunofluorescent staining of 53BP1 and γ-H2AX in TOP2A-expressing HTETOP cells treated with 100 μM dexrazoxane for 24 h. Quantitative data are mean values from three experiments. *P < 0.05, **P < 0.01, significantly different as indicated.
Figure 2
Figure 2
Dexrazoxane activates DDR. Phosphorylations of Chk1 at Ser317 (pChk1) and Chk2 at Thr68 (pChk2) (A), and of ATR at Ser428 (pATR) and ATM at Ser1981 (pATM) (B) were detected by Western blot. Total Chk1, Chk2, ATM or ATR was used as loading controls. TOP2A-expressing HTETOP cells were exposed to 100 μM dexrazoxane (DRZ) for the indicated time periods. (C) HTETOP cells were exposed to ATR inhibitor VE-821 (10 μM for 30 min), followed by 24 h treatment with 100 μM dexrazoxane. pChk1 (Ser317) and total Chk1 protein levels were detected by Western blot. (D) Total p53 protein level determined by Western blot in TOP2A-expressing HTETOP cells incubated with 100 μM dexrazoxane for the indicated time periods. Quantitative data are mean values from three experiments. (E) Caspase 3/7 activity in TOP2A-expressing HTETOP cells following 100 μM dexrazoxane for the indicated time periods. n = 5. *P < 0.05, **P < 0.01, significantly different as indicated. All Western blots represent at least three independent experiments. RLU, relative light units.
Figure 3
Figure 3
Dexrazoxane, but not ICRF-161, induces TOP2A-dependent ATF3 overexpression. HTETOP cells expressing (+TOP2A) or depleted of TOP2A (−TOP2A) by tetracycline pre-treatment were treated with increasing concentrations of dexrazoxane (DRZ) for 24 h (A) or with 100 μM dexrazoxane for the indicated time periods (B). ATF3 and TOP2A protein levels were analysed by Western blot. (C) ATF3 protein expression determined by Western blot in TOP2A-expressing HTETOP cells following 24 h of ICRF-161 treatment with indicated concentrations. (D) ATF3 mRNA expression assessed by TaqMan in HT1080 cells incubated with 100 μM dexrazoxane for 24 h. n = 3, *P < 0.05, significantly different as indicated.
Figure 4
Figure 4
The effects of ATM, JNK, p38 and DNA-PK on the induction of ATF3 and γ-H2AX by dexrazoxane. Protein levels of ATF3 (A) and γ-H2AX (B) following dexrazoxane (DRZ; 100 μM, 24 h) in the presence or absence of ATM inhibitor KU55933 (20 μM), JNK inhibitor SP600125 (20 μM) or p38 inhibitor SB203580 (20 μM) were assessed by Western blot in TOP2A-expressing HTETOP cells. The inhibitors were administered 30 min prior to dexrazoxane treatment. Quantitative data are mean values from three experiments. **P < 0.01, significantly different as indicated. (C) γ-H2AX Western blot was performed in dexrazoxane-treated (100 μM for 24 h) ATM mutant (GM05849) and wild-type (GM637) cells. (D) HTETOP cells were first incubated with DNA-PK inhibitor NU7026 (10 μM) for 30 min then with dexrazoxane (100 μM) for 4 h. Western blots were used to evaluate ATF3 and γ-H2AX protein levels. *P < 0.05, **P < 0.01, significantly different as indicated.
Figure 5
Figure 5
The effect of ATF3 on p53 induction and apoptosis. (A) ATF3 expression 48 h after the transfection with siRNA targeting GFP or two different loci of ATF3 mRNA assessed by Western blot. (B) p53 and ATF3 protein levels assessed by Western blot in TOP2A-expressing HTETOP cells incubated for 24 h with 100 μM dexrazoxane (DRZ) beginning at 24 h after ATF3-2 siRNA transfection. Quantitative data are mean values from three experiments. *P < 0.05, significantly different as indicated. (C) FACS analysis of Annexin V staining in cells pre-treated with or without ATF3-2 siRNA for 24 h followed by exposure to 100 μM dexrazoxane for another 24 h. n = 5, *P < 0.05, **P < 0.01, significantly different as indicated.
Figure 6
Figure 6
The effect of ATF3 on DSB formation in TOP2A-expressing HTETOP cells in response to dexrazoxane (DRZ) or IR. (A) γ-H2AX protein levels were assessed by Western blot in TOP2A-expressing HTETOP cells. ATF3 siRNA was transfected 24 h before 100 μM dexrazoxane exposure for 24 h. (B) γ-H2AX levels in scrambled (SCR) or ATF3-2 siRNA-transfected HTETOP cells treated 24 later with 100 μM dexrazoxane for another 24 h. Cells were subsequently washed with PBS, fed with fresh medium and γ-H2AX levels were assessed at the indicated time points after washing. *P < 0.05, significantly different as indicated. (C) The quantification of γ-H2AX/53BP1 foci determined by immunofluorescent staining. The treatment was the same as in (B). n = 3, *P < 0.05, significantly different as indicated. (D) γ-H2AX levels in SCR or ATF3-2 siRNA-transfected cells exposed 24 h later to 10 Gy of IR (Φ: without IR exposure). γ-H2AX levels were determined at the indicated time points after IR exposure. *P < 0.05, significantly different as indicated. All histograms represent mean values from three independent experiments.
Figure 7
Figure 7
HTETOP cell response to dexrazoxane (DRZ), including DSB induction, DDR activation, ATF3 accumulation and apoptosis.
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