Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons

Abstract

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

FIGURE 1.

Dose-response analysis of S139 phosphorylation of H2AX by different types of topoisomerase II inhibitors. A, logarithmically growing rat cardiomyoblast cells (H9c2) were treated with increasing concentrations of different types of topoisomerase II poisons. Two h after addition of the anthracycline derivative doxorubicin (Doxo) (0.1–10 μm) or the podophyllotoxin etoposide (Eto) (1–100 μm), the level of γH2AX was determined by Western blot analysis. For quantitative densitometric analysis, the relative level of γH2AX was set to 1.0 in untreated control. ERK2 protein expression was determined as internal loading control. B, logarithmically growing rat cardiomyoblast cells (H9c2) were treated with increasing concentrations of doxorubicin for 2 h before the level of γH2AX was determined by Western blot analysis. Autoradiographies were quantified densitometrically. *, p ≤ 0.05 compared with untreated control. #, p ≤ 0.05 compared with treatment with 2 μm doxorubicin. C, logarithmically growing rat cardiomyoblast cells (H9c2) were treated with 0.1–10 μm anthracycline derivative epirubicin for 2 h before the level of γH2AX was determined by Western blot analysis. D, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated with a high dose of doxorubicin (10 μm, 1 h) before etoposide (10 μm) was added. After a further incubation period of 2 h, the level of γH2AX was determined by Western blot analysis. Error bars, S.D.

FIGURE 2.

Time kinetic analysis of H2AX phosphorylation by doxorubicin. A, logarithmically growing H9c2 cells were exposed to 1 μm (A) or 10 μm (B) doxorubicin (Doxo). After the indicated periods of time, the level of γH2AX protein was determined by Western blot analysis. Shown is the result of a representative experiment. For densitometric analysis, the amount of γH2AX in untreated control was set to 1.0. ERK2 protein expression was determined as internal protein loading control. B, H9c2 cells were treated with the indicated concentrations of doxorubicin for 2 h before the levels of p-ATM and p-ATR were determined by Western blot analysis. For loading control (Con), protein expression of talin1 was determined.

FIGURE 3.

γH2AX foci formation, DNA strand break induction, and formation of DNA-topo II covalent complexes by different types of topoisomerase II inhibitors. A, logarithmically growing rat cardiomyoblast cells (H9c2) were treated with increasing concentrations of the anthracycline derivative doxorubicin (Doxo) or the podophyllotoxin etoposide (Eto). Two h after drug addition, the number of γH2AX foci was determined by immunohistochemistry. ***, p ≤ 0.001. B, the level of DNA strand break induction was monitored by both the alkaline (left) and neutral (right) comet assay. For control, cells were irradiated with 5 Gy (IR). Data shown are the mean ± S.E. (error bars) from two to six independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. C, logarithmically growing rat cardiomyoblast cells (H9c2) were treated with increasing concentrations of doxorubicin or etoposide. Two h after drug addition, the formation of DNA-topo IIα covalent complex was analyzed as described under “Experimental Procedures.” Nuclei were counterstained with DAPI. The histogram shows the percentage of topo IIα-positive cells. Shown is the result of a single experiment with 50 nuclei being analyzed per treatment. **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 4.

Effect of inhibition of Rho signaling on S139 H2AX phosphorylation following exposure to topoisomerase II inhibitors. A, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with lovastatin (Lova) (20 μm) before doxorubicin (Doxo) (1 μm) or etoposide (Eto) (10 μm) was added. After incubation period of 2 h, the level of γH2AX protein was analyzed by Western blotting. The relative amount of γH2AX protein in untreated cells (Con) was set to 1.0. Shown is a representative result from at least three independent experiments. B, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with different concentrations of lovastatin before doxorubicin (1 μm) or etoposide (10 μm) was added. After an incubation period of 2 h, the level of γH2AX protein was analyzed by Western blotting. Relative amount of γH2AX in corresponding non-lovastatin-treated cells was set to 1.0. Shown is a representative result from two or three independent experiments. C, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated for 3 h with the Rac1-specific inhibitor NSC23766 (NSC) (100 μm) (64) before doxorubicin (1 μm) or etoposide (10 μm) was added. After an incubation period of 2 h, the level of γH2AX protein was analyzed by Western blotting. Relative amount of γH2AX in untreated cells was set to 1.0. Shown is a representative result from two or three independent experiments. D, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with lovastatin (20 μm) or for 3 h with the Rac1 inhibitor NSC23766 (100 μm) before doxorubicin (1 μm) or etoposide (1 μm, 10 μm) was added. After an incubation period of 2 h, the formation of γH2AX foci was analyzed by immunohistochemistry as described under “Experimental Procedures.” Shown is a representative result. E, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with lovastatin (L) (20 μm) or for 3 h with the Rac1 inhibitor NSC23766 (N) (100 μm) before doxorubicin (D) (1 μm) or etoposide (E) (10 μm) was added. After further incubation period of 2 h, the formation of γH2AX foci was analyzed by immunohistochemistry as described under “Experimental Procedures.” Shown are the mean ± S.E. (error bars) of two independent experiments with 50 nuclei being analyzed per experiment. **, statistically significant (p ≤ 0.01).

FIGURE 5.

The inhibitory effects of lovastatin and NSC23766 doxorubicin-induced DDR are additive. A and B, logarithmically growing H9c2 cells were single pretreated with lovastatin (Lova) (overnight) or NSC23766 (NSC) (3 h) or were simultaneously pretreated with lovastatin plus NSC23766. Two h after doxorubicin (Doxo) treatment, the level of H2AX phosphorylation was analyzed by Western blotting (A) or immunohistochemistry (B). Relative γH2AX levels detected by Western blot analysis were quantitated densitometrically and set to 1.0 in untreated cells. Data shown under B are the mean ± S.E. (error bars) derived from one experiment with 50 nuclei being analyzed per condition. *, p ≤ 0.05. C, after pretreatment with lovastatin (overnight, 20 μm) or NSC23766 (3 h, 100 μm), medium was removed and replaced by fresh medium. After a postincubation period of 3 h in the absence of Rac1 inhibitors, doxorubicin (Doxo) (1 μm) was added, and H2AX phosphorylation was determined 2 h later by Western blotting. Shown is the result of a representative experiment. Relative γH2AX level in untreated control was set to 1.0.

FIGURE 6.

Inhibition of Rac1 signaling reduces the level of DNA strand breaks and attenuates the formation of the DNA-topo II covalent complex following treatment with topo II inhibitors. A, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with the lovastatin (20 μm) (Lova) or for 3 h with the Rac1-specific inhibitor NSC 23766 (NSC) (100 μm) before doxorubicin (Doxo) (1 μm) or etoposide (Eto) (10 μm) was added. After incubation period of 2 h, formation of DNA strand breaks was analyzed by the comet assay as described under “Experimental Procedures.” Shown are the mean ± S.D. (error bars) from two to six independent experiments. For reason of control, cells were irradiated with 5 Gy (IR), and DNA strand breaks were analyzed 30 min later. *, statistically significant (p < 0.05). B, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with lovastatin (L) (20 μm) or for 3 h with the Rac1-specific inhibitor NSC23766 (N) (100 μm) before doxorubicin (1 μm) or etoposide (10 μm) was added. After an incubation period of 2 h, the formation of covalent DNA-topo IIα complexes was analyzed by laser scanning microscopy. Relative fluorescence in doxorubicin- or etoposide-treated cells was set to 1.0. Data shown are the mean ± S.E. (error bars) from two independent experiments with 50 nuclei being evaluated per experimental condition. *, p ≤ 0.05.

FIGURE 7.

Lovastatin and NSC23766 do not affect doxorubicin-stimulated activation of ATM. A and B, logarithmically growing H9c2 cells were pretreated overnight with lovastatin (20 μm) before doxorubicin (1 μm) was added. After a further incubation period of 2 h, phosphorylation status of ATM (p-ATM), checkpoint kinase-1 (p-Chk1), and H2AX (γH2AX) (A) as well as of ATR (p-ATR) and checkpoint kinase-2 (p-Chk2) (B) was monitored. Protein expression of ERK2, β-actin, and talin1 was analyzed as internal protein-loading controls (Con). C, H9c2 cells were pretreated for 3 h with the Rac1-specific inhibitor NSC23766 before doxorubicin (1 μm) or etoposide (10 μm) was added. After a further incubation period of 2 h, phosphorylation status of ATM, ATR, and H2AX was analyzed. ERK2 and talin1 protein expression was determined as internal protein loading control. Shown are representative data from two independent experiments.

FIGURE 8.

Inhibition of the Rac1 effector PAK partially blocks DDR stimulated by topo II poisons. Logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated for 2 h with PAK inhibitor IPA3 (10 and 30 μm) before doxorubicin (Doxo) (1 μm) or etoposide (Eto) (10 μm) was added. After a further incubation period of 2 h, H2AX phosphorylation was analyzed by Western blotting (A) or immunohistochemistry (B) as described under “Experimental Procedures.” 1, 10 μm IPA3; 2, 30 μm IPA3. **, p ≤ 0.01; ***, p ≤ 0.001; ns, not significant. Error bars, S.E.

FIGURE 9.

DDR stimulated to the topo I poison topotecan is not inhibited by lovastatin. A and B, logarithmically growing rat cardiomyoblast cells (H9c2) were exposed to increasing concentrations of topoisomerase type I inhibitor topotecan (TPT). After an incubation period of 2 h, H2AX phosphorylation and the formation of γH2AX foci were analyzed by Western blotting and immunohistochemistry, respectively, as described under “Experimental Procedures.” Shown are results from a representative experiment. For control, cells were irradiated with 5 Gy. ***, p ≤ 0.001; ns, not significant. C and D, logarithmically growing rat cardiomyoblast cells (H9c2) were pretreated overnight with lovastatin (20 μm) before topotecan was added at different concentrations. After further incubation period of 2 h, H2AX phosphorylation was analyzed by Western blotting (C) and immunohistochemistry (D) as described under “Experimental Procedures.” Data shown are from representative experiments. +, pre-treatment with lovastatin; *, p ≤ 0.05; ***, p ≤ 0.001; ns, not significant. Error bars, S.E.

FIGURE 10.

Model of the genoprotective effect of pharmacological inhibition of Rac1 signaling in rat cardiomyoblast cells. At therapeutically relevant low doses, topo II poisons promote irreversible interaction of topo II proteins with DNA (DNA-topo II complex), eventually leading to the formation of DNA DSBs and activation of the DDR, as reflected by γH2AX. Meanwhile, DSB formation and subsequent DDR do not occur at a high concentration of the anthracycline derivative doxorubicin. Pharmacological targeting of Rac1 signaling by statins (e.g. lovastatin) or a Rac1-specific small molecule inhibitor (e.g. NSC23766) attenuates DSB formation and subsequent ATM-regulated H2AX phosphorylation by counteracting the formation of the DNA-topo II complex. The data are indicative of a genoprotective function of statins, which is in line with a previous report demonstrating that statins maintain genomic stability (61).