Transient ATM kinase inhibition disrupts DNA damage-induced sister chromatid exchange

Abstract

Cells derived from ataxia telangiectasia (A-T) patients exhibit defective cell cycle checkpoints because of mutations in the gene encoding ATM (ataxia telangiectasia mutated). After exposure to ionizing radiation (IR), A-T cells exhibit sensitivity to IR-induced cellular damage that results in increased chromosome aberrations and cell death (radiosensitivity). ATM is a member of a family of kinases that become activated in response to DNA damage. We showed that even transient inhibition of ATM kinase for 1 hour, initiated 15 minutes after cellular irradiation, resulted in an accumulation of persistent chromosome aberrations and increased cell death. Using reversible inhibitors of DNA-PK (DNA-dependent protein kinase), another kinase involved in responding to DNA damage, and ATM, we showed that these two kinases acted through distinct DNA repair mechanisms: ATM resolved DNA damage through a mechanism involving sister chromatid exchange (SCE), whereas DNA-PK acted through nonhomologous end joining. Furthermore, because DNA damage-induced SCE occurred in A-T fibroblasts that lack functional ATM protein, and the inhibitors of ATM kinase had no effect on DNA damage-induced SCE in A-T fibroblasts, we showed that the consequences of short-term inhibition of the kinase activity of ATM and adaptation to ATM protein disruption were distinct. This suggests that A-T fibroblasts have adapted to the loss of ATM and have alternative mechanisms to initiate SCE.

Fig. 1

Temporal dynamics of reversibility of inhibition of ATM. (A) IMR90 fibroblasts were treated with 10 μM KU55933 from −15 to +90 min, +15 to +75 min, or +15 to +90 min after 2-Gy IR and harvested at +90 min. The kinase activity of ATM activity was restored when KU55933 was removed from +75 to +90 min after 2-Gy IR. (B) Diagram of the experimental paradigm for samples evaluated in (A). Thick bars indicate when KU55933 was present. (C) IMR90 fibroblasts were treated with KU55933 from +15 to +75 min after 2-Gy IR, KU55933 was removed, and nocodazole was added to collect fibroblasts that entered into mitosis. Fibroblasts were collected at +75 min, 4, 8, and 12 hours after IR for flow cytometric analysis of DNA content (% mitosis reflects 4N fibroblasts positive for phosphorylated histone H3 at +75 min). Recovery from the G₂-M checkpoint was not perturbed by addition of KU55933 from +15 to +75 min after 2-Gy IR. This experiment was performed in triplicate three times.

Fig. 2

Transient inhibition of ATM and DNA-PK activities radiosensitizes cells. (A) DNA-PK activity was inhibited in IMR90 fibroblasts when 5 μM NU7441 was added to the fibroblasts at the indicated times before 5-Gy IR. (B) DNA-PK activity was undiminished when NU7441 was removed from the fibroblasts 5 min before 5-Gy IR. (C and D) Diagram of the experimental paradigm for samples evaluated in (A) and (B). In (C), only the paradigm for samples with NU7441 treatment is shown. In (D), only the paradigm for the experimental treatments [four rightmost in (B)] is shown. Thick bars indicate when NU7441 was present. (E) The kinase activity of ATM was inhibited in IMR90 fibroblasts after irradiation when 10 μM KU55933 was added 5 min before the 5 Gy IR. The kinase activity of ATM was undiminished 15 min after irradiation when KU55933 was removed 5 min before irradiation. (F) Cell survival was assayed after 1 hour of inhibition of ATM or DNA-PK activities from +15 to +75 min after irradiation. Inhibition of DNA-PK had a greater effect on cell survival than did ATM inhibition at both 2.5 and 5 Gy. (G) Cell survival was assayed after 4 hours of inhibition of ATM or DNA-PK activities from +15 min to +4 hours 15 min after 5 Gy IR. DNA-PK inhibition caused greater radiosensitization than did ATM inhibition. Experiments shown in (F) and (G) were performed in triplicate three times.

Fig. 3

Transient inhibition of either ATM or DNA-PK activities results in an IR dose–dependent and linear accumulation of persistent chromosome aberrations in late S-, G₂-, and M-phase fibroblasts. (A) IMR90 fibroblasts were treated with 5 μM NU7441 from +15 to +75 min after 2-Gy IR and fibroblasts were harvested with calyculin A 48 hours after IR and analyzed for chromosome aberrations (table S2). (B) The damage that occurred when DNA-PK was inhibited appears to increase linearly with IR dose. The linear correlation coefficients indicate a high degree of correlation. (C) IMR90 fibroblasts were treated with 10 μM KU55933 from +15 to +75 min after 2-Gy IR and fibroblasts were isolated by applying calyculin A 48 hours after IR and analyzed for chromosome aberrations (table S1). (D) The damage that occurred when ATM was inhibited appears to increase linearly with IR dose. The linear correlation coefficients indicate a high degree of correlation.

Fig. 4

Transient inhibition of the activity of ATM, DNA-PK, or both results in an accumulation of persistent chromosome aberrations in late S- and G₂-phase fibroblasts. (A) IMR90 fibroblasts were treated with 10 μM KU55933 or 5 μM NU7441, or both, from +15 to +75 min after 2-Gy IR. M-phase fibroblasts (colcemid treated) were harvested or late S-, G₂-, and M-phase fibroblasts (calyculin treated) were harvested 48 hours after IR, and samples were analyzed for chromosome aberrations (table S3). The difference between irradiated and unirradiated controls of calyculin harvests is shown (right); errors have been summed. (B) IMR90 fibroblasts were treated with 10 μM KU55933 or 5 μM NU7441, or both, from +15 to +4 hours 15 min after 2-Gy IR, M-phase fibroblasts (colcemid treated) were harvested, or late S-, G₂-, and M-phase fibroblasts (calyculin treated) were harvested 48 hours after IR and samples were analyzed for chromosome aberrations (table S4). The difference between irradiated and unirradiated controls of calyculin harvests is shown (right); errors have been summed. The experiments described in (A) and (B) were performed twice. (C) Artemis-defective fibroblasts were treated with 10 μM KU55933 from +15 to +75 min after 2-Gy IR and late S-, G₂- and M-phase fibroblasts were harvested with calyculin A 48 hours after IR and analyzed for chromosome aberrations (table S5).

Fig. 5

Transient inhibition of the kinase activity of ATM affects the kinetics of DNA repair. Fibroblasts were exposed to KU55933 from +15 to +75 min after 2-Gy IR and analyzed at the indicated times. (A) The numbers of 53BP1 or γ-H2AX nuclear foci were greatest at 1 hour after IR, irrespective of treatment with KU55933. No difference in the appearance or resolution of either 53BP1 or γ-H2AX foci was evident in cells treated or not treated with KU55933. Representative images at 4 hours after IR are presented at right. (B) A higher percentage of fibroblasts were positive for RPA34 foci at 12 hours after IR when ATM was inhibited from +15 to +75 min. Representative images at 12 hours after IR are presented at right. These experiments were performed three times.

Fig. 6

Transient inhibition of ATM abrogates IR-induced SCE. (A) Treatment with 10 μM KU55933 from +15 to +75 min after 2-Gy IR had no affect on IR-induced SCEs in GM09607 A-T fibroblasts at 20 hours. (B) Treatment with 10 μM KU55933 from +15 to +75 min after 2-Gy IR abrogated IR-induced SCE in IMR90 fibroblasts at 20 hours. The difference between irradiated and unirradiated controls is shown (right); errors have been summed. (C) There is no difference in the number of SCEs when comparing 10 μM KU55933 treatments from +15 to +75 min and +15 min to 4 hours 15 min after 2-Gy IR. (D) Treatment with 10 μM KU55933 from +15 to +75 min after 2-Gy IR abrogated IR-induced SCE in IMR90 fibroblasts at 12 hours. Treatment with 10 μM KU55933 from −45 to +15 min after 2-Gy IR had no effect on IR-induced SCE in IMR90 fibroblasts at 12 hours. For each experimental condition, the number of SCEs per cell was counted in 50 cells and each experiment was performed twice.

Fig. 7

ATM inhibition disrupts camptothecin-induced SCE. (A) Treatment of IMR90 fibroblasts with 10 μM KU55933 and 100 nM camptothecin from 0 to 1 hour disrupts camptothecin-induced SCE at 12 hours. Cells were treated with camptothecin from 0 to 1 hour and KU55933 from 0 to 12 hours (B) Treatment with 1 μM KU60019 and 100 nM camptothecin had no effect on camptothecin-induced SCEs in GM09607 A-T fibroblasts at 12 hours (left), but disrupts camptothecin-induced SCEs in IMR90 fibroblasts at 12 hours (right). Cells were treated with camptothecin from 0 to 1 hour and with 1 μM KU60019 from 0 to 12 hours.