Tachpyridine, a metal chelator, induces G2 cell-cycle arrest, activates checkpoint kinases, and sensitizes cells to ionizing radiation

Abstract

Iron is critical for cell growth and proliferation. Iron chelators are being explored for a number of clinical applications, including the treatment of neurodegenerative disorders, heart disease, and cancer. To uncover mechanisms of action of tachpyridine, a chelator currently undergoing preclinical evaluation as an anticancer agent, cell-cycle analysis was performed. Tachpyridine arrested cells at G2, a radiosensitive phase of the cell cycle, and enhanced the sensitivity of cancer cells but not nontransformed cells to ionizing radiation. G2 arrest was p53 independent and was accompanied by activation of the checkpoint kinases CHK1 and CHK2. G2 arrest was blocked by UCN-01, a CHK1 inhibitor, but proceeded in CHK2 knock-out cells, indicating a critical role for CHK1 in G2 arrest. Tachpyridine-induced cell-cycle arrest was abrogated in cells treated with caffeine, an inhibitor of the ataxia-telangiectasia mutated/ataxia-telangiectasia-mutated and Rad3-related (ATM/ATR) kinases. Further, G2 arrest proceeded in ATM-deficient cells but was blocked in ATR-deficient cells, implicating ATR as the proximal kinase in tachpyridine-mediated G2 arrest. Collectively, our results suggest that iron chelators may function as antitumor and radioenhancing agents and uncover a previously unexplored activity of iron chelators in activation of ATR and checkpoint kinases.

Figure 1.

Tachpyridine induces G₂/M arrest in HeLa cells. Cells were treated with 5, 7.5, and 10 μM tachpyridine for 24 hours, and cell-cycle distribution was determined by flow cytometry.

Figure 2.

Tachpyridine induces cell-cycle arrest in the G₂ phase of the cell cycle. HeLa cells treated with 7.5 μM tachpyridine for 18 hours were double-labeled for the M-phase marker phosphorylated H3 and for DNA content, followed by flow cytometry analysis. Untreated controls (top) show 12% of cells in G₂/M by propidium iodide (PI) staining and 2.2% of those cells in mitosis. Cells treated with 50 ng/mL nocodazole, an M-phase arrestor, show 96% of cells in G₂/M by PI staining and 87% of those cells in mitosis (middle). Tachpyridine-treated cells (bottom) display 41% of cells in G₂/M by PI staining, and only 0.03% of cells in mitosis, consistent with the induction of a G₂- and not M-phase arrest.

Figure 3.

Tachpyridine induces G₂/M arrest and radiosensitizes HCT 116 TP53^+/+ and HCT 116 TP53^-/- cells but does not radiosensitize normal diploid fibroblasts. (A) HCT 116 TP53^+/+ or (B) HCT 116 TP53^-/- cells were treated with 10 μM tachpyridine for 24 hours, and cell-cycle distribution was determined by flow cytometry. (C) HCT 116 TP53^+/+ or (D) HCT 116 TP53^-/- cells were pretreated with 7.5 μM tachpyridine for 12 hours, followed by irradiation at 1, 2, and 4 Gy, and clonogenic survival was determined as described in “Materials and methods.” (E) HCT 116 TP53^+/+ cells were pretreated with 7.5 μM tachpyridine for 12 hours, followed by irradiation at 1, 2, and 4 Gy, and viability was determined by the MTT assay as described in “Materials and methods.” (F) MRC5 fibroblast cells were pretreated with 25 or 30 μM tachpyridine for 12 hours, followed by irradiation at 6 and 10 Gy, and viability was determined by the MTT assay. Data shown are the mean and SE of 3 independent experiments performed in triplicate.

Figure 4.

Tachpyridine activates cell-cycle checkpoint kinases CHK1 and CHK2 and induces phosphorylation of p53 on serine-15. (A) HeLa cells were treated with 7.5 μM tachpyridine and analyzed by Western blotting using phosphospecific antibodies against activated CHK1 (S-345) and CHK2 (T-68). β-Actin was included as a loading control. (B) HeLa cells were treated with 10 μM tachpyridine and analyzed by Western blotting using a phosphospecific antibody against phosphorylated p53 (S-15). Equivalent loading of protein was confirmed by Ponceau S staining of the transfer membrane (not shown).

Figure 5.

Caffeine inhibits tachpyridine-induced G₂ arrest and activation of tachpyridine-induced CHK1 but not CHK2. (A) HeLa cells were either not pretreated or pretreated for 12 hours with 5 mM caffeine, followed by 18 hours of treatment with 7.5 μM tachpyridine, and analyzed by flow cytometry. (B) HeLa cells were either not pretreated or pretreated for 12 hours with 5 mM caffeine, followed by treatment with 7.5 μM tachpyridine over a time course of 3 to 24 hours, and analyzed by Western blotting (U indicates untreated control at 24 hours; caff, cells treated with caffeine alone for 24 hours).

Figure 6.

G₂/M arrest proceeds in ATM-deficient cells but is inhibited in ATR-deficient cells. (A) Cells were treated with 10 μM tachpyridine for 24 hours and analyzed by flow cytometry. (B) Western blotting confirms absence of ATM in ATM-deficient cells. (C) ATR^flox/- cells were treated with Adeno-Cre at a multiplicity of infection of 10 for 2 days to eliminate ATR expression. Cells were then treated with 10 μM tachpyridine for 24 hours and analyzed by flow cytometry. (D) Western blotting was used to assess ATR levels in HCT 116 parental cells, HCT 116 ATR^flox-/- cells, and HCT 116 ATR^flox-/- cells treated with Adeno-Cre.

Figure 7.

Working model of the mechanism of tachpyridine-induced G₂ arrest. Tachpyridine activates ATR (and possibly ATM), which phosphorylates p53 on serine 15, followed by phosphorylation of CHK1 and CHK2. The CHK kinases then signal G₂ arrest through phosphorylation of key substrates important for G₂ to M progression. Signaling events not required for G₂ arrest are indicated by gray dashed lines.