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, 20 (6), 665-75

BNIP3 Is Essential for Mediating 6-thioguanine- And 5-fluorouracil-induced Autophagy Following DNA Mismatch Repair Processing

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BNIP3 Is Essential for Mediating 6-thioguanine- And 5-fluorouracil-induced Autophagy Following DNA Mismatch Repair Processing

Xuehuo Zeng et al. Cell Res.

Abstract

DNA mismatch repair (MMR) processes the chemically induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine (6-TG) and 5-fluorouracil (5-FU). MMR processing of these drugs has been implicated in activation of a prolonged G2/M cell cycle arrest for repair and later induction of apoptosis and/or autophagy for irreparable DNA damage. In this study, we investigated the role of Bcl2 and adenovirus E1B Nineteen-kilodalton Interacting Protein (BNIP3) in the activation of autophagy, and the temporal relationship between a G2/M cell cycle arrest and the activation of BNIP3-mediated autophagy following MMR processing of 6-TG and 5-FU. We found that BNIP3 protein levels are upregulated in a MLH1 (MMR(+))-dependent manner following 6-TG and 5-FU treatment. Subsequent small-interfering RNA (siRNA)-mediated BNIP3 knockdown abrogates 6-TG-induced autophagy. We also found that p53 knockdown or inhibition of mTOR activity by rapamycin cotreatment impairs 6-TG- and 5-FU-induced upregulation of BNIP3 protein levels and autophagy. Furthermore, suppression of Checkpoint kinase 1 (Chk1) expression with a subsequent reduction in 6-TG-induced G2/M cell cycle arrest by Chk1 siRNA promotes the extent of 6-TG-induced autophagy. These findings suggest that BNIP3 mediates 6-TG- and 5-FU-induced autophagy in a p53- and mTOR-dependent manner. Additionally, the duration of Chk1-activated G2/M cell cycle arrest determines the level of autophagy following MMR processing of these nucleoside analogs.

Figures

Figure 1
Figure 1. BNIP3 protein levels are upregulated in a MMR-dependent manner in response to 6-TG
(A) HCT116 cells stably transfected with MLH1 cDNA (MLH1+, MMR+) were treated with 3 µmol/L 6-TG for 24 h, harvested, and subjected to immunoblot analysis with antibodies against p62/SQSTM1 at day 3 after 6-TG treatment. (B) HCT116 cells stably transfected with an empty vector (MLH1, MMR) or with MLH1 cDNA (MLH1+, MMR+) were treated with 3 µmol/L 6-TG for 24 h, harvested, and subjected to immunoblot analysis with antibodies against BNIP3 at day 3 after 6-TG treatment. (C) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24 h. Total RNA was isolated at day 3 after 6-TG treatment. Quantitative real-time PCR was then performed as described in the Materials and Methods. The expression levels of BNIP3 mRNA were normalized to those of the endogenous control 18S rRNA and the mean ΔCt obtained in untreated cells for BNIP3 mRNA was used as calibrator. The relative quantitation values from triplicate reactions were plotted on a log10 scale as mean ± standard error. (D) HCT116 (MLH1+, MMR+) cells were transfected with control luciferase (cont) or BNIP3 siRNA oligos, as described in the Materials and Methods. Two days after the siRNA transfection, the cells were harvested and analyzed by immunoblot analysis with antibodies against BNIP3. (E) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24 h, harvested, and subjected to immunoblot analysis with antibodies against cleaved PARP at day 3 after 6-TG treatment.
Figure 2
Figure 2. siRNA-mediated silencing of BNIP3 expression impairs 6-TG and 5-FU–induced autophagy
(A) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24h. Control luciferase (cont) or BNIP3 siRNA oligos were then transfected into the cells, as described in the Materials and Methods. At day 3 after exposure to 6-TG, the cells were harvested and analyzed by immunoblot analysis with antibodies against BNIP3 (upper panel) and LC3 (lower panel). (B) Control luciferase (cont) or BNIP3 siRNA oligos were transfected into HCT116 (MLH1+, MMR+) cells, as described above. The cells were then incubated with 5 µmol/L 5-FU for 48h, harvested and subjected to SDS-PAGE and immunoblot analysis with anti-BNIP3 (upper panel) and anti-LC3 (lower panel).
Figure 3
Figure 3. shRNA-mediated suppression of p53 expression abrogates 6-TG and 5-FU-induced up-regulation of BNIP3 protein levels
(A) HCT116 cells stably transfected with an empty vector (MLH1, MMR) or with MLH1 cDNA (MLH1+, MMR+) were treated with 3 µmol/L 6-TG for 24h, harvested, and subjected to immunoblot analysis with antibodies against p53 at day 3 after 6-TG treatment. (B) small hairpin RNA–mediated stable silencing of p53 expression was performed, as described in the Materials and Methods. p53 expression was tested by immunoblot analysis with anti-p53. Control and p53 knockdown HCT116 (MLH1+, MMR+) cells were then treated with 3 µmol/L 6-TG for 24 h (C) or 5 µmol/L 5-FU for 48 h (D). 72h after 6-TG exposure or 48h after 5-FU addition, the cells were harvested and subjected to SDS-PAGE and immunoblot analysis with anti-BNIP3.
Figure 4
Figure 4. Inhibition of mTOR by rapamycin impairs 6-TG and 5-FU-induced up-regulation of BNIP3 protein levels
(A) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24h. Then, 0, 0.2, 1, or 5 nmol/L rapamycin was added for another 3d to inhibit mTOR activity. At day 3 after exposure to 6-TG, cells were harvested and analyzed by immunoblot analysis with antibodies against phospho-p70S6K (S6K1) (Thr389) (upper panel) and BNIP3 (lower panel). (B) HCT116 (MLH1+, MMR+) cells were cotreated with 5 µmol/L 5-FU and 5 nmol/L rapamycin for 48h. The treated cells were harvested and analyzed by immunoblot analysis with anti-BNIP3 (upper panel) and anti-LC3 (lower panel).
Figure 5
Figure 5. 6-TG induces a G2/M cell cycle arrest peaking at day 1 while activation of autophagy and apoptosis exhibits a peak response at day 3 in MMR+ cells
HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24h. The cells were then harvested and stained with propidium iodide for cell cycle analysis (A), as described in the Materials and Methods, or subjected to immunoblot analysis with anti-phospho-Chk1 (Ser345) (B) daily for up to 5d after 6-TG treatment. The amount of phospho-Chk1 (Ser345) and tubulin was quantified using NIH Image J and the ratio of phosphor-Chk1 (Ser345) to tubulin following 6-TG treatment was graphed in the lower panel. (C) HCT116 (MLH1+, MMR+) cells were stably transfected with pGFP-LC3, as described in the Materials and Methods. The GFP-LC3–expressing cells were treated with 3 µmol/L 6-TG for 24h. The cells were examined under fluorescence microscopy and the percentage of GFP-LC3–positive cells with GFP-LC3 punctate dots was determined for up to 5d after 6-TG treatment. A minimum of 250 cells per sample was counted. (D) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24h. The cells were then harvested, stained with propidium iodide, and then subjected to flow cytometry for DNA content analysis as described above. The percentage of cells in subG1 was then graphed. Open square, untreated; closed square, 6-TG treated.
Figure 6
Figure 6. siRNA-mediated silencing of Chk1 expression promotes 6-TG-induced autophagy
(A) Control luciferase (cont) or Chk1 siRNA were transfected into HCT116 (MLH1+, MMR+) cells, as described in the Materials and Methods. Chk1 protein levels were determined by immunoblot analysis with anti-Chk1. (B) HCT116 (MLH1+, MMR+) cells were treated with 3 µmol/L 6-TG for 24h. Control luciferase (cont) or Chk1 siRNA oligos were then transfected to the cells as described above. At day 3 after exposure to 6-TG, the cells were harvested, stained with propidium iodide for cell cycle analysis (B) as described in the Materials and Methods or subjected to SDS-PAGE and immunoblot analysis with anti-LC3 (C). The amount of LC3-II and actin was quantified using NIH Image J and the ratio of LC3-II to actin was graphed in the lower panel,

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