Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin

Abstract

Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (∼30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway.

FIGURE 1.

Ape1 suppression in shRNA-treated cells. TK6 or HCT116 cells were infected with the indicated retroviral vectors, and after 2 days, puromycin (1.5 μg/ml) was added to the culture medium for 5–7 days for selection. Abbreviations used are as follows: Con, uninfected; sLUC, luciferase-specific shRNA vector; sAPE1, APE1-specific shRNA vectors; sLUC+LUC, sLUC, co-expressed with LUC expression retroviral vector. After an additional 3 days of selection, cell samples were subjected to immunoblotting with an Ape1-specific antibody, which was quantified by phosphorimaging and normalized to immunoblotting for β-actin (bar graphs). The upper panels show a representative immunoblot (see “Experimental Procedures” for details) and the lower panels the quantification from three independent experiments. Standard deviations are shown, and the asterisk indicates significant difference from control with p < 0.05.

FIGURE 2.

Cytotoxicity induced by IR or BLM in Ape1-deficient TK6 and HCT116 cells. Cells were untreated (Con) or infected with retroviral siRNA expression vectors for luciferase (sLUC) or APE1 (sAPE1). Additionally, in E and F, the yeast Apn1 protein was co-expressed with sLUC (sLUC+Apn1) or sAPE1 (sAPE1+Apn1) and luciferase was co-expressed with sLUC (sLUC+LUC) as a control for the activation of RNAi pathways. A and B, TK6 cells were treated with increasing doses of x-rays (100 keV, 1–4 Gy) or BLM (10–60 μg/ml, 60 min). C and D, HCT116 cells treated with 2.5–10 Gy x-rays or 25–150 μg/ml BLM for 60 min. E and F, TK6 cells treated with 1.5 Gy x-rays, or 12 μg/ml BLM for 60 min. The cells were washed twice, and the incubation continued for 4 days. One thousand cells per sample were then reseeded in 60-mm dishes or a 96-well plate, and the incubation was continued in fresh medium for 96 h. The cell number was determined by trypan blue cell counting or a commercial cell counting kit (cell counting kit-8). The data were quantified from three independent experiments. Standard deviations are shown, and asterisk indicates significant difference from control with p < 0.05.

FIGURE 3.

Apoptosis induced by IR or BLM in Ape1-deficient TK6 cells. TK6 cells were untreated (Con) or infected with retroviral shRNA expression vectors for luciferase (sLUC), APE1 (sAPE1), or co-expressed with Apn1 (sLUC+Apn1 and sAPE1+Apn1). The cells were treated with x-rays (1.5 Gy) or BLM (12 μg/ml) for 60 min. The cells were washed twice, and the incubation was continued in fresh medium for 24 h. Apoptosis was determined by the annexin V-FITC and propidium iodide assay. The panel shows a representative experiment, and the data were quantified from three independent experiments. The number in each panel indicates the percentage of apoptotic (annexin V-positive) cells and standard deviations. Asterisk indicates significant difference from the untreated control with p < 0.05. The PI staining indicates cell membrane leaking, a hallmark of early necrosis, whereas annexin V-positive indicates early apoptosis. The PI (+)/annexin V (−) (upper left quadrant in each panel) corresponds to necrosis; PI(−)/annexin V (+) (lower right quadrant) indicates apoptosis; PI(+)/annexin V (+) (upper right quadrant) indicates late cell death that could be due to either necrosis or apoptosis; PI(−)/annexin V (−) (lower left quadrant) corresponds to normal viable cells.

FIGURE 4.

DSB formation by IR and BLM and subsequent cellular processing. TK6 cells were untreated (Con) or infected with retroviral shRNA expression vectors for luciferase (sLUC), APE1 (sAPE1), or sAPE1 co-expressed with Apn1 (sAPE1+Apn1), or Apex (sAPE1+Apex). A and C, cells treated with x-rays (1.5 Gy). B and D, cells were treated with 12 μg/ml BLM for 60 min and then washed incubated in fresh medium for the indicated times. DSB were quantified as the tail moment in a neutral comet assay (A and B). Apoptosis was determined by the annexin V-FITC/PI assay (C and D). The data were quantified from three independent experiments. Standard deviations are shown, and asterisk indicates significant difference from control with p < 0.05.

FIGURE 5.

Formation of abasic sites in Ape1-deficient cells. TK6 cells were untreated (Con) or infected with retroviral shRNA expression vectors for luciferase (sLUC) and APE1 (sAPE1) or co-expressed with Apn1 (sLUC+Apn1 and sAPE1+Apn1). The cells were treated with 1.5 Gy x-rays (A) or BLM at 12 μg/ml for 60 min (B). At 1 h after finishing the treatment, the cells were subjected to DNA extraction, and the levels of abasic sites were determined using an aldehyde-reactive probe. C, kinetics of abasic site levels following IR (1.5 Gy x-rays). The data were quantified from three independent experiments. Standard deviations are shown, and asterisk indicates significant difference from untreated control with p < 0.05. The data were used to generate the least squares lines shown in the figure.

FIGURE 6.

Effect of p53 deficiency on BLM sensitivity in TK6 and HT116 cells. A, level of p53 protein was examined by immunoblotting in TK6 cells (transfected with vector only (WT) or with E6 protein (E6) or E6 with ectopic expression of p53 (ex53)) and in HCT116 cells (WT; homozygous p53 deficiency (null); null with ectopic expression of p53 (ex53)). The upper panels show p53, and the lower panels show β-actin as a loading control. B and C, BLM sensitivity of cells treated with control siRNA-LUC (sLUC) or APE1-specific siRNA (sAPE1) as a function of p53 status (p53d, p53-deficient). BLM treatment was as described for Fig. 2. D, BLM-induced apoptosis in TK6 cells as a function of p53 status. Cells were infected with the control (sLUC) or APE1-specific siRNA (sAPE1), challenged with BLM (30 μg/ml, 1 h), or untreated (UT) and 24 h later assayed for apoptosis by annexin V-FITC and propidium iodide staining. The data were quantified from three independent experiments. Standard deviations are shown, and asterisk indicates significant difference from sLUC control with p < 0.05.