Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing

Abstract

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.

Keywords: Ape1; DNA Damage; DNA Damage Response; DNA Enzyme; DNA Repair; DNA-binding Protein; High LET; Radiation Biology; Radiosensitivity.

FIGURE 1.

Up-regulation of Ape1 sensitizes cells to high LET radiation-induced killing. A, the Ape1 levels were detected by immunoblotting at 30 h after transfecting the Ape1-containing vector or control vector into the MEF cell lines. WT, wild type MEF cells; NHEJd, NHEJ-deficient Ku80^−/− MEF cells. β-Actin was used as the internal control. B, MEF cell sensitivities to low LET (L) or high LET (H) radiation at different doses as labeled were examined using a clonogenic assay. Data are the mean and S.D. (error bars) obtained from three independent experiments. **, p < 0.01. C, Ogg1 levels were detected by immunoblotting at 30 h after transfecting the Ogg1-containing vector or control vector into the MEF Ogg1^−/− cells. D, the sensitivities of the MEF cells that transfected with either control vector without Ogg1 (Ogg1−/−) or vector encoding Ogg1 (+Ogg1) to low LET or high LET radiation at different doses, as labeled, were examined using a clonogenic assay. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. E, the Ape1 levels were detected by Western blot at 30 h after transfecting the vector control, vector encoding Ape1 to the human cells (WT MRC5SV), control RNA, or siRNA against Ogg1 shown in the left frame. The Ogg1 levels are shown in the right frame. β-Actin was used as the internal control. F, the sensitivities of human cell MRC5SV (WT) or 180BRM (NHEJd; NHEJ-deficient) to low LET or high LET at different doses as labeled were examined using a clonogenic assay. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. G, the RBE of NHEJd cells (M, Ku80^−/− MEF cells; H, transformed 180BRM human fibroblast cells) in cell killing with or without Ape1 overexpression.

FIGURE 2.

Up-regulation of Ape1 results in more unrepaired DNA DSBs in high LET- than in low LET-irradiated cells. A, left, image of γ-H2AX foci in wild type cells at 1 h after 1 Gy of high or low LET radiation; right, percentage of γ-H2AX foci-positive WT MEF cells transfected with control plasmid (CP) or the plasmid encoding Ape1 (AP) at different times (1, 4, and 24 h) after 1 Gy of high LET or low LET IR. B, NHEJ-deficient (NHEJd) (Ku80^−/−) MEF cells in the same assay as described in A. C, percentage of γ-H2AX foci-positive Ogg1^−/− MEF transfected with control vector or Ogg1^−/− MEF cells with Ogg1 re-expressed (Ogg1+) (with or without overexpression of Ape1, as described in A, at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. (error bars) from two separate experiments (examining 300 cells for each experiment). *, p < 0.05. D, quantitation of γ-H2AX foci-positive cells in WT human MRC5SV cells transfected with control plasmid or the plasmid encoding Ape1 at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. from two separate experiments (300 cells examined during each experiment). *, p < 0.05. E, quantitation of γ-H2AX foci-positive cells in NHEJ-deficient human 180BRM cells transfected with control plasmid or the plasmid encoding Ape1 at different times (1, 4, and 24 h) after 1 Gy of high or low LET IR. Each point represents the mean ± S.D. from two separate experiments (300 cells examined during each experiment). *, p < 0.05.

FIGURE 3.

Up-regulation of Ape1 results in more Mre11 bound to chromatin DNA in high LET-irradiated cells. A, the levels of Mre11 or γ-H2AX associated with chromatin (Chr bound) were examined in the WT MEF cells transfected with control plasmid (Control) or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The levels of Mre11 or H2A in whole cell lysates (WC lysates) were used as a loading control. The data represent the mean ± S.D. (error bars) from three independent experiments; *, p < 0.05. B, the levels of Mre11 or γ-H2AX associated with chromatin were examined in NHEJd (Ku80^−/−) MEF cells transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments; *, p < 0.05. C, the levels of Mre11 or γ-H2AX associated with chromatin were examined in Ogg1^−/− MEF cells transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments. D, the levels of Mre11 or γ-H2AX associated with chromatin were examined in Ogg1⁺ cells (Ogg1^−/− cells with Ogg1 re-expressed) transfected with control plasmid or the plasmid encoding Ape1 at 1 h after exposure to 10 Gy of low or high LET IR. The data represent the mean ± S.D. from three independent experiments. *, p < 0.05.

FIGURE 4.

Up-regulation of Ape1 results in more DNA signals that were detected in the Mre11 immunoprecipitated complex in high LET-irradiated cells. A, the outline of a flow chart to detect DNA fragments in the Mre11-DNA complex. B, image of the DNA levels in the Mre11-DNA complex from non-irradiated (NR), low LET-irradiated, or high LET-irradiated Ogg1^−/− MEF cells co-transfected with the control vector (Ogg1−/−) or the vector encoding Ogg1 (Ogg1+) with another control plasmid (CP) or the plasmid encoding Ape1. One-tenth of the immunoprecipitation (IP) samples were used as the internal loading control. C, quantitation of the image data representing the mean ± S.D. (error bars) from three independent experiments. **, p < 0.01.

FIGURE 5.

Physiological levels of Ape1 contribute to high LET IR-induced RBE in cell killing. A, outline of the plasmid constructs of the enzymatically overactivated or inactivated mouse Ape1. B, description of the design for siRNA-resistant mouse Ape1 vector. The pink sites are mutated nucleotides. C, endogenous or exogenous Ape1 was detected by immunoblotting with the Ape1 or HA antibody in wild type MEF cells transfected with wild type (WT), inactivated (In), or overactivated (Ac) Ape1 combined with control (Ct) RNA or siRNA against Ape1 (siApe1). D, the relative Ape1 enzymatic activity in the MEF cells expressing different types of Ape1 was analyzed by comparing with the enzymatic activity of the cells transfected with wild type Ape1 when the endogenous Ape1 was knocked down without IR (NR). The cells were collected at 1 h after exposure to 4 Gy of low LET (L4 Gy) or 2 Gy of high LET (H2 Gy) radiation. The data were derived from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, the cell sensitivities to low LET (L) or high LET (H) irradiation at the indicated dose as labeled were examined using a clonogenic assay. Data are the mean and S.D. (error bars) obtained from three independent experiments. *, p < 0.05; **, p < 0.01.

FIGURE 6.

Small DNA DSB fragments (∼40 bp) do not affect HRR efficiency in vivo. A, HRR reporter plasmid design. pDR-GFP-40 was generated by introducing a second I-SceI digestion site 40 bp upstream from the original I-SceI site within the GFP coding region. B, the digestion efficiency of I-SceI in vitro. Top, efficiency of I-SceI digestion of the plasmid with one digestion site (1 dig site) or two digestion sites (2 dig sites) for I-SceI as described in A. The digestion efficiency was analyzed by PCR with proper primers. Bottom, based on the image of the gel, as shown in the top, we analyzed the in vitro digestion efficiency of I-SceI. Data represent the mean ± S.D. (error bars) from three independent experiments. **, p < 0.01. C, HRR efficiency was examined in human 293FT cells integrated with the substrate of I-SceI after transfecting with I-SceI plasmid. Data are the mean and S.D. obtained from three independent experiments. **, p < 0.01. D, a model explaining how high LET IR generates DSBs in mammalian cells. High LET radiation-induced small DNA fragments are generated from a direct energy transfer track to break DNA strands and from subsequent Ape1 enzymatic modification in the clustered damage DNA sites. These small DNA fragments interfere with Ku-dependent NHEJ but do not affect HRR.