Overexpression of exogenous Bcl2 suppresses APE1 endonuclease activity in association with decreased AP site repair. A, Bcl2/pCIneo DNA construct was stably transfected into H1299 human lung cancer cells. Expression levels of Bcl2 were determined by Western blotting. B, ³²P-labeled 26-mer AP site mimetic oligonucleotide was incubated with nuclear extract (5 μg) isolated from vector-only control cells or three stable clones expressing similar levels of exogenous Bcl2. APE1 endonuclease activity was analyzed by autoradiography. C, vector-only control cells or three stable clones expressing similar levels of exogenous Bcl2 were treated with NNK (5 μm) for 60 min. The cells were washed and incubated in regular culture medium for various times up to 72 h. The AP sites were analyzed using an AP site counting kit. The data represent the means ± S.D. of three separate determinations. D, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract (5 μg) isolated from Bcl2-overexpressing H1299 cells in the absence or presence of increasing concentrations (0.5–2 ng/ml) of purified APE1. APE1 endonuclease activity was analyzed by autoradiography.

Bcl2 directly interacts with APE1 via the BH domains of Bcl2, and deletion of any of the BH domains from Bcl2 results in loss of the ability of Bcl2 to suppress APE1 endonuclease activity as well as AP site repair. A, schematic representation of the BH domains in Bcl2 protein. B, purified recombinant APE1 (10 ng) was incubated with purified WT, ΔBH1, ΔBH2, ΔBH3, or ΔBH4 Bcl2 deletion mutants (10 ng each) in 1% CHAPS lysis buffer at 4 °C for 2 h. The APE1-associated Bcl2 was co-immunoprecipitated (IP) with APE1 antibody. The APE1-associated Bcl2 was analyzed by Western blot. C, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with APE1 (1 ng/ml) in the absence or presence of purified, recombinant WT, ΔBH1, ΔBH2, ΔBH3, or ΔBH4 Bcl2 mutant protein (1 ng/ml) for 15 min. APE1 activity was analyzed by autoradiography. Lane 1 is the 26-mer AP site mimetic oligonucleotide-only control without treatment. D, WT, ΔBH1, ΔBH2, ΔBH3, and ΔBH4 Bcl2 mutants were stably transfected into H1299 cells. Expression levels of Bcl2 were analyzed by Western blot. E, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract isolated from vector-only cells or H1299 cells expressing WT or each of the BH deletion Bcl2 mutants. APE1 endonuclease activity was analyzed by autoradiography. Lane 1 is the 26-mer AP site mimetic oligonucleotide-only control without treatment. F, vector-only control cells or H1299 cells expressing WT or each of the BH deletion Bcl2 mutants were treated with NNK (5 μm) for 60 min. The cells were washed and incubated in regular culture medium for various times up to 72 h. The AP sites were analyzed using an AP site counting kit. The data represent the means ± S.D. of three separate determinations.

Depletion of endogenous Bcl2 expression by RNA interference results in elevated APE1 endonuclease activity and accelerated AP site repair. A, H460 cells expressing high levels of endogenous Bcl2 were transfected with Bcl2 siRNA (i.e. 10 nm) or control siRNA. Expression levels of Bcl2 were analyzed by Western blot. B, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract isolated from H460 parental cells or H460 cells expressing control siRNA or Bcl2 siRNA. APE1 endonuclease activity was analyzed by autoradiography. Lane 1 is the 26-mer AP site mimetic oligonucleotide-only control without treatment. C, H460 cells expressing control siRNA or Bcl2 siRNA were treated with NNK (5 μm) for 60 min. The cells were washed and incubated in regular culture medium for various times up to 72 h. AP sites were analyzed using an AP site counting kit. The data represent the means ± S.D. of three separate determinations.

Expression of endogenous Bcl2 in human lung cancer cells is associated with decreased APE1 endonuclease activity. A, expression levels of endogenous Bcl2 and APE1 in various human lung cancer cells were analyzed by Western blot. B, schematic representation of AP oligonucleotide endonuclease assay. Radiolabeled 26-mer oligonucleotide containing a tetrahydrofuran AP site mimic (F) at position 15 in the labeled strand reacts with APE1 protein or nuclear extract isolated from cells in assay buffer. After the reaction is stopped, the fragments are denatured and analyzed on a DNA gel. If there is no AP endonuclease activity at the AP site, the unreacted product migrates as a 26-mer, whereas AP endonuclease activity is shown as a 14-mer fragment. C, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract (5 μg) isolated from various human lung cancer cells. AP endonuclease activity was measured and analyzed by autoradiography in various human lung cancer cells. Lane 1 is the 26-mer AP site mimetic oligonucleotide-only control without treatment.

DNA-damaging agent NNK promotes Bcl2 nuclear accumulation and interaction with APE1. A, H460 cells expressing high levels of endogenous Bcl2 were treated with NNK (5 μm) for 60 min. Subcellular distribution of Bcl2 was examined by immunofluorescent staining using a rabbit antibody against human Bcl2 and Alexa 594 (red)-conjugated anti-rabbit secondary antibodies for 60 min. The samples were observed under a fluorescent microscope (Zeiss). B, H460 cells were treated with NNK (5 μm) for various times. Subcellular fractionation was performed to isolate nuclear and mitochondrial fractions. The levels of Bcl2 in the nuclear or mitochondrial fraction were analyzed by Western blotting using a Bcl2 antibody. C, H460 cells were treated with increasing concentrations of NNK for 1 h. A co-immunoprecipitation (IP) experiment was performed in isolated nuclear extract using APE1 antibody. Bcl2 and APE1 were analyzed by Western blot.

Overexpression of Bcl2 in cells inhibits formation of the APE1·XRCC1 complex, and purified Bcl2 directly disrupts the APE1· XRCC1 interaction in association with inhibition of APE1 endonuclease activity in vitro. A, vector-only control cells or Bcl2-overexpressing H1299 cells were treated with NNK (5 μm) for 60 min and disrupted in 1% CHAPS lysis buffer. Co-immunoprecipitation (IP) was performed using an agarose-conjugated APE1 antibody. The APE1-associated XRCC1 and total APE1 were then analyzed by Western blot. B, the APE1·XRCC1 complex was co-immunoprecipitated from H1299 parental cells that express undetectable levels of endogenous Bcl2 using an agarose-conjugated APE1 antibody and incubated with increasing concentrations of purified Bcl2 (0.5–2 ng/ml) in 1% CHAPS lysis buffer at 4 °C for 2 h. The samples were centrifuged at 14,000 × g for 5 min. The resulting supernatant and immunocomplex beads were subjected to SDS-PAGE. Bcl2 and XRCC1 that bound to APE1, total APE1, or nonbound XRCC1 present in the supernatant were then analyzed by Western blot. C, ³²P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract isolated from H1299 parental cells in the absence or presence of increasing concentrations (0.1–5 ng/ml) of purified, recombinant Bcl2. APE1 endonuclease activity was analyzed by autoradiography. Lane 1 is the 26-mer AP site mimetic oligonucleotide-only control without treatment.