Distinct Roles of Ape1 Protein in the Repair of DNA Damage Induced by Ionizing Radiation or Bleomycin

Hua Fung,Bruce Demple

doi:10.1074/jbc.m110.146498

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.

Highlights

For nonsurgical cancer therapy, tumor DNA is a frequent target for treatment with ionizing radiation (IR)2 or the radiomimetic drug bleomycin (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
When these occur in clusters affecting both DNA strands, highly cytotoxic double strand breaks (DSB) can be formed either directly from closely opposed single strand breaks or indirectly via the cleavage activity of DNA repair enzymes (2– 4)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions—The immortalized but untransformed TK6 human B-lymphoblastoid cell line and the derived p53-deficient TK6-E6 cells were the generous gifts of Prof. For BLM treatment, the exponentially growing cells were treated with the indicated concentrations for 1 h at 37 °C For both IR and BLM treatments, the cells were either harvested immediately after the treatment or the medium was exchanged to fresh medium, and the incubation was continued at 37 °C to allow for repair. Apoptosis Assays—IR- or BLM-treated and untreated TK6 cells (about 105 cells) were harvested, immediately stained with annexin V-FITC and propidium iodide (PI), and assayed by flow cytometry following the protocol of the manufacturer (BD Biosciences, ApoAlert kits). Samples of 1 ␮g of DNA were subjected to an AP site quantification assay based on the aldehyde-reactive probe (ARP kit, Kamiya Biomedical Co., Seattle) according to the manufacturer’s instructions. Statistical Analysis—Statistical significance between means was determined by using the standard t test and graphed with the mean Ϯ S.E

RESULTS

Untreated sLUC

DISCUSSION