Abstract
Introduction: The base excision repair (BER) pathway is considered to be one of the most important pathways involved in the repair of DNA damage induced by ionizing radiation (IR). The DNA-(apurinic or apyrimidinic site) lyase (APEX1) and the DNA repair enzyme XRCC1 are two of its key players. Inconsistent data exist for the association between APEX1 and XRCC1 expression and cellular radiosensitivity. Some investigators have demonstrated a positive association, while others have shown little or no correlation. This study aims to investigate the effects of a decrease in the expression of APEX1 and XRCC1 on (i) growth characteristics, (ii) the survival and the cellular sensitivity to ionizing radiation, and (iii) the radiation-induced DNA damage. Gene expression profiles were determined after silencing and after additional irradiation with 5 Gy to identify possible alternative backup repair pathways and differences in the radiation response between normal and cancer cells. Thus, the study will elucidate whether imbalances in BER are associated with altered radiosensitivity. Such imbalances can be used to predict the radiation response after irradiation in cancer patients and will contribute to the understanding of normal tissue toxicity due to genetic variation in BER genes. Methods: Gene defects were analyzed in two different cell types, the breast adenocarcinoma cell line, MCF7, and a healthy counterpart, the human mammary epithelial cells HMEpC. The APEX1 and/or the XRCC1 gene were silenced in both cell types by applying the RNAi knockdown technique. Clonogenic assay and Sulforhodamine B assay were performed to assess growth characteristics and sensitivity to radiation. DNA damage after irradiation was evaluated with the DNA single-strand break specific alkaline comet assay and the gamma-H2AX assay, which uses an antibody specific for the phosphorylated form of the variant histone H2AX (gamma-H2AX) at DNA double-strand breaks. Gene expression profiles were determined on Illumina BeadChip Human Sentrix-8 arrays. Results: At the time of irradiation, APEX1 and XRCC1 mRNA amounts were decreased by more than 80 % in silenced cells, which led to reduced protein levels of up to 90 % in MCF7 cells, and up to 86 % in HMEpC. The silencing did not affect the IR-induced p53 response measured by quantification of CDKN1A mRNA levels. In MCF7, a reduced plating efficiency and growth capability was determined in XRCC1 knockdown cells. Silencing of APEX1 also resulted in a reduced growth compared to controls. No difference was observed in DNA repair rates. However, APEX1-silenced cells showed a trend towards lower initial single-strand breaks after irradiation, whereas XRCC1-silenced cells showed a trend towards a higher damage. In HMEpC, the silencing did not affect growth or the initial damage induction. The functional consequences caused by the simultaneous knockdown of APEX1 and XRCC1 were comparable to the controls in both cell types. Further, the irradiation of APEX1- and/or XRCC1-silenced cells and control showed no significant differences regarding radiation sensitivity in both cell types. However, these observations are accompanied by a cell type-specific deregulation of DNA repair genes and genes involved in the cell cycle, the cellular growth, and the proliferation. In particular, genes from the nucleotide excision repair (NER) and mismatch repair (MMR) pathway were induced in silenced cells after irradiation. Conclusions: We have established a cell model to study the effect of a deficiency in BER on cellular radiosensitivity. After silencing APEX1 and/or XRCC1, we did not observe an altered radiosensitivity compared to controls. The response to radiation and the efficiency of BER depend on all BER components and their multiple interactions within the cell. An imbalance in one of the enzymes may be compensated by other components. Based on our results, we assume that i) the APEX1-independent pathway and a possible XRCC1-independent pathway are used in situations with reduced availability of one of the proteins, and ii) other pathways such as NER and MMR are able to serve as a backup repair mechanism to repair radiation-induced DNA damage in double knockdown cells. This study suggests that APEX1 and XRCC1 are useful targets to modulate cellular responses to DNA damaging agents including IR. Our dataset offers several scientific directions for future studies with the aim to further investigate the mechanisms responsible for the different functions of APEX1 and XRCC1 in BER in primary and cancer cells.
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