The roles of BER/SSBR key proteins in genotoxic response in human cells

Abstract

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) defines genotoxic and genotoxicity as exogenous agents and processes which alter the structure, information content or integrity of DNA, including those which cause DNA damage, obstructing normal replication in a physical or non-physical manner. Even though genotoxic agents are harmful and may be life threatening, medicine has been using them for decades as a backbone of anticancer therapy. Nevertheless, as science evolved so did our knowledge of the biological characteristics and vulnerabilities of malignant tumors. The novel cancer treatments are based on the genetic make-up of cancer and designed to attack specific molecular targets. One of the most recently developed targeted therapies are poly(ADP-ribose) polymerase inhibitors (PARPi), mostly inhibiting PARP1 and/or PARP2. PARP1 (aka ARTD1) acts as a primary sensor of genotoxic stress. PARP1 detects DNA strand breaks and subsequently catalyses the formation of poly(ADP-ribose) (PAR) using NAD+ as a substrate, which leads to the recruitment of the scaffold protein XRCC1 during BER and SSBR repair and the assembly of multi-protein complexes to promote DNA repair. Beside PARP1, the role of PARP2 in BER/SSBR and its capacity in XRCC1 recruitment was demonstrated on several occasions. To better understand the roles and interplay of the above proteins in genotoxic stress response, HeLa PARP1 knockout (KO), XRCC1 KO and PARP1/XRCC1 double knockout (DKO) cells were employed and analysed in a comprehensive manner upon established anti-cancer treatments, i.e., X-irradiation and topoisomerase-1 inhibitor camptothecin (CPT). Beside the well-studied and commonly used high-dose rate radiation (HDR), HeLa KO/DKO cells were exposed to low-dose rate X-irradiation (LDR) because it is precisely these conditions that are the most relevant for the exposure of the general population. Obtained results revealed that double or single depletion of PARP1 and XRCC1 significantly diminished clonogenic survival of HeLa cells upon CPT or HDR/LDR treatments. Interestingly, the analysis of NAD+ levels and DNA damage and repair after treatment with CPT revealed a hypersensitivity phenotype of XRCC1 KO cells compared to PARP1 KO cells — an effect that could be rescued by the additional genetic deletion of PARP1 or by pharmacological PARP inhibition. In contrast to CPT results, an increase in NAD+ levels could be detected in all given genotypes, which was most prominent after 16 h both types of ionising radiation (IR) exposure. Since PARP2 acts as a XRCC1 recruiter in DNA damage repair, further in the course of the study PARP2 KO and PARP1/PARP2 DKO HeLa and PARP1 KO, XRCC1 KO, PARP2 KO, PARP1/XRCC1 DKO and PARP1/PARP2 DKO hTERT RPE-1 cells were added to the list of genotypes and the full set was analysed with regard to the response to bleomycin toxicity through a variety of biological endpoints. In concordance with the CPT and IR data, PARP1 and/or XRCC1 depletion sensitised HeLa cells to bleomycin toxicity. Moreover, NAD+ analysis and DNA damage induction and repair revealed XRCC1 KO hypersensitivity to bleomycin in both cell lines as seen upon CPT exposure and again rescued via simultaneous PARP1 depletion. Remarkably, PARP2 depletion rescued HeLa and RPE-1 cells from bleomycin toxicity, which was evident in higher survival rates upon long- and short-term exposure, low DNA strand break induction and particularly efficient repair, stable NAD+ levels and protection from ROS formation. Interestingly, the protective effects of PARP2 depletion were abrogated by simultaneous PARP1 depletion. Apart from their roles in the genotoxic stress response, deficiency of all the above proteins affected cellular function in non-stress conditions in reproducible manner: PARP1 depletion in both KO and DKO variants significantly reduced replication capacity of both cell lines. In HeLa cells, PARP1 deficiency reduced colony formation capability, and deficiency of the respective protein in any given genotype decreased the size of the colonies.