Targeting truncated APE1 in mitochondria enhances cell survival after oxidative stress

The high steady-state level of mitochondrial DNA (mtDNA) oxidative lesions is assumed to be the result of high susceptibility to DNA damage attack and limited DNA repair capacity in mitochondria. As a key enzyme of base excision repair (BER), human apurinic/apyrimidinic endonuclease (APE1) is often scarce in mitochondria, and mitochondria-targeted APE1 with robust repair activity represents a promising therapeutic candidate. In this study, overexpression vectors of mitochondria-targeted truncated APE1 (mtAPE1) and that of full-length APE1 (flAPE1) were constructed and transfected to human umbilical vein endothelial cells to test their protective effects after hydrogen peroxide-induced oxidative stress. The overexpression of truncated APE1 was achieved at protein and enzyme activity levels in mitochondria of mtAPE1-transfected cells. In parallel, enhanced mtDNA repair capacity and increased cell survival were observed. MtAPE1 transfection also prevented apoptosis by blocking mitochondria-dependent pathways. In contrast, flAPE1 transfection rendered slight elevation of nuclear APE1 protein level and nuclear APE activity but no benefits for cell resistance to oxidative stress. The present results suggest that overexpression of the truncated APE1 in mitochondria appears to be a viable approach to protecting healthy cells from some deleterious effects of oxidative stress.     

The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures--specifically neuronal cells--from IR-induced damage, while the redox activity of APE1 does not appear to be involved.     

Diverse Small Molecule Inhibitors of Human Apurinic/Apyrimidinic Endonuclease APE1 Identified from a Screen of a Large Public Collection

The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.     

H(2)O(2) induces translocation of APE/Ref-1 to mitochondria in the Raji B-cell line

Reactive oxygen species (ROS) are generated as by-products of respiration and are used as signal transducing intermediates in out-in signaling pathways. ROS are also generated during inflammatory responses and it has been shown that hydrogen peroxide may trigger activation of B-lymphocytes, similar to cross-linking of surface immunoglobulins. On the other hand, both exogenous and endogenous generated ROS are a major source of nuclear and mitochondrial DNA (mtDNA) damage. The base excision repair (BER) enzyme APE/Ref-1 normally repairs small nuclear DNA lesion such as oxidized or alkylated bases. It is not clear though whether DNA repair mechanisms able to abolish oxidative damage from nuclear DNA are present into mitochondria too. Here we show by confocal microscopy and Western blot analysis that in the B-lymphocyte Raji cell line a fraction of APE/Ref-1 rapidly re-localizes into mitochondria following H(2)O(2) activation. Targeting of APE/Ref-1 to mitochondria is not associated with cytochrome-c loss or apoptosis induction. These findings indicate that the APE/Ref-1 translocates to mitochondria in response to oxidative stress and thereby it might exert a protective function.     

Processing oxidatively damaged bases at DNA strand breaks by APE1

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3′ to 5′ exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3′-end of a DNA strand during replication or repair. Importantly, 3′-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we use X-ray crystallography to characterize the exo activity of APE1 on 3′-8-oxoG substrates. These structures support a unified APE1 exo mechanism that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein: DNA contacts that specifically mediate 8-oxoG removal by APE1.     

Role of the DNA Base Excision Repair Protein,APE1 in Cisplatin,Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.     

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53^(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.     

Intracellular trafficking and regulation of mammalian AP-endonuclease 1 (APE1), an essential DNA repair protein

AP endonuclease (APE), with dual activities as an endonuclease and a 3' exonuclease, is a central player in repair of oxidized and alkylated bases in the genome via the base excision repair (BER) pathway. APE acts as an endonuclease in repairing AP sites generated spontaneously or after base excision during BER. It also removes the 3' blocking groups in DNA generated directly by ROS or after AP lyase reaction. In contrast to E. coli and lower eukaryotes which express two distinct APEs of Xth and Nfo types, mammalian genomes encode only one APE, APE1, which is of the Xth type. However, while the APEs together are dispensable in the bacteria and simple eukaryotes, APE1 is essential for mammalian cells. We have shown that apoptosis of mouse embryo fibroblasts triggered by APE1 inactivation can be prevented by ectopic expression of repair competent but not repair-defective APE1. The mitochondrial APE (mtAPE) is an N-terminal truncation product of APE1. A significant fraction of APE1 is cytosolic, and oxidative stress induces its nuclear and mitochondrial translocation. Such age-dependent increase in APE activity in the nucleus and mitochondria is consistent with the hypothesis that aging is associated with chronic oxidative stress.     

APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability

XRCC1 protein is essential for mammalian viability and is required for the efficient repair of single strand breaks (SSBs) and damaged bases in DNA. XRCC1-deficient cells are genetically unstable and sensitive to DNA damaging agents. XRCC1 has no known enzymatic activity and is thought to act as a scaffold protein for both SSB and base excision repair activities. To further define the defects leading to genetic instability in XRCC1-deficient cells, we overexpressed the AP endonuclease APE1, shown previously to interact with and be stimulated by XRCC1. Here, we report that the overexpression of APE1 can compensate for the impaired capability of XRCC1-deficient cells to repair SSBs induced by oxidative DNA damage, both in vivo and in whole-cell extracts. We show that, for this kind of damage, the repair of blocked DNA ends is rate limiting and can be performed by APE1. Conversely, APE1 overproduction resulted in a 3-fold increase in the sensitivity of XRCC1-deficient cells to an alkylating agent, most probably due to the accumulation of SSBs. Finally, the overproduction of APE1 results in increases of 40% in the frequency of micronuclei and 33% in sister chromatid exchanges of XRCC1⁻ cells. These data suggest that the spontaneous generation of AP sites could be at the origin of the SSBs responsible for the spontaneous genetic instability characteristic of XRCC1-deficient cells.