Cisplatin-Mediated Upregulation of APE2 Binding to MYH9 Provokes Mitochondrial Fragmentation and Acute Kidney Injury.

Cisplatin chemotherapy is standard care for many cancers but is toxic to the kidneys. How this toxicity occurs is uncertain. In this study, we identified apurinic/apyrimidinic endonuclease 2 (APE2) as a critical molecule upregulated in the proximal tubule cells (PTC) following cisplatin-induced nuclear DNA and mitochondrial DNA damage in cisplatin-treated C57B6J mice. The APE2 transgenic mouse phenotype recapitulated the pathophysiological features of C-AKI (acute kidney injury, AKI) in the absence of cisplatin treatment. APE2 pulldown-MS analysis revealed that APE2 binds myosin heavy-Chain 9 (MYH9) protein in mitochondria after cisplatin treatment. Human MYH9-related disorder is caused by mutations in MYH9 that eventually lead to nephritis, macrothrombocytopenia, and deafness, a constellation of symptoms similar to the toxicity profile of cisplatin. Moreover, cisplatin-induced C-AKI was attenuated in APE2-knockout mice. Taken together, these findings suggest that cisplatin promotes AKI development by upregulating APE2, which leads to subsequent MYH9 dysfunction in PTC mitochondria due to an unrelated role of APE2 in DNA damage repair. This postulated mechanism and the availability of an engineered transgenic mouse model based on the mechanism of C-AKI provides an opportunity to identify novel targets for prophylactic treatment of this serious disease. SIGNIFICANCE: These results reveal and highlight an unexpected role of APE2 via its interaction with MYH9 and suggest that APE2 has the potential to prevent acute kidney injury in patients with cisplatin-treated cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/713/F1.large.jpg.

An APE1 inhibitor reveals critical roles of the redox function of APE1 in KSHV replication and pathogenic phenotypes.

APE1 is a multifunctional protein with a DNA base excision repair function in its C-terminal domain and a redox activity in its N-terminal domain. The redox function of APE1 converts certain transcription factors from inactive oxidized to active reduced forms. Given that among the APE1-regulated transcription factors many are critical for KSHV replication and pathogenesis, we investigated whether inhibition of APE1 redox function blocks KSHV replication and Kaposi's sarcoma (KS) phenotypes. With an shRNA-mediated silencing approach and a known APE-1 redox inhibitor, we demonstrated that APE1 redox function is indeed required for KSHV replication as well as KSHV-induced angiogenesis, validating APE1 as a therapeutic target for KSHV-associated diseases. A ligand-based virtual screening yielded a small molecular compound, C10, which is proven to bind to APE1. C10 exhibits low cytotoxicity but efficiently inhibits KSHV lytic replication (EC50 of 0.16 µM and selective index of 165) and KSHV-mediated pathogenic phenotypes including cytokine production, angiogenesis and cell invasion, demonstrating its potential to become an effective drug for treatment of KS.

Accelerated processing of solitary and clustered abasic site DNA damage lesions by APE1 in the presence of aqueous extract of Ganoderma lucidum.

The stimulatory effect of the aqueous extract of G. lucidum, a basidiomycetes class fungus in the APE1-enzyme-mediated processing of solitary and bistranded clustered abasic sites DNA damages is presented. Abasic sites are considered the most common type of DNA damage lesions. Our study shows enhanced activity of APE1 in the processing of abasic sites in the presence of the polysaccharides fraction of G. lucidum. Remarkable increase in the amount of single-strand breaks (SSBs) and double-strand breaks (DSBs) from solitary and bistranded clustered abasic sites respectively with APE1 in the presence of the extract was found. This trend is maintained when abasic sites in DNA oligomers are exposed to fibroblast cell extracts in the presence of the extract. While DNA conformational alteration is negligible, APE1 enzyme shows characteristic changes in the alpha helix and beta strand ratio after incubation with G. lucidum extract. The enhanced reactivity of APE1 at the molecular level in the presence of G. lucidium is attributed to this effect. This study potentially amplifies the scope of the use of G. lucidum, which was earlier shown to have only reactive oxygen species (ROS) scavenging properties with regards to DNA damage inhibition.

Inactivation by oxidation and recruitment into stress granules of hOGG1 but not APE1 in human cells exposed to sub-lethal concentrations of cadmium.

The induction of mutations in mammalian cells exposed to cadmium has been associated with the oxidative stress triggered by the metal. There is increasing evidence that the mutagenic potential of Cd is not restricted to the induction of DNA lesions. Cd has been shown to inactivate several DNA repair enzymes. Here we show that exposure of human cells to sub-lethal concentrations of Cd leads to a time- and concentration-dependent decrease in hOGG1 activity, the major DNA glycosylase activity responsible for the initiation of the base excision repair (BER) of 8-oxoguanine, an abundant and mutagenic form of oxidized guanine. Although there is a slight effect on the level of hOGG1 transcripts, we show that the inhibition of the 8-oxoguanine DNA glycosylase activity is mainly associated with an oxidation of the hOGG1 protein and its disappearance from the soluble fraction of total cell extracts. Confocal microscopy analyses show that in cells exposed to Cd hOGG1-GFP is recruited to discrete structures in the cytoplasm. These structures were identified as stress granules. Removal of Cd from the medium allows the recovery of the DNA glycosylase activity and the presence of hOGG1 in a soluble form. In contrast to hOGG1, we show here that exposure to Cd does not affect the activity of the second enzyme of the pathway, the major AP endonuclease APE1.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) in human ovarian cancer and indentification of the therapeutic potential of APE1/Ref-1 inhibitor.

Resistance to platinum is a major limitation for the treatment of ovarian cancer. In an effort to overcome the platinum resistance problem in ovarian cancer treatment, we explored the correlation between cisplatin resistance and the human AP endonuclease (APE1 or Ref-1). APE1/Ref-1 is a multifunctional protein that is not only an essential enzyme in base excision repair pathway, but also acts as a major redox-signaling factor that has a wide variety of important cellular functions including transcription factor regulation, oxidative signaling and cell cycle control. In this study, we examined APE1/Ref-1 expression by immunohistochemistry in sections of ovarian cancers from 78 patients who were administered standard adjuvant chemotherapy based on platinum post-operatively. Altered levels and subcellular APE1/Ref-1 expression was found in patients not responding to platinum-based chemotherapy comparing with those who responded to platinum-based chemotherapy. Meanwhile, we detected the APE1/Ref-1 expression in A2780 and CP70 cell lines which have different sensitivity to cisplatin. We found similar altered APE1/Ref-1 expression in them. We hypothesized that the APE1/Ref-1 expression is responsible in part for the cisplatin resistance. To answer this hypothesis, we decreased the APE1/Ref-1 level by silencing RNA targeting technology in A2780 and CP70 cell lines. The A2780 cells treated with APE1-siRNA had IC50 values ranging from 6.70 to 1.74 microM cisplatin compared with 15.81 microM for control A2780 cells. The CP70 cells treated with APE1-siRNA had 1.62-4.63-fold enhancement in cisplatin sensitivity. The apoptosis assays using TUNEL analysis showed that decreased APE1/Ref-1 level resulted in increased apoptosis levels in A2780 and CP70 cell lines compared with the control-treated cells. These data suggest that APE1/Ref-1 levels play an important role in the sensitization of ovarian cancer cells to apoptosis. In vitro studies revealed that it is possible to substantially enhance the cisplatin cytotoxicity by decreasing APE1/Ref-1 level in cisplatin-resistant cell lines.

[Effects of Ruanmailing Oral Liquid on spatial learning and memory ability and expression of APE/Ref-1 in hippocampal CA1 region in rats with experimental vascular dementia].

OBJECTIVE: To study the effects of Ruanmailing Oral Liquid, a compound traditional Chinese herbal medicine, on spatial learning and memory ability and expression of apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) in hippocampal CA1 region in rats with experimental vascular dementia (VaD). METHODS: VaD was induced in rats by permanent occlusion of bilateral common carotid arteries. Forty-five VaD rats were randomly divided into untreated group, nimodipine group, low-dose Ruanmailing group and high-dose Ruanmailing group. Another 15 rats underwent a sham operation consisting of similar skin incision and manipulation but without occlusion of carotid arteries. From the next day after occlusion, the rats were intragastrically administered with normal saline, nimodipine suspension or Ruanmailing Oral Liquid respectively for 30 days. Morris water maze experiment was adopted to test learning and memory of rats in each group. Expression of APE/Ref-1 protein in the hippocampal CA1 region was measured by immunohistochemical method. RESULTS: Escape latency was significantly shortened and number of entries in the target area of rats was significantly increased in the high-dose Ruanmailing group as compared with those in the untreated group (P<0.01). Compared with the untreated group, count of APE/Ref-1 positive cells was significantly increased in the hippocampal CA1 region in the high- and low-dose Ruanmailing groups (P<0.01). Compared with the low-dose group and the nimodipine group, the count of APE/Ref-1 positive cells was remarkably increased in the hippocampal CA1 region in rats of the high-dose Ruanmailing group (P<0.01). There was no statistical difference between the low-dose Ruanmailing group and the nimodipine group. CONCLUSION: Ruanmailing Oral Liquid can improve the learning and memory ability and enhance the lowered expression level of APE/Ref-1 in the hippocampal CA1 region of rats with VaD.

Radiation-induced HIF-1alpha cell survival pathway is inhibited by soy isoflavones in prostate cancer cells.

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-kappaB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-kappaB and HIF-1alpha. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1alpha survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1alpha. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (AR+) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1alpha activation by radiation and nuclear translocation of HIF-1alpha. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1alpha and NF-kappaB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1alpha and NF-kappaB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-kappaB and HIF-1alpha. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1alpha, APE1/Ref-1 and NF-kappaB.

Apurinic/apyrimidinic endonuclease/redox effector factor-1(APE/Ref-1): a unique target for the prevention and treatment of human melanoma.

Management of melanoma is a growing and challenging public health issue requiring novel and multidisciplinary approaches to achieve more efficient prevention and therapeutic benefits. The aim of this article is to show the critical role of APE/Ref-1 on melanomagenesis and progression. APE/Ref-1 serves as a redox-sensitive node of convergence of various signals as well as a DNA-repair enzyme, and its activation protects melanocytes and melanoma cells from chronic oxidative stress and promotes cell survival via mediation of downstream pathways. APE/Ref-1 is a strong candidate as a potential drug-treatable target for the prevention and treatment of human melanoma. Lead compounds exhibiting inhibitory effects on APE/Ref-1 are also reviewed. We anticipate potential clinical benefit in the future through inhibition of APE/Ref-1 and/or Ref-1-mediated signaling.

Combined treatment with temozolomide and methoxyamine: blocking apurininc/pyrimidinic site repair coupled with targeting topoisomerase IIalpha.

PURPOSE: Methoxyamine has been shown to potentiate the cytotoxic effect of temozolomide both in vitro and in human tumor xenograft models. We postulate that the enhanced cytotoxicity is mediated by methoxyamine-bound apurininc/pyrimidinic (MX-AP) site, a key lesion formed by the combination of temozolomide and methoxyamine. When located within topoisomerase IIalpha (topo II) cleavage sites in DNA, MX-AP sites act as dual lethal targets, not only functionally disrupting the base excision repair (BER) pathway but also potentially poisoning topo II. EXPERIMENTAL DESIGN: Using oligonucleotide substrates, in which a position-specific MX-AP site is located within topo II cleavage sites, we examined the effect of MX-AP site on both AP endonuclease- and topo II-mediated DNA cleavage in vitro. RESULTS: MX-AP sites were refractory to the catalytic activity of AP endonuclease, indicating their ability to block BER. However, they were cleaved by either purified topo II or nuclear extracts from tumor cells expressing high levels of topo II, suggesting that MX-AP sites stimulate topo II-mediated DNA cleavages. In cells, treatment with temozolomide and methoxyamine increased the expression of topo II and enriched the formation of gammaH2AX foci, which were colocalized with up-regulated topo II, confirming that DNA double-strand breaks marked by gammaH2AX foci are associated with topo II in cells. CONCLUSIONS: Our findings identify a molecular mechanism of cell death whereby MX-AP sites that cumulated in cells due to resistance to BER potentially convert topo II into biotoxins, resulting in enzyme-mediated DNA scission and cell death.

Age-related loss of the DNA repair response following exposure to oxidative stress.

Young (4- to 6-month-old) and aged (24- to 28-month-old) mice were exposed to 2-nitropropane (2-NP), a DNA oxidizing agent, and the ability to induce DNA polymerase beta (beta-pol) and AP endonuclease (APE) was determined. In contrast to the inducibility of these gene products in response to oxidative damage in young mice, aged mice showed a lack of inducibility of beta-pol and APE. APE protein level and endonuclease activity were both reduced 40% (p<.01) in response to 2-NP. Accordingly, the accumulation of DNA repair intermediates in response to 2-NP differed with age. Young animals accumulated 3'OH-containing DNA strand breaks, whereas the aged animals did not. A role for p53 in the difference in DNA damage response with age is suggested by the observation that the accumulation of p53 protein in response to DNA damage in young animals was absent in the aged animals. Our results are consistent with a reduced ability to process DNA damage with age.

Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe.

The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation. We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1. We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts. Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE.

Cyclophilin A modulates the sensitivity of HIV-1 to host restriction factors.

Many mammalian species express restriction factors that confer host resistance to retroviral infection. Here we show that HIV-1 sensitivity to restriction factors is modulated by cyclophilin A (CypA), a host cell protein that binds the HIV-1 capsid protein (CA). In certain nonhuman primate cells, the CA-CypA interaction is essential for restriction: HIV-1 infectivity is increased >100-fold by cyclosporin A (CsA), a competitive inhibitor of the interaction, or by an HIV-1 CA mutation that disrupts CypA binding. Conversely, disruption of CA-CypA interaction in human cells reveals that CypA protects HIV-1 from the Ref-1 restriction factor. These findings suggest that HIV-1 has co-opted a host cell protein to counteract restriction factors expressed by human cells and that this adaptation can confer sensitivity to restriction in unnatural hosts. Manipulation of HIV-1 CA recognition by restriction factors promises to advance animal models and new therapeutic strategies for HIV-1 and AIDS.