The redox function of APE1 is found only in mammals and not in other vertebrates, as demonstrated by the lack of redox function of the Zebrafish APE1 (zAPE1). The acquisition of the redox function in APE1 proteins is discussed in a recent publication (39). The two major functions of APE1, redox and repair, are completely independent in their actions, as shown by the observation that a mutation of the Cys at position 65 (C65) abolishes the redox function but does not affect the repair function (86), whereas mutation of a variety of amino acids required for DNA repair activity, such as Histidine 309 (H309) and others (87, Vascotto et al., unpublished observations), do not affect the redox function. While the DNA repair active site of APE1 has been clearly delineated (44, 90), the redox domain is much less defined. The only Cys residue required for full redox function is C65, which is buried within the APE1 protein (44, 90), as recently confirmed by Georgiadis who mutated in the zAPE1 a Thr (T58) to a Cys located at the same position of the Cys in the mammalian APE1, resulting in the acquisition of redox activity of the mutated protein (39).

The vital role of APE1 seems to be due to its fundamental activity in the base excision repair (BER) pathway of DNA lesions (35). However, the biological relevance in eukaryotic transcriptional regulation of gene expression as a redox co-activator of different transcription has yet to be fully elucidated (126).

Another interesting function is associated to its ability to indirectly bind to the negative calcium response elements (nCaRE) of some promoters (i.e., parathyroid hormone (PTH) and APE1 promoters) acting as a transcriptional repressor. In particular, binding to nCaRE-B, in the PTH gene promoter, requires Ku70 (Ku86) as an additional factor in the complex (17), while binding to nCaRE-B2 in the APE1 promoter involves hnRNPL interaction (77). This activity seems to be regulated by acetylation of APE1 through the CBP/p300 HAT action (7). A more recent finding has identified APE1's role in mediating production of single-strand DNA (ssDNA) breaks in gene promoters during repair of targeted base oxidation lesions caused by oxygen radicals generated during physiologic signaling (10, 153, 154). Thus, defects in the APE1-mediated step in BER pathway could be linked to altered gene expression besides altering transcription factor state. This is discussed later in this review.

APE1 subcellular distribution, in different mammalian cell types, is mainly nuclear and is critical in controlling cellular proliferative rate (35, 57, 67). Interestingly, the expression of APE1 is strictly connected to different tumorigenic processes (126). The necessity of APE1 for cellular survival and its frequent overexpression in tumor cells strongly suggest a fundamental role of this protein in preventing cell death and in controlling cellular proliferation. However, APE1 abilities to activate transcription factors, such as p53 and Egr-1 (14, 30, 38, 54, 64, 107, 117), mainly involved in controlling cell-cycle arrest and apoptotic programs, leave open the debate concerning the mechanisms responsible for controlling the different functions in several contexts. APE1 protein is also localized within mitochondria in different cell types but, at present, the role in this organelle has not been completely elucidated (15, 88, 122), as well as the molecular mechanism regulating its mitochondrial localization (15).

The BER is the most used pathway to cope with the single base lesion. The BER pathway is also involved in repairing the DNA single-strand breaks (SSB) induced by free radical agents. One of the key enzymes of the BER pathway in mammals is APE1. The basic reactions of the BER pathway have been extensively reviewed (26, 58, 81, 88, 126). They require the coordinated activity of a number of enzymes including: (a) a DNA glycosylase capable of excising a specific modified base; (b) an AP endonuclease, such as APE1, which cleaves the 5′ phosphodiester bond, generating 3′OH and 5′dRP termini; (c) an exonuclease activity (β-polymerase, FEN, APE1); (d) a DNA polymerase (β-polymerase, XRCC1 or δ/-polymerase with PCNA); and, finally (e) a ligation activity (DNA ligases I and III, XRCC1), (Fig. 1B). The three ways by which APE1 produces the 3′OH terminus for priming and the types of evolutionarily conserved amino acids necessary for these functions have been already reviewed (26, 58 and other articles in this issue of ARS).

All the steps of the BER pathway are finely orchestrated, both from the thermodynamic and the kinetic point of view, to provide an accurate repair of the damaged base and to avoid the generation of intermediate products that are toxic for the cell (58). This implies the coordinated interaction of the various players in the BER process and, when necessary, their further interaction with the DNA replication machinery, as demonstrated by the co-immunoprecipitation of the BER repair proteins with cyclin A and DNA replication proteins (103). In this context, it is noteworthy that RPA proteins are able to suppress the APE1 endonuclease activity in ssDNA of a replicative fork but not in a transcription bubble or in dsDNA (27) and that Cockayne syndrome B protein potentiates the APE1 activity on fully paired AP-DNA but much more on bubble AP-DNA, suggesting a role for this protein in the transcription-repair pathway (143).

Of particular interest in the definition of new functions and targets of APE1 is the finding that, after UVA irradiation, APE1 co-localizes with Ogg1 in the nuclear speckles, organelles associated to transcription and RNA processing. This localization is abolished in the presence of antioxidants (12), suggesting that ROS are the driving force of this localization. Another interesting APE1 function, connected to its capacity to bind to SSB, is that of inhibiting the same binding by PARP1, with consequent inactivation of the poly-ADP-ribosylation and prevention of necrosis (121).

A good example of how APE1 may use its dual nature to produce a cut in the DNA strand and to activate other proteins is that of the VEGF gene which contains a hypoxic response element (HRE) target of HIF-1α. Oxygen deprivation induces formation of an abasic site in the response element followed by HIF-1α binding. The data suggest that the abasic site is the target of APE1 activity with consequent increased flexibility of DNA which causes HIF-1 binding (10) and transcriptional activation of the VEGF gene (46, 153).

The regulatory functions of the different APE1 activities can be fine-tuned and implemented via three different mechanisms: (a) increase in APE1's level after transcriptional activation (107, 108, 111, 126); (b) relocalization of APE1 from the cytoplasm to the nucleus (126); and (c) modulation of APE1's post-translational modifications (PTM), such as acetylation and phosphorylation. As recently demonstrated, in addition to redox regulation, acetylation appears to have a fine-tuning role in affecting APE1's different activities (7, 30, 67).

Both in vivo and in vitro studies demonstrated that different oxidative or toxic agents and/or intracellular produced ROS efficiently and rapidly (within minutes to hours, depending on the specific ROS-generating stimuli) promote a transient increase in APE1 protein levels, which is inhibited by cycloheximide (111, 129). Different transcription factors, including Sp-1 (36), Egr-1 (107), STAT3 (53), CREB (48), and Jun/ATF4 (37) are involved in the inducible expression of APE1. APE1 itself may inhibit its own expression through the binding to nCa-RE sequences within the APE1 distal promoter, thus constituting an autoregulatory functional loop (68). In addition, APE1 expression is linked in a positive autoregulatory loop with Egr-1 (107), and in a negative inhibitory loop with p53 (151). Interestingly, protein upregulation is always associated with an increase in both redox and AP endonuclease activity, followed by an increase in cell resistance toward oxidative stress and DNA damaging agents (49, 87, 88, 111, 126), strengthening the conclusion that an upregulation of APE1 protein levels has profound biological consequences.

The activation of APE1 is also obtained by a process independent from de novo synthesis and involves cytoplasm to nucleus translocation after exposure of cells to oxidative stress conditions (111, 126, 127) or upon physiologic increase in intracellular ROS production (108). Nuclear localization of APE1 is controlled by the first 20 amino acids at the N-terminal sequence, as determined by Jackson et al. (69) and nuclear import is controlled through a bipartite NLS comprising residues 1–7 and 8–13 with the involvement of an importin system. In fact, the first 20 residues directly bind to karyopherin α1 and α2. Data obtained by treatment of cells with the nuclear export inhibitor leptomycin B suggested the presence of a nuclear export signal (NES) that may reside in a Leu-rich region (L291, L292, L295 residues, which are exposed in the 3-D structure) (69). Recently, it has been shown that the region comprising amino acids 64–80 contains a NES (110). Thus, both nuclear import and export may control subcellular distribution of APE1. In addition, the interaction with specific nuclear proteins could be a means to maintain APE1 within the nucleus. This hypothesis, recently proposed by Jung et al. based on their data showing that nuclear localization of APE1 was dependent on GADD45a nuclear protein expression (71), deserves further experimental support.

During the last few years, several lines of evidence have been accumulating, demonstrating that functional triggering of membrane-bound receptors (such as those for TSH, CD40L, ATP, IL-2, etc.) can lead to APE1 functional activation through intracellular generation of sublethal doses of ROS (126). Noteworthy is the observation that APE1 is also directly responsible for the control of the intracellular ROS levels through its inhibitory effect on Rac1 (2, 52, 102, Vascotto et al., unpublished observations), the regulatory subunit of a membrane nonphagocytic NADPH oxidase system. This enzyme, composed of multiple membrane-associated (Mox and p22^phox) and cytosolic components (p67^phox, p47^phox, and Rac1), catalyzes the transformation of the molecular oxygen to the superoxide anion by transferring an electron from the substrates NADH or NADPH (3). Since we have recently demonstrated that NADPH-mediated ROS production induced by P2Y triggering was able to promote APE1 functional activation (108), we propose the existence of an autoregulatory loop between these two systems. This mechanism may be of therapeutic relevance for endothelial, fibroblastic, and smooth muscle cells, and should be analyzed in diseases of the vascular system where an overactivation of the NADPH oxidase system is involved (19), as well as in the angiogenesis process (133), where an additional autoregulatory loop between APE1 and VEGF may be inferred (10, 153). This observation could be therapeutically relevant in the treatment of tumor progression and cancer metastasis.

Based on the above-mentioned considerations, APE1 seems to act as an intracellular signaling tool involved both in modulating the cellular response to acute and chronic oxidative stress conditions, and also in controlling the endogenous ROS levels during the physiological generation of ROS as intracellular signaling molecules. Since the cell system must be able to discriminate between different ROS-generating stimuli, APE1 behaves as an integrating signaling molecule.

APE1 is an abundant protein (~10⁴–10⁵ copies/cell) within eukaryotic cells and with a relatively long half-life [~8h (Vascotto et al., unpublished observations)]. Therefore, the fine-tuning of the multiple functions of this pleiotropic protein may reside in the impact that PTMs have on the function of APE1 and on the modulation of the APE1-interactome under different conditions. Whereas for the former hypothesis some experimental evidences have been obtained [acetylation of K6/K7 residues (7, 30)], very little information is now available on the protein interacting partners of APE1. Pioneering in silico studies discovered that several different phosphorylation sites were scattered throughout the molecule. These potential phosphorylation sites included consensus sequences for casein kinase I and II (CKI and CKII), for protein kinase C (PKC), and for GSK3 (Fig. 1A) (34, 148). Initial in vivo studies confirmed a role for PKC in phosphorylating APE1 in response to PMA or to alkylating agents (i.e., MMS) leading to AP-1 activation (62). However, these studies have not been repeated nor followed up. Therefore, the role of phosphorylation on APE1 is still not clear.

APE1 is a site for redox regulation by the dithiol-reducing enzyme Trx (61, 109, 132, 141), through Cys35 and Cys32 in the catalytic center of Trx, and involving the Cys65 redox sensitive site of APE1 (139, 141, 146). The Trx-mediated redox regulation of APE1 is required for the functional activation of p53 (132) and AP-1 (61). Though the biological relevance of Cys65 residue seems determined, it is not currently known whether this Cys residue undergoes PTM in vivo.

Qu et al. (110) demonstrated that two (Cys93 and Cys310) of the seven Cys residues of APE1 can undergo S-nitrosation in response to nitric oxide stimulation, leading to nucleus to cytoplasm relocalization of the protein in a CRM1-independent process, possibly as a consequence of demasking a putative nuclear export signal (aa 64–80). S-nitrosation may therefore constitute a specific molecular switch to strictly control the intracellular distribution of APE1 between nucleus and cytoplasm, and provides a new working hypothesis for the cytoplasmic accumulation of APE1 observed in more aggressive tumors (126). Unfortunately, no detail is available about the functional implications of S-nitrosation on the different biological functions of APE1. Accordingly, since both NO and APE1 are associated with tumorigenesis and neurodegenerative diseases, future work is needed to address whether nitrosative stress leads to genomic instability, and may be the target for designing new therapeutic strategies.

An interesting post-translational processing that has been recently described is proteolysis occurring at residue Lys31. This PT regulation of APE1 protein is responsible for enhanced cell death mediated by granzyme A (GzmA) (29) and granzyme K (GzmK) (52). APE1 is associated with the endoplasmic reticulum in a macromolecular complex of 270–420kDa containing evolutionarily conserved proteins called SET, pp32, and HMG2. GzmA cleaves APE1 after Lys31, giving rise to a protein form called NΔ33APE1, and alters its ability to be actively accumulated within nuclei of cells (15, 69, and our unpublished observations) and to interact with XRCC1 (136). However, some authors claimed that truncated APE1 may loose its AP-endonuclease activity (29) and acquire a nonspecific DNAse function (150). This peculiar processing is not limited to immune cells but may constitute a general molecular device for redirecting APE1 to mitochondria (125), as suggested by Chattopadhyay et al. (15) and Mitra et al. (88), in spite of the intriguing finding of a proteolysis occurring at the level of Asn33 rather than Lys31. Again, if the removal of the terminal 31–33 amino acids is responsible for APE1 to move to the mitochondria to function in mitochondrial BER as an AP endonuclease, it is hard to understand this truncated protein having nonspecific DNAse activity (150) unless it is very cell-type specific. Accordingly, previous work by many investigators has never observed a nonspecific nuclease activity with the cleavage of the first 61 amino acids (66) and additional data clearly showed that the truncated APE1 protein has an unaltered AP-endonuclease activity (15, and our unpublished observations), at least in vitro.

While it is known that nuclear accumulation of APE1 triggers the activation of several transcription factors, the functional role of acetylation is barely understood. Acetylation of both histones and regulatory proteins is commonly catalyzed by the histone acetyltransferase (HAT) p300/CBP, and can be reversed by histone deacetylases (HDACs), which in turn control the acetylation level of transcription factors or co-activators (50, 74). Bhakat et al. have reported that the balance between the acetyltransferase activity of p300/CBP and the deacetylase activity of HDAC1 maintains APE1's acetylation at Lys residues 6 and 7 (K6, K7) in response to Ca²⁺ levels, thus controlling expression of target genes (7). More recently, we found that exposure of HeLa cells to H₂O₂ and to histone deacetylase inhibitors increases acetylation of APE1 at residues Lys6/Lys7, leading to Egr-1-mediated induction of the tumor suppressor PTEN gene expression (30). Our data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress and underline the double-face nature of APE1 which plays a role in both pro-survival and in cell cycle arrest mechanisms. Interestingly, despite the very low homology degree in the N-terminal region (<40%), K6 or K6/K7 are much more conserved, thus reinforcing their primary role during phylogenesis.

One of the genes that emerged from our analysis and is involved in the control of cell growth processes, as well as in the antioxidant response, is Egr-1. This transcription factor resulted as a good candidate target of APE1, since its expression was significantly reduced in APE1 knocked-down cells both basally and upon serum treatment after starvation (Fig. 4A). We therefore investigated the functional effect of APE1 silencing in controlling the expression of one of the most well-known Egr-1 target genes, the tumor suppressor PTEN, which is induced upon UV- and oxidative stress-cellular damage (5, 137). An impairment of Egr-1 expression upon APE1 silencing affected the inducible expression of PTEN gene with a transcriptional mechanism, confirming the dual nature of APE1 itself (Fig. 4B and C). Thus, the lack of APE1 impairs the adaptive cellular response to damaging agents. These findings further enlighten the double nature of APE1 to be involved in both the processes of cell growth and in growth arrest upon cellular damage. Interestingly, this kind of dual role nature in transcriptional regulation and DNA repair for proteins is consistently present in the five major DNA repair pathways, that is, homologous recombinational repair (HRR), nonhomologous end joining (NHEJ), nucleotide excision repair (NER), BER and mismatch repair (MMR), as in the cases of BRCA1, ATM, and p53 itself (for review, refer to Ref. 6). The existence and the correct regulation of a mechanism shifting cells from DNA repair to apoptosis is central to avoid progression to cancer, preventing clonal expansion of cells in which unrepaired damage would lead to mutation and to carcinogenesis. In this regard, it is interesting to note that a number of DNA repair genes (i.e., GADD45, BRCA1) were downregulated as a consequence of APE1 silencing, suggesting the existence of a cross-regulation of the expression between individual partners of different pathways and underlining the central role of APE1 in DNA repair processes with a different function besides the well-known AP-endonuclease activity. This hypothesis will require further investigation.

The interesting finding that, besides its activity as a redox coactivator for Egr-1 transcriptional activity (64, 107), APE1 is also able to regulate Egr-1 expression levels reinforces the previously suggested hypothesis (107) of the existence of a functional loop between APE1 and Egr-1 in reciprocally modulating each other. This finding strengthens the biological complexity typical of this multifunctional protein and could well be a molecular tool by which APE1 may regulate the expression of DNA repair genes such as GADD45 (130).

In addition to the canonical 37kDa APE1 protein, and the truncated NΔ33APE1 form, in some circumstances the appearance of a lower mobility band of ~50kDa has been detected (our unpublished data). This form would account for a PTM that introduces a substantial modification to the wild-type protein. A recently identified PTM, typically occurring on Lys residues of several nucleoproteins, is sumoylation (40, 56). The addition of a small ubiquitin-like modifier (SUMO) molecule to the target protein accounts for an increase of ~12kDa of the apparent molecular mass of the target protein itself. Many SUMO-modified proteins function in regulation of transcription, chromatin structure, maintenance of the genome stability, and signal transduction. Upon sumoylation, interactions that are dependent on other post-translational modifications or on unmodified lysine are lost. Sumoylation also promotes novel interactions, in some cases associated with a conformational change in the target protein. The effects of PTM by SUMO to compete for target lysines enhance or inhibit interactions with other proteins (or other binding partners, such as DNA) or induce conformational changes. These effects are not mutually exclusive, but might not all occur on the same substrate. Thus, we investigated the possibility that APE1 may undergo sumoylation. APE1 protein contains 29 Lys residues, 15 of them lie in the N-terminal domain, and 14 residues lie in the C-terminal domain of the protein. Protein sequence analysis using three different softwares for prediction of sumoylation sites (SUMOPlot, SUMO sp, and SUMO PSFS) revealed several “low probability” putative sumoylation sites and a “high probability” putative sumoylation site, which contains a canonical sumoylation ψ-K-X-D/E motif (Fig. 5A and B). Putative sumoylation sites do not distribute homogenously along the whole sequence, but concentrate at the N-terminal domain, which is not present in functionally related proteins from other organisms and is required for the redox activity of APE1. Co-localization experiments on cells transfected with GFP-SUMO-1 clearly demonstrated that endogenous APE1 can be modified by wild-type SUMO-1 but not by a deletion mutant (i.e., Δ6SUMO-1), which looses its ability to modify target proteins (Fig. 5C). In vitro sumoylation experiments also clearly demonstrated that APE1 may undergo sumoylation (unpublished data). Our inability to identify the Lys residue target of sumoylation, by mutagenizing the putative sumoylation sites identified during in silico analysis (unpublished data), may suggest that multiple Lys residues could be the simultaneous target of this PTM. Additional work, which is difficult as sumoylated APE1 represents a very tiny fraction in comparison to the nonsumoylated protein, is required to understand the role of sumoylation in controlling APE1 functions.

In summary, all of these studies have confirmed the dual role nature of APE1, as a prototypical example of an apparent biological paradox. While a number of reports clearly demonstrated the antiapoptotic roles as well as the positive effect on cell proliferation (for a review, see Ref. 126, and 35, 67, 88), other data underlined its potential role in controlling proapoptotic functions through p53-mediated activation of p21 (38, 54, 117), leading to the arrest of cell cycle by inhibiting cyclin-dependent kinase function (11), and cyclin G, having pro-apoptotic activity (97). However, it is becoming increasingly evident that the antiapoptotic roles of APE1 are ascribable to its DNA repair functions (35) rather than to its activities as a transcriptional co-activator. Thus, it is tempting to speculate that the latter function—activation of transcription factors (i.e., p53 and Egr-1) ensuring efficient cell-cycle arrest—may act in concert with the previous (repair of DNA damage) to protect cells from accumulation of oxidative damage (Fig. 6) and be a later evolutionarily acquired function since only mammals and not just vertebrates appear to have the redox signaling function (39). This may have been an advantageous addition to APE1's interactions and functions that benefited mammalian cells, given the potentially toxic nature of their environment. This may have subsequently led to additional signaling and regulatory interactions and functions. Obviously, for a proper modulation of these two interconnected functions, a fine-tuned regulation of APE1 activities is required. Thus, a better understanding of the processes controlling APE1 subcellular distribution, of the post-translational modifications occurring on the protein itself, of the mechanisms controlling protein half-life, and of the different interacting partners recruited as a function of cellular response, is required to fully address this paradoxical issue.

Whether the alterations of APE1 subcellular localization are causally responsible or only associated with tumor progression is not clear at present. However, our recent findings in HCC, in which a chronic cellular oxidative stress conditions (91) in a milieu of inflammation (106) plays a central role in tumor progression implying functional activation of pro-survival transcription factors such as NF-κB and STAT3 (43, 84), supports the latter conclusion. To definitely address this point, it is mandatory that the interactome network of APE1 as well as the role of PTM in controlling the cytoplasmic localization of the protein must be unveiled. An interesting working hypothesis for a role of APE1 in the inflammatory process comes from recent works on APE1-mediated IL-8 production in Helicobacter pylori infections (96). These authors demonstrated that APE1 is required for H. pylori infection-mediated induction of IL-8 production by gastric cells through the involvement of NF-κB and AP-1 transcription factors (96). Thus, APE1 seems to play also a leading role in the production of inflammatory cytokines. Future work will be needed to extend these observations and to open new applicative perspectives in molecular medicine.

Use of specific small molecule inhibitors, such as one that blocks APE1 redox, but not repair, will be important to delineate the distinct roles of APE1 in various cancers, other diseases, and normal cellular functions. Likewise, an APE1 specific repair inhibitor will help to elucidate that role (4). Ultimately, using APE1 redox inhibitors with APE1 specific endonuclease repair inhibitors will give a clearer picture of the multiple activities of APE1.

APE1 was originally identified as the primary target of E3330 (3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl-2-propionic acid), a small molecule redox inhibitor (155). E3330 was immobilized on beads and APE1 was identified from a nuclear extract of a leukemia cell line as a protein that specifically bound to E3330. Using surface plasmon resonance (SPR), an equilibrium constant (K_D) of 1.6 nM was obtained for the binding of E3330 to APE1, suggesting a specific interaction. E3330 was also shown to block the ability of APE1 to reduce NF-κB, thus interfering with the redox activity of APE1 (45, 60, 89, 118). The proposed binding site on APE1 is somewhat puzzling. The amino acid residues 72–80 form a ridge on the surface of the molecule with no obvious cavities or binding pockets that are large enough to bind E3330. Currently, we are pursuing studies to delineate the binding site or region of E3330 on APE1.

Our recent data have demonstrated that E3330 blocks the redox function of APE1 with AP-1 as the downstream target in vitro (86). Additionally, using a transactivation assay for AP-1 or HIF-1α targets in ovarian cancer cells, increasing amounts of E3330 led to decreased activation of a luciferase reporter downstream of AP-1 and HIF-1α (86). While E3330 blocked APE1's redox function, it had no effect on APE1 repair endonuclease activity nor other members of the BER pathway (86). These studies demonstrate the specificity of E3330 for APE1's redox, but not for its repair function. This is supported by our recently published data in ovarian cancer studies in xenografts demonstrating that the knockdown of APE1 results in the blocking of cell growth and proliferation, but not necessarily cell death (31). This is the first time that cancer cell killing has been reported using a small molecule inhibitor of APE1 redox function.

E3330 was also shown to have single agent inhibition of cell growth using a variety of cancer cell lines including, ovarian, colon, lung, breast, brain, pancreatic, prostate, and multiple myeloma cancers (Kelley et al. unpublished observations). In stark contrast, we do not see significant growth inhibition in our studies with normal cells such as hematopoietic embryonic cells (155) or in human CD34+progenitor cells (Kelley et al. unpublished observations). These data are novel in that they implicate the redox role of APE1 in cancer, but not “normal”, cell survival.

Recent studies using this APE1 redox inhibitor in angiogenesis model systems have been instructive. Proliferation of endothelial cells (EC) is an important index for angiogenic ability of EC in vitro. E3330 inhibited retinal vascular endothelial cells (RVEC) growth in a dose-dependent manner (86). Additionally, an in vitro angiogenesis assay was also used to determine the effect of E3330 on RVEC formation of capillary-like structures on Matrigel. The inhibitory effect of E3330 was similar to the proliferation assay with the complete loss of tube formation at low micromolar concentrations. These results demonstrate that blocking APE1's redox function attenuates RVEC proliferation and capillary formation in vitro and these findings implicate the use of an APE1 redox inhibitor in antiangiogenic translational studies. Further mechanistic studies as to how this is occurring are in progress, although preliminary studies indicate the redox inhibitory effect is not necessarily related to cell killing, but to a block in cell proliferation or cytostatic effect similar to that observed using APE1 siRNA in vivo (31).