New perspectives in cancer biology from a study of canonical and non-canonical functions of base excision repair proteins with a focus on early steps.

Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.

Histone H3 Lysine 56 Acetylation Enhances AP Endonuclease 1-Mediated Repair of AP Sites in Nucleosome Core Particles.

Deciphering factors modulating DNA repair in chromatin is of great interest because nucleosomal positioning influences mutation rates. H3K56 acetylation (Ac) is implicated in chromatin landscape regulation, impacting genomic stability, yet the effect of H3K56Ac on DNA base excision repair (BER) remains unclear. We determined whether H3K56Ac plays a role in regulating AP site incision by AP endonuclease 1 (APE1), an early step in BER. Our in vitro studies of acetylated, well-positioned nucleosome core particles (H3K56Ac-601-NCPs) demonstrate APE1 strand incision is enhanced compared with that of unacetylated WT-601-NCPs. The high-mobility group box 1 protein enhances APE1 activity in WT-601-NCPs, but this effect is not observed in H3K56Ac-601-NCPs. Therefore, our results suggest APE1 activity on NCPs can be modulated by H3K56Ac.

APE1/Ref-1 facilitates recovery of gray and white matter and neurological function after mild stroke injury.

A major hallmark of oxidative DNA damage after stroke is the induction of apurinic/apyrimidinic (AP) sites and strand breaks. To mitigate cell loss after oxidative DNA damage, ischemic cells rapidly engage the base excision-repair proteins, such as the AP site-repairing enzyme AP endonuclease-1 (APE1), also named redox effector factor-1 (Ref-1). Although forced overexpression of APE1 is known to protect against oxidative stress-induced neurodegeneration, there is no concrete evidence demonstrating a role for endogenous APE1 in the long-term recovery of gray and white matter following ischemic injury. To address this gap, we generated, to our knowledge, the first APE1 conditional knockout (cKO) mouse line under control of tamoxifen-dependent Cre recombinase. Using a well-established model of transient focal cerebral ischemia (tFCI), we show that induced deletion of APE1 dramatically enlarged infarct volume and impaired the recovery of sensorimotor and cognitive deficits. APE1 cKO markedly increased postischemic neuronal and oligodendrocyte degeneration, demonstrating that endogenous APE1 preserves both gray and white matter after tFCI. Because white matter repair is instrumental in behavioral recovery after stroke, we also examined the impact of APE1 cKO on demyelination and axonal conduction and discovered that APE1 cKO aggravated myelin loss and impaired neuronal communication following tFCI. Furthermore, APE1 cKO increased AP sites and activated the prodeath signaling proteins, PUMA and PARP1, after tFCI in topographically distinct manners. Our findings provide evidence that endogenous APE1 protects against ischemic infarction in both gray and white matter and facilitates the functional recovery of the central nervous system after mild stroke injury.

Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.

APEX1 regulation of aldosterone synthase gene transcription is disrupted by a common polymorphism in humans.

RATIONALE: The genetic mechanisms underlying hypertension are unclear, but relative aldosterone excess, present in ≈10% of hypertensive patients, is known to be a heritable trait. This phenotype associates with a T/C single nucleotide polymorphism (SNP) at position -344 of the aldosterone synthase gene (CYP11B2). However, deletion of this SNP has no effect on gene transcription. We have identified another T/C SNP at -1651, in tight linkage disequilibrium with the -344 SNP and here investigate its functional effect on CYP11B2 transcription. OBJECTIVE: We assessed the effect on transcriptional activity of the -1651 T/C SNP in vivo and in vitro and propose the mechanism by which transcriptional activity is altered. METHODS AND RESULTS: We demonstrated that the SNP at -1651 exerts significant allele-dependent effects on CYP11B2 transcription. We confirm binding of the transcriptional repressor APEX1 to -1651T, which is associated with reduced transcriptional activity in relation to the less strongly bound -1651C. We show that inhibiting APEX1 by small molecule inhibition or small interfering RNA (SiRNA) leads to increased CYP11B2 transcription. In addition, overexpression of APEX1 is associated with reduced transcriptional activity. Finally, we also show that -1651T associates with lower excretion rates of aldosterone metabolites in human subjects. CONCLUSIONS: We conclude that APEX1 is a novel transcriptional repressor of CYP11B2 and that differential APEX1 binding at -1651 of CYP11B2 results in altered gene expression. This mechanism may contribute to the observed relationship between CYP11B2 genotype and aldosterone phenotype in a subgroup of hypertensive patients.

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present a possible regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome. The 1082B precursor transcript and its 788 unique piRNAs are repressed by the Alkbh1 dioxygenase and the testis-specific transcription repressor Tzfp. We observe a remarkable >1000-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1- and Tzfp-deficient murine testes. Repression of cluster 1082B is further supported by the identification of a 10-bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of LINE1 and IAP transcripts in the Alkbh1- and Tzfp-deficient mice leads us to propose a potential role for the 1082B-encoded piRNAs in transposon control.

UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways.

Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.

Apurinic/apyrimidinic endonucelase 1 maintains adhesion of endothelial progenitor cells and reduces neointima formation.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that processes DNA-repair function and controls cellular response to oxidative stress. Endothelial progenitor cells (EPCs) are recruited to oxidative stress-rich injured vascular walls and positively contribute to vascular repair and endothelialization. We hypothesized that APE1 functions for EPCs-mediated inhibition of neointima formation in injured vasculature. EPCs isolated from bone marrow cells of C57BL6 mice (12-16 wk old) were able to survive in the presence of hydrogen peroxide (H2O2; up to 1,000 µM) due to the highly expressed reactive oxygen species (ROS) scavengers. However, adhesion capacity of EPCs was significantly inhibited by H2O2 (100 µM) even though an intracellular ROS was retained at small level. An APE1-selective inhibitor or RNA interference-mediated knockdown of endogenous APE1 in EPCs aggravated the H2O2-mediated inhibition of EPCs-adhesion. In contrast, when APE1 was overexpressed in EPCs using an adenovirus harboring the APE1 gene (APE-EPCs), adhesion was significantly improved during oxidative stress. To examine in vivo effects of APE1 in EPCs, APE-EPCs were transplanted via the tail vein after wire-mediated injury of the mouse femoral artery. The number of adherent EPCs at injured vascular walls and the vascular repair effect of EPCs were enhanced in APE-EPCs compared with control EPCs. Among the cellular functions of EPCs, adhesion is especially sensitive to oxidative stress. APE1 enhances in vivo vascular repair effects of EPCs in part through the maintenance of adhesion properties of EPCs. APE1 may be a novel and useful target gene for effective cellular transplantation therapy.

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge.

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.

Alteration of the microRNA expression profile in human osteosarcoma cells transfected with APE1 siRNA.

Apurinic/apyrimidinic endonuclease1 (APE1), which has the dual functions of DNA repair and redox regulation, is considered to be a promising potential target in cancer treatment. Microarray and qRT-PCR were used to confirm the change of miRNA followed by analysis with comprehensive bioinformatics-based analysis. Both microarray and qRT-PCR demonstrated that 13 microRNAs (miRNAs) were significantly changed (>2-fold) in APE1 knockdown HOS cells; seven of them (hsa-miR-451, hsa-miR-1290, hsa-miR-765, hsa-miR-483-5p, hsa-miR-513a-5p, hsa-miR-129-5p and hsa-miR-31) were up-regulated and the other six (hsa-miR-29b, hsa-miR-197, has-let-7b, hsa-miR-324-5p, hsa-let-7i and hsa-miR-484) were down-regulated. Furthermore, pathway analysis showed that these miRNAs and their target genes affected by the expression of APE1 were involved in pathways relating to developmental processes, regulation of cellular processes, cell signaling (such as TGF-ß, Wnt, MAPK and the p53 signaling pathway) and cancers. There are putative binding sites of NF-κB, p53, HIF-1α, AP-1, PEBP2, ATF, NF-Y, Pax-2,CREB and c-Myb in the promoters of several down regulated miRNAs, indicating that APE1 may regulate miRNAs via transcription factors. Our data suggest that our understanding of the biological functions of APE1 will inevitably expand due to the novel pathways that APE1 uses to regulate gene expression through miRNAs.

A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase beta.

DNA repair is required to maintain genome stability in stem cells and early embryos. At critical junctures, oxidative damage to DNA requires the base excision repair (BER) pathway. Since early zebrafish embryos lack the major polymerase in BER, DNA polymerase ß, repair proceeds via replicative polymerases, even though there is ample polb mRNA. Here, we report that Polb protein fails to appear at the appropriate time in development when AP endonuclease 1 (Apex), the upstream protein in BER, is knocked down. Because polb contains a Creb1 binding site, we examined whether knockdown of Apex affects creb1. Apex knockdown results in loss of Creb1 and Creb complex members but not Creb1 phosphorylation. This effect is independent of p53. Although both apex and creb1 mRNA rescue Creb1 and Polb after Apex knockdown, Apex is not a co-activator of creb1 transcription. This observation has broad significance, as similar results occur when Apex is inhibited in B cells from apex(+/-) mice. These results describe a novel regulatory circuit involving Apex, Creb1 and Polb and provide a mechanism for lethality of Apex loss in higher eukaryotes.

Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia.

Inducible DNA repair via the base-excision repair pathway is an important prosurvival mechanism activated in response to oxidative DNA damage. Elevated levels of the essential base-excision repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 correlate closely with neuronal survival against ischemic insults, depending on the CNS region, protective treatments, and degree of insult. However, the precise mechanisms by which this multifunctional protein affords protection and is activated by upstream signaling pathways in postischemic neurons are not well delineated. Here we show that intracerebral administration of pituitary adenylate cyclase-activating polypeptide (PACAP), an endogenously occurring small neuropeptide, induces expression of APE1 in hippocampal neurons. Induction of APE1 expression requires PKA- and p38-dependent phosphorylation of cAMP response-element binding and activating transcription factor 2, which leads to transactivation of the APE1 promoter. We further show that PACAP markedly reduces oxidative DNA stress and hippocampal CA1 neuronal death following transient global ischemia. These effects occurred, at least in part, via enhanced APE1 expression. Furthermore, the DNA repair function of APE1 was required for PACAP-mediated neuroprotection. Thus, induction of DNA repair enzymes may be a unique strategy for neuroprotection against hippocampal injury.

Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: implication of PARP-1, APEX-1 and ERK1/2 catalytic activities.

Human Apolipoprotein D (apoD) is upregulated under several stress conditions and pathological situations such as neurodegenerative diseases and cancers. We previously showed that apoD mRNA expression is induced in growth-arrested cells and demonstrated the specific binding of nuclear proteins to the region -514 to -475 of the promoter. Such region contains a pair of Serum Responsive Elements (SRE), an Ets-Binding Site (EBS) and a Glucocorticoid Responsive Element (GRE). In this study, we show that Parp-1, HnRNP-U, CBF-A, BUB-3, Kif4, APEX-1 and Ifi204 bind these regulatory elements of the apoD promoter. Specific binding of HnRNP-U and Parp-1 was confirmed by Electrophoretic Mobility Shift Assay (EMSA). In a biotin pull-down assay, Kif4 and BUB-3 bind preferentially the SRE1 and the EBS-GRE sites, respectively, while APEX-1 seems recruited indirectly to these elements. We found that the mRNA expression of some of these binding factors is upregulated in growth-arrested cells and that these proteins also transactivate the apoD promoter. In agreement with these results, mutants of APEX-1 and of Parp-1 defective for their DNA-binding and catalytic activities could not transactivate the promoter. The knockdown of Parp-1 and HnRNP-U and the use of specific inhibitors of MEK1/2 and of Parp-1 also inhibited the induction of apoD gene expression. Moreover, ERK1/2 was found activated in a biphasic manner post serum-starvation and the inhibition of Parp-1 causes a sustained activation of ERK2 but not ERK1 for up to 2h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.

Low-dose carcinogenicity of 2-amino-3-methylimidazo[4,5-f ]quinoline in rats: Evidence for the existence of no-effect levels and a mechanism involving p21(Cip / WAF1).

The carcinogenicity of the low amounts of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and to investigate mechanisms by which IQ exerts its carcinogenic effects. A total of 1595 male F344 rats were divided into seven groups and administered with IQ at doses of 0, 0.001, 0.01, 0.1, 1, 10 and 100 p.p.m. in the diet for 16 weeks. We found that IQ doses of 1 p.p.m. and below did not induce preneoplastic lesions in either the liver or the colon, while IQ doses of 10 and 100 p.p.m. induced preneoplastic lesions in both of these organs. These results demonstrate the presence of no-effect levels of IQ for both liver and colon carcinogenicity in rats. The finding that p21(Cip/WAF1) was significantly induced in the liver at doses well below those required for IQ mediated carcinogenic effects suggests that induction of p21(Cip/WAF1) is one of the mechanisms responsible for the observed no-effect of low doses of IQ. Furthermore, IQ administration caused significant induction of CYP1A2 at doses of 0.01-10 p.p.m., but administration of 100 p.p.m. IQ induced CYP1A1 rather than CYP1A2. This result indicates the importance of dosage when interpreting data on the carcinogenicity and metabolic activation of IQ. Overall, our results suggest the existence of no-effect levels for the carcinogenicity of this genotoxic compound.

Similar frequency and signature of untargeted substitutions induced by abasic site analog under reduced human APE1 conditions.

Abasic sites are formed in cells by various factors including environmental mutagens and considered to be involved in cancer initiation, promotion, and progression. A chemically stable abasic site analog (tetrahydrofuran-type analog, THF) induces untargeted base substitutions as well as targeted substitution and large deletion mutations in human cells. The untargeted substitutions may be initiated by the cleavage of the DNA strand bearing THF by the human apurinic/apyrimidinic endonuclease 1 (APE1) protein, the major repair enzyme for THF and abasic sites. To examine the effects of lower APE1 levels, the protein was knocked down by siRNA in human U2OS cells. A plasmid containing a single THF modification outside the supF gene was introduced into the knockdown cells, and the untargeted substitution mutations in the reporter gene were analyzed. Unexpectedly, the knockdown had no evident impact on their frequency and spectrum. The G bases of 5'-GpA-3' dinucleotides on the modified strand were quite frequently substituted, with and without the APE1 knockdown. These results suggested that the DNA strand cleavage by APE1 is not essential for the THF-induced untargeted base substitutions.

Conformational transitions in human AP endonuclease 1 and its active site mutant during abasic site repair.

AP endonuclease 1 (APE1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5' to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2'-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 x 10(4)-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis.

The role of mammalian ribonucleases (RNases) in cancer.

Ribonucleases (RNases) are a group of enzymes that cleave RNAs at phosphodiester bonds resulting in remarkably diverse biological consequences. This review focuses on mammalian RNases that are capable of, or potentially capable of, cleaving messenger RNA (mRNA) as well as other RNAs in cells and play roles in the development of human cancers. The aims of this review are to provide an overview of the roles of currently known mammalian RNases, and the evidence that associate them as regulators of tumor development. The roles of these RNases as oncoproteins and/or tumor suppressors in influencing cell growth, apoptosis, angiogenesis, and other cellular hallmarks of cancer will be presented and discussed. The RNases under discussion include RNases from the conventional mRNA decay pathways, RNases that are activated under cellular stress, RNases from the miRNA pathway, and RNases with multifunctional activity.

p66(Shc) has a pivotal function in impaired liver regeneration in aged mice by a redox-dependent mechanism.

Liver regeneration involves complicated processes and is affected by various patho-physiological conditions. This study was designed to examine the molecular mechanisms underlying the aging-associated impairment of liver regeneration. Male C57BL/6J mice were used as young and aged mice (<10 weeks and >20 months old, respectively). These mice were subjected to 70% partial hepatectomy (PH). Liver regeneration and liver injury/stresses were evaluated chronologically after PH. Post-hepatectomy liver regeneration was markedly impaired in aged mice. Though the extent of hepatocyte proliferation in the regenerating liver was similar in aged and young mice, cell growth was absent in aged mice. Oxidative stress (OS) was observed immediately after hepatectomy, followed by marked apoptosis in aged mice. Signaling molecules regarding cell proliferation (mitogen-activated protein kinase, STAT3, p46/52(Shc)) and anti-oxidation (catalase, superoxide dismutase, Ref-1, glutathione peroxidase) were expressed/activated after hepatectomy in livers of both aged and young mice. Akt was not activated in aged-mouse liver, but its expression was similar to that in young mice. p66(Shc), known as an age-/oxidant-associated protein, was strongly phosphorylated. By knocking down p66(Shc), the impairment of liver regeneration was normalized. OS immediately after hepatectomy induced subsequent liver injury (apoptosis), and deletion of p66(Shc) suppressed both OS and hepatocyte apoptosis in the regenerating liver of aged mice. Though we need additional data in other animal models to fully understand the mechanism, p66(Shc) may have a pivotal function in the impairment of liver regeneration in aged mice by triggering OS and subsequent apoptosis. This data may provide a clue to understanding the mechanism underlying the association between aging and the impairment of liver regeneration.

Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers.

BACKGROUND: Altered DNA repair may be associated with aggressive tumour biology and impact upon response to chemotherapy and radiotherapy. We investigated whether expression of human AP endonuclease (APE1), a key multifunctional protein involved in DNA BER, would impact on clinicopathological outcomes in ovarian, gastro-oesophageal, and pancreatico-biliary cancer. METHODS: Formalin-fixed human ovarian, gastro-oesophageal, and pancreatico-biliary cancers were constructed into TMAs. Expression of APE1 was analysed by IHC and correlated to clinicopathological variables. RESULTS: In ovarian cancer, nuclear APE1 expression was seen in 71.9% (97 out of 135) of tumours and correlated with tumour type (P=0.006), optimal debulking (P=0.009), and overall survival (P=0.05). In gastro-oesophageal cancers previously exposed to neoadjuvant chemotherapy, 34.8% (16 out of 46) of tumours were positive in the nucleus and this correlated with shorter overall survival (P=0.005), whereas cytoplasmic localisation correlated with tumour dedifferentiation (P=0.034). In pancreatico-biliary cancer, nuclear staining was seen in 44% (32 out of 72) of tumours. Absence of cytoplasmic staining was associated with perineural invasion (P=0.007), vascular invasion (P=0.05), and poorly differentiated tumours (P=0.068). A trend was noticed with advanced stage (P=0.077). CONCLUSIONS: Positive clinicopathological correlations of APE1 expression suggest that APE1 is a potential drug target in ovarian, gastro-oesophageal, and pancreatico-biliary cancers.

TRIM5alpha.

TRIM5alpha protein blocks retroviral replication at early postentry stage reducing the accumulation of reverse transcriptase products. TRIM5alpha proteins of Old World primates restrict HIV-1 infection whereas TRIM5alpha proteins of most New World monkeys restrict SIV(mac) infection. TRIM5alpha protein has a RING domain, B-box 2 domain, coiled-coil domain, and PRYSPRY domain. The PRYSPRY domain of TRIM5alpha determines viral specificity and restriction potency by mediating recognition of the retroviral capsid. The coiled-coil domain is essential for TRIM5alpha oligomerization, which contributes to binding avidity for the viral capsid. The RING domain and B-box 2 domain are required for efficient restriction activity of TRIM5alpha protein but the mechanisms remain to be defined.