Dysregulation of human apurinic/apyrimidinicendonuclease 1 (APE1) expression in advancedretinoblastomaJob Sudhakar,1,2 Vikas Khetan,3 Srinivasan Madhusudan,4

Subramanian Krishnakumar1

1Larsen and Toubro OcularPathology Department, VisionResearch Foundation, SankaraNethralaya, Chennai, India2Birla Institute of technologyand Science (BITS), Pilani,Rajasthan, India3Bhagwan MahaveerVitreoretinal Services, MedicalResearch Foundation, SankaraNethralaya, Chennai, India4Laboratory of MolecularOncology, Academic Unit ofOncology, School of MolecularMedical Sciences, University ofNottingham, NottinghamUniversity Hospitals,Nottingham, UK

Correspondence toDr S Krishnakumar,Department of OcularPathology, Vision ResearchFoundation, Sankara Nethralaya,Chennai 600006, India;drkrishnakumar_2000@yahoo.com

Received 14 August 2013Revised 15 November 2013Accepted 2 December 2013Published Online First2 January 2014

To cite: Sudhakar J,Khetan V, Madhusudan S,et al. Br J Ophthalmol2014;98:402–407.

ABSTRACTBackground Retinoblastoma (RB) is a childhood eyetumour. Dysregulation of DNA repair may not onlyinfluence pathogenesis but could also adversely impacton response to cytotoxic chemotherapy frequently usedin RB therapy. We studied the expression of humanapurinic/apyrimidinic endonuclease (APE1), a keymultifunctional protein involved in DNA base excisionrepair in RB.Methods Expression of APE1 was evaluated byimmunohistochemistry in a series of 55 RBs and inretina. In tumours, APE1 expression was analysed incytoplasm and nucleus independently and correlatedwith histopathological features, including invasion,differentiation and International IntraocularRetinoblastoma Classification groups. Relative APE1mRNA and protein expressions were evaluated by real-time PCR and western blot. The expression of APE1 intumour groups was compared with retinal tissue.Results APE1 cytoplasmic expression was observed in98% and nuclear positivity was observed in 83% oftumours analysed. Tumour cells invading the optic nerveshowed predominant cytoplasmic immunoreactivity. Aninverse correlation between cytoplasmic and nuclearpositivity was observed. Real-time PCR revealed anincrease in APE1 transcripts compared with retina.Western blot revealed a decreased protein concentrationcompared with retinal tissue.Conclusions This is the first study of APE1 expressionin RB. Our observation suggests that subcellularlocalisation of APE1 is altered in RB. APE1 could be apotential drug target in RB.

INTRODUCTIONRetinoblastoma (RB) is the most common primaryintraocular tumour usually seen in children below5 years. Untreated RB invades sclera and opticnerve and eventually leads to death of the patients.The most common chemotherapeutic regimen con-stitutes a triple therapy, including carboplatin, vin-cristine and etoposide in multiple cycles;1 however,therapeutic failure is not uncommon in RB. Themechanisms of drug resistance in RB are complex2

and are yet to be fully understood.Recent evidence suggests that proficient DNA

repair in cancer cells that allow repair of DNAdamage induced by cytotoxic agents is an import-ant factor determining therapeutic resistance.3

DNA base excision repair (BER) is involved inthe repair of DNA bases that have been damagedby alkylation, oxidation or ring saturation andalso in processing deaminated bases and DNA

single-strand breaks. Although there is more thanone subpathway of BER, in most cases excision of adamaged base by a DNA glycosylase enzyme leadsto the formation of a potentially cytotoxic apuri-nic/apyrimidinic (AP) site intermediate. This is atarget for AP endonuclease, which cleaves thephosphodiester backbone on the 50 site of the APsite via a hydrolytic mechanism.The major AP endonuclease in human cells, apuri-

nic/apyrimidinic endonuclease-1 (APE1), accountsfor over 95% of the total AP endonuclease activity inmost cultured human cell lines.4 APE1 is a ubiquitousmultifunctional protein involved in DNA repair activ-ity and also in the regulation of redox function andtranscription.5 6 Depletion of intracellular APE1 bysiRNAs or shRNAs sensitises mammalian cells to avariety of DNA-damaging agents.7–9 On the otherhand, overexpression of APE1 conferred protectiveeffect against chemotherapeutics and radiation.10 Inhuman tumours, APE1 expression has prognostic/predictive significance in several solid tumours,7 11 12

and its overexpression was associated with cisplatinresistance.13

As APE1 is a promising drug target, severalgroups have developed small molecule inhibitorsfor therapeutic application. Preclinical studies ofinhibitors targeting either the DNA repair domainor the redox domain of APE1 potentiate the cyto-toxicity of chemotherapeutics.13 14 In the currentstudy, we investigated APE1 expression in RB. Weprovide the first evidence of APE1 overexpressionin the RB cytoplasm compared with retinal tissue,implying that APE1 may be involved in the patho-genesis of RB. Given the recent interest in thedevelopment of small molecule inhibitors of APE1as an anticancer strategy and based on the expres-sion pattern in RB, our study provides preliminaryevidence that APE1 is a potential target in RB.

MATERIALS AND METHODSThis study was reviewed and approved by the localethics committee of Vision Research Foundation,Sankara Nethralaya, India, and the committeedeemed it in conformation with the generallyaccepted principles of research and in accordancewith the Helsinki declaration (study code 301-2012-P). RB tissues were collected from globe of patientswho underwent enucleation as primary therapy. Awritten consent was obtained from patient’s parentsfor the use of tumour for research. Normal retinawas collected from cadaveric eyes received at ourinstitute’s eye bank. After removal of cornea for

402 Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

transplant, the retina was carefully detached and used for RNAand protein extraction.

Clinical informationA total of 55 tumours were evaluated in this study. Among thetumours analysed, there were 17 invasive and 38 non-invasivetumours. Based on differentiation, there were 17 undifferenti-ated, 14 poorly differentiated, 7 moderately differentiated and17 well-differentiated tumours.15 Based on the clinical presenta-tion, the tumours were grouped as per International IntraocularRetinoblastoma Classification (IIRC).16 There were 36 group Eand 19 group D tumours. Among group E tumours, there were9 undifferentiated, 11 poorly differentiated, 4 moderately differ-entiated, 12 well differentiated, 14 high-risk invasive and 22non-invasive tumours. Similarly, among group D tumours, therewere 9 undifferentiated, 2 poorly differentiated, 3 moderatelydifferentiated, 5 well differentiated, 5 high-risk invasive and 14non-invasive tumours. Demography of clinical and histopatho-logical characteristics is given in table 1, and the respective clin-ical details are summarised in table 2.

ImmunohistochemistryRB paraffin sections (5 mm thick) were dewaxed and rehy-drated. Antigen retrieval was performed by pressure cookermethod in citrate buffer (pH 6.0). Immunohistochemistry wasperformed using Novolink Mini polymer detection system kit(RE7290-K) supplied by Leica Microsystems, Newcastle, UK. Inbrief, the deparaffinised slides were flooded with deionisedwater for 5 min to neutralise proteins, washed in 50 mM Trisbuffered saline (TBS) pH 7.4, incubated with protein block,washed in TBS and primary antibody (anti-APE1 rabbit poly-clonal antibody, Novus Biologicals) diluted in TBS (1/400) wasadded and incubated for 2 hours at room temperature. The sec-tions were washed in TBS and subjected to postprimary block-ing and further incubated with polymer and washed in TBS.The reaction was revealed by 3,30diaminobenzidine tetrahy-drochloride and counterstained with haematoxylin. Negativecontrol included the omission of the primary antibody and sub-stitution with non-immune serum.17 18

Immunochemistry analysisEvaluations of immunostaining in RB tissues were objectivelyperformed by two investigators independently and wereblinded to the clinicopathological characteristics of patients.Immunoreactivity was recorded in percentage for both nuclearand cytoplasmic staining independently. In brief, randomly 10

viable tumour fields were scanned for immunoreactivity underhigh-power objective (400×). Any appreciable brown colourwas considered positive immunoreactivity. Difference in immu-nostaining evaluation between investigators was observed infour tumours, which were reviewed and settled over consensus.

The non-parametric Mann–Whitney U test was used to deter-mine the significance of immunoreactivity with tumour invasionand differentiation groups. Pearson’s correlation was used todetermine the correlation. p Values<0.05 were considered sig-nificant. The analyses were performed using the SPSS softwarepackage for Windows (V.10.0; SPSS Inc, Chicago, Illinois, USA).

cDNA synthesis and quantitative real-time PCRRNA was extracted by the guanidine isothiocyanate and chloro-form method (TRI Reagent; Sigma-Aldrich, St. Louis, Missouri,USA). Fresh tumours were collected in RNase free vials and storedin –80°C or snap frozen in liquid nitrogen until processing. Onemillilitre of TRI reagent was added to approximately 250 mg ofRB tissue/retinal tissue and RNA was extracted as recommendedby the supplier. All RNA samples were treated with Turbo DNase(Ambion, Austin, Texas, USA). cDNA from RB tissue and retinaltissue were used for relative quantitation by real time PCR ana-lyses. First-strand cDNA synthesis was performed from 1 mg ofRNA in a 20 ml reaction mixture using reverse transcriptase andrandom primers supplied in SuperScript II, Invitrogen (Carlsbad,California, USA). APE1 and 18S transcripts were amplified usingTaqMan_Gene expression assays (APE1; Hs00959050_g1) and(18S; Hs99999901_s1) probes supplied by Applied Biosystems,Foster City, California, USA. Relative gene expression was calcu-lated by 2−ΔΔCt method.19 The expression levels of 18S were usedto normalise the relative expression levels.

Western blotApproximately 250 mg of RB tissues was homogenised in radioim-munoprecipitation assay (RIPA) buffer. The RB/retina tissue lysatewere then sonicated, and the supernatant was collected to whichProtease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Missouri,USA) was added. Protein estimation was carried out by Lowry’smethod. Precisely, 30 μg of each sample was run on 12%SDS-PAGE and electrophoretically transferred onto a nitrocellu-lose membrane. Uniformity of transfer was checked with 0.1%Ponceau and non-specific sites were blocked with 5% non-fat drymilk in PBS. The blots were incubated with primary antibody inwash buffer containing 5% skimmed milk (1/2000 dilution) forovernight at 4°C, washed in Tris-buffered saline containing 0.1%Tween 20 (TBS-T) and incubated in horseradish peroxidase-conjugated secondary antibody supplied by Amersham Biosciences(Amersham, Buckinghamshire, UK) at a dilution of 1/2000 for2 h. After intermittent washes with TBS-T, the immunoreactivebands were detected by horseradish peroxidase substrate,3,30,5,50-tetramethylbenzidine (Sigma-Aldrich, St. Louis, USA).β-Actin was used as the loading control. Relative protein levels ofAPE1 were determined by an optical density measurement using asoftware ImageJ-1.39u against retina as control.

RESULTSAPE1 expression in retinal tissue and RBsNon-neoplastic retina in the globe with RB showed nuclearpositivity in ganglion cells, inner and outer nuclear layers. Novisible cytoplasmic immunoreactivity was observed (figure 1A).In tumours, both nuclear and cytoplasmic staining was predom-inantly observed (figure 1B). Among the 55 tumours analysed,APE1 cytoplasmic expression was seen in 54 tumours (98%)and nuclear positivity was observed in 46 tumours (83%)

Table 1 Clinical and histopathological demography of the cohort

Tumourfeatures

Invasive RB(n=17)

Non-invasive RB(n=38)

Group D(n=19)

Group E(n=36)

WD 5 12 5 12MD 1 6 3 4PD 5 8 2 11UD 6 12 9 9IIRC Grp D 3 16 – –

IIRC Grp E 14 22 – –

Inv – – 3 14NI – – 16 22

Grp, group; IIRC, International Intraocular Retinoblastoma Classification; Inv, invasion;MD, moderately differentiated; NI, no invasion; PD, poorly differentiated; RB,retinoblastoma; UD, undifferentiated; WD, well differentiated.

Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166 403

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

Table 2 Clinicopathological features and APE1 reactivity in primary retinoblastoma

S. no. AgeClinicopathological information (laterality andinvasion of choroids and optic nerve information)

% APE1 nuclearpositivity (%)

% APE1 cytoplasmicpositivity (%)

1 11 months/F OD, UD, Grp E, Focal Ch Inv, ON Inv beyond LC 50 502 2 years/M OS, WD, Grp E, Focal Ch Inv, Postlam ON Inv, Focal RPE invasion 0 403 15 months/F OD, MD, Grp E, Focal RPE invasion, Ch Inv, Lam and Postlam On Inv 0 504 5 months/M OS, WD, Grp E, Prelam and Lam ON, Ch Inv 5 305 2 years/M OS, WD, Grp E, Full thickness Ch and scleral Inv 5 906 7 years/M OD, UD, Grp E, ON Inv, SE involvement 10 607 3 years/M OD, PD, Grp E, Diff Ch Inv, tumour cells in aqueous chamber 10 808 4 years/M OS, UD, Grp E, Diff Ch Inv and ON Inv at proximal end 10 809 2 years/M OD, UD, Grp E, Diff Ch Inv, Lam and postlam ON Inv 20 7010 3 years/M OD, PD, Grp E, Focal RPE Inv, Focal Ch and ON Inv 20 7011 2 years/F OD, PD, Grp E, ON Inv with extensive meningeal infiltration and SE involvement 20 8012 18 months/M OD, PD, Grp E, Prelam and Postlam ON Inv 30 6013 2 years/M OS, UD, Grp D, Extension into ON beyond LC 30 7014 3 years/M OS, WD, Grp D, Focal RPE Inv, Focal Ch Inv, Prelam and lam ON Inv. 40 6015 1 years/M OS, WD, Grp E, OS, WD, Invasion into ON head 50 4016 2 years/M OS,PD, Grp E, posterior scleral Inv 60 4017 6 years/F OD, UD, Grp D, Diff Ch and orbital Inv 70 6018 1 years/M OD, PD, Grp D, NI 10 5019 2 years/M OS, UD, Grp E, Focal Ch Inv 0 020 9 months/M OS, WD, Grp E, NI 0 1021 4 years/F OD, UD, Grp D, NI 0 2022 9 months/M OS, WD, Grp D, NI 0 4023 2 years/M OS, PD, Grp E, Diff Invasion of Ch and sclera 0 7024 4 years/F OS, MD, Grp E, minimal RPE Inv 0 7025 4 years/F OD, UD, Grp D, NI 0 9026 4 years/M OD, PD, Grp D, NI 5 4027 3 years/M OD, WD, Grp D, NI 5 6028 2 years/M OS, PD, Grp E, NI 10 4029 1 years/F OS, UD, Grp D, NI 10 5030 10 months/M OS, MD, Grp D, NI 10 6031 2 years/M OD, PD, Grp E, NI 10 6032 3 months/F OS, WD, Grp D, Focal Ch Inv 10 6033 1 years/M OS, WD, Grp D, NI 10 70

34 2 years/M OS, UD, Grp E, NI 10 8035 1 years/F OS, PD, Grp E, NI 20 4036 2 months/M OS, WD, Grp E, NI 20 8037 2 years/M OD, MD, Grp D, NI 20 8038 3 years/F OD, WD, Grp E, Focal RPE Inv 20 8039 3 years/M OD, UD, Grp D, NI 20 8040 1 years/F OD, WD, Grp E, NI 25 7541 2 years/M OS, UD, Grp E, Prelam ON Inv 30 7042 4 months/M OS, WD, Grp E, Focal Ch Inv, lam and Prelam ON Inv, 30 8043 2 years/M OS, MD, Grp D, NI 40 2044 7 months/F OD, PD, Grp E, Multiple focal RPE Inv, Prelam ON Inv 40 4045 24 years/M OD, PD, Grp E, Focal Ch Inv 40 6046 3 years/F OS, MD, Grp E, Focal RPE Inv and Prelam ON Inv 40 6047 3 years/M OS, UD, Grp D, NI 40 6048 2 years/F OD, MD, Grp E, Focal Ch Inv 50 2049 3 years/M OS, UD, Grp D, NI 50 2050 3 years/F OD, UD, Grp E, NI 50 5051 4 years/M OD, UD, Grp E, NI 50 5052 9 months/F OS, WD, Grp E, Focal Ch Inv, Prelam ON Inv 60 4053 6 months/M OS, WD, Grp E, NI 70 3054 3 years/M OD, WD, Grp E, NI 80 2055 2 years/M OD, UD, Grp D, Focal Ch Inv 90 10

APE1, human apurinic/apyrimidinic endonuclease; Ch Inv, choroidal invasion; Diff, diffuse; F, female; Focal Ch Inv, focal choroidal invasion; Grp, IIRC group; LC, laminar cribrosa; M,male; MD, moderately differentiated; NI, no invasion; OD, oculus dexter (right eye); ON, optic nerve; OS, oculus sinister (left eye); PD, poorly differentiated; Postlam, postlaminar;Prelam, prelaminar; RPE, retinal pigment epithelium; SE, surgical end; WD, well differentiated.

404 Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

(figure 1D; table 2). In invasive tumours, cells invading theoptic nerve showed predominant cytoplasmic immunoreactivity(figure 1C). The immunoreactivity intensity varied in varioustumours, and the distribution is given in table 3.

Clinicopathological correlations in tumoursAmong the 55 tumours analysed, an inverse correlation wasobserved between cytoplasmic and nuclear APE1 immunoreac-tivity (r=−0.315, p=0.019). The same was not observed ingroups based on invasion and differentiation. Further, we

observed no significant correlation in APE1 expression betweeninvasive and non-invasive groups, and undifferentiated and dif-ferentiated groups.

Expression of APE1 in IIRC groupsAmong the 55 tumours analysed, there were 19 group D and 36group E tumours. Both the nuclear and cytoplasmic immunor-eactivity of APE1 did not reveal any significance between groupD and group E tumours.

APE1 transcripts and relative expressionA total of 13 tumours were screened for APE1 expression invarious RB groups (figure 2). The median fold change of APE1transcripts in undifferentiated RB was high (14.5-fold; n=5) com-pared with well-differentiated RB (4.27-fold; n=8). A similarincrease in median fold change was observed in invasive (n=8)tumours compared with non-invasive (n=8) tumours with10.62-fold and 2.60-fold, respectively. However, these differencesdid not reveal any statistical significance. A relative increased APE1transcript was observed in all the tumours compared with retina,except one well-differentiated tumour (figure 3).

Expression of APE1 protein in RBThe presence of APE1 expression was confirmed by blot in sixtumours and retinal tissue extracts. A specific 37 kDa proteinwas observed in all the tissues analysed. The western blot ana-lysis showed varied levels of APE1 protein in tumours. Theamount of APE1 protein in tumours was relatively close toretina (figure 4).

DISCUSSIONAPE1 is a multifunctional protein that is essential for DNA BERand is also involved in apoptosis, redox regulation of

Figure 1 Immunohistochemistry of human apurinic/apyrimidinic endonuclease (APE1) in retina and retinoblastoma (RB). (A). Intense staining andunstained cytoplasm was observed in non-neoplastic retina in a globe with RB (B). Tumour shows strong nuclear and expression of APE1 in the RBtumours. (C). Tumour cells invading optic nerve show cytoplasmic immunoreactivity with negative expression. (D). Strong nuclear expression of APE1in well-differentiated RB tumours.

Table 3 Distribution summary of APE1 immunoreactivity in RB

% Immunoreactivity

Invasive tumours(n=17)

Non-invasive tumours(n=38)

Nuclear Cytoplasmic Nuclear Cytoplasmic

0 2 0 7 15 2 0 2 010 3 0 8 220 3 0 5 525 0 0 1 030 2 1 2 140 1 3 5 650 2 2 4 460 1 4 1 770 1 3 1 475 0 0 0 180 0 3 1 690 0 1 1 1

APE1, human apurinic/apyrimidinic endonuclease; RB, retinoblastoma.

Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166 405

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

transcription factors, cell cycle control and RNA metabolism.APE1 levels have been found to be elevated in many cancerssuch as cervical, rhabdomyosarcoma, germ cell tumours, glio-blastoma, and ovarian and lung cancers.7 11 12 Further, APE1may also predict resistance to cytotoxic therapy.

In the present study, we analysed the expression of APE1 inRB and in retinal tissue by immunohistochemistry, real-timePCR and western blot. Although retina showed nuclear stainingonly, we observed nuclear as well as cytoplasmic staining in thetumours analysed. This suggests that APE1 expression is dysre-gulated in RB. Whether this altered subcellular localisationreflects altered biochemical function in RB is unknown.However, our data are consistent with previous studies in othercancers such as breast,20 hepatocellular,21 ovarian,22 lung23 24

and prostate,5 where altered subcellular localisation has beenconsistently observed. In another study in lung cancer, the cyto-plasmic APE1 was found to induce Cox-2 by redox activity ofNF-κB by in vivo and in vitro studies, and it also showed thatcytoplasmic APE1 can activate cancer cells towards invasion,tumour growth and metastasis.23 Moreover, inverse correlationbetween cytoplasmic and nuclear expression observed in ourstudy provides additional evidence that the dysregulation mayhave functional consequences in RB cells. In subgroup analyses,we did not observe any significant correlation in APE1

Figure 2 Relative human apurinic/apyrimidinic endonuclease (APE1)transcripts in retinoblastoma (RB).APE1 transcripts were analysed in 13tumours and normalised with 18Stranscripts. Relative fold change wasplotted against adult retina byreal-time PCR. Tumours 1–8 are fromdifferentiated RB and 9–13 fromundifferentiated. Error bars representSD from two independent values witheach experiment done in triplicates.

Figure 3 Relative human apurinic/apyrimidinic endonuclease (APE1)transcripts distribution in invasive and differentiation groups inretinoblastoma (RB). The median fold change of APE1 transcripts inundifferentiated RB was high (14.5) compared with well-differentiatedRB (4.27). A similar increase in median fold change was observed ininvasive tumours (10.62) compared with non-invasive tumours (2.60).The top and bottom quartiles and the median values are depicted asbox plot.

Figure 4 Human apurinic/apyrimidinic endonuclease (APE1)protein expression in retinoblastoma(RB). Western blot analysis was donewith APE1 antibody and reprobed withβ-actin antibody as loading control.Bars represent APE1 expression inundifferentiated RB (lanes 1–6) andretina (lane 7). Error bars represent SDfrom three independent values.

406 Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

expression between tumour invasion and differentiation. This islikely due to smaller numbers of tumours in our study. A largerstudy is warranted to investigate this hypothesis.

Further, we observed a relative increase in APE1 mRNAexpression in most of the RB analysed. Undifferentiatedtumours are known to contain multiple high-risk factors, includ-ing invasion in RB.25 We have observed an increased mRNAlevel in undifferentiated tumours compared with differentiatedtumours. This indicates that APE1 may play a significant role inthe development of RB and its risk factor(s). Based on the rela-tive quantification of APE1 mRNA and protein in RB, it isevident that APE1 mRNAs are relatively high compared withprotein. Therefore, in RB targeting APE1 mRNA, using anti-sense oligonucleotides could be a more significant approachthan targeting protein.

This is the first evidence of dysregulation of BER molecule inRB. APE1 has the potential in the development of drug resist-ance, aggressiveness and metastasis. Further investigations arerequired to understand the mechanism and the potential of tar-geting RB.

Acknowledgements We thank Ms Shalini Subramanian for the technical help inimmunohistochemistry.

Contributors JS, SK and SM planned the work. JS performed the experiments.VK provided clinical information and tumour samples. All authors were involved inanalysis and preparation of the manuscript.

Funding This work was supported by the Indian Council of Medical Research(ICMR), India (grant identification number: 5/4/6/3/NE/06-II).

Competing interests None.

Patient consent Obtained.

Ethics approval This study was reviewed and approved by the local ethicscommittee of Vision Research Foundation, Sankara Nethralaya, India, and thecommittee deemed it in conformation with the generally accepted principles ofresearch and in accordance with the Helsinki declaration (study code 301-2012-p).

Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES1 Kim JW, Abramson DH, Dunkel IJ. Current management strategies for intraocular

retinoblastoma. Drugs 2007;67:2173–85.2 Krishnakumar S, Mallikarjuna K, Desai N, et al. Multidrug resistant proteins:

P-glycoprotein and lung resistance protein expression in retinoblastoma. Br JOphthalmol 2004;88:1521–6.

3 Bobola MS, Finn LS, Ellenbogen RG, et al. Apurinic/apyrimidinic endonucleaseactivity is associated with response to radiation and chemotherapy inmedulloblastoma and primitive neuroectodermal tumors. Clin Cancer Res2005;11:7405–14.

4 Demple B, Herman T, Chen DS. Cloning and expression of APE, the cDNA encodingthe major human apurinic endonuclease: definition of a family of DNA repairenzymes. Proc Natl Acad Sci USA 1991;88:11450–4.

5 Kelley MR, Cheng L, Foster R, et al. Elevated and altered expression of themultifunctional DNA base excision repair and redox enzyme Ape1/ref-1 in prostatecancer. Clin Cancer Res 2001;7:824–30.

6 Fishel ML, Kelley MR. The DNA base excision repair protein Ape1/Ref-1 as atherapeutic and chemopreventive target. Mol Aspects Med 2007;28:375–95.

7 Wang D, Luo M, Kelley MR. Human apurinic endonuclease 1 (APE1) expression andprognostic significance in osteosarcoma: enhanced sensitivity of osteosarcoma toDNA damaging agents using silencing RNA APE1 expression inhibition. Mol CancerTher 2004;3:679–86.

8 Yang ZZ, Chen XH, Wang D. Experimental study enhancing the chemosensitivity ofmultiple myeloma to melphalan by using a tissue-specific APE1-silencing RNAexpression vector. Clin Lymphoma Myeloma 2007;7:296–304.

9 Jiang Y, Guo C, Vasko MR, et al. Implications of apurinic/apyrimidinic endonucleasein reactive oxygen signaling response after cisplatin treatment of dorsal rootganglion neurons. Cancer Res 2008;68:6425–34.

10 Abbotts R, Madhusudan S. Human AP endonuclease 1 (APE1): from mechanisticinsights to druggable target in cancer. Cancer Treat Rev 2010;36:425–35.

11 Puglisi F, Aprile G, Minisini AM, et al. Prognostic significance of Ape1/ref-1subcellular localization in non-small cell lung carcinomas. Anticancer Res2001;21:4041–9.

12 Al-Attar A, Gossage L, Fareed KR, et al. Human apurinic/apyrimidinic endonuclease(APE1) is a prognostic factor in ovarian, gastro-oesophageal and pancreatico-biliarycancers. Br J Cancer 2010;102:704–9.

13 Luo M, Kelley MR. Inhibition of the human apurinic/apyrimidinic endonuclease(APE1) repair activity and sensitization of breast cancer cells to DNA alkylatingagents with lucanthone. Anticancer Res 2004;24:2127–34.

14 Madhusudan S, Smart F, Shrimpton P, et al. Isolation of a small molecule inhibitorof DNA base excision repair. Nucleic Acids Res 2005;33:4711–24.

15 Eagle RC Jr. High-risk features and tumor differentiation in retinoblastoma:a retrospective histopathologic study. Arch Pathol Lab Med 2009;133:1203–9.

16 Linn Murphree A. Intraocular retinoblastoma: the case for a new groupclassification. Ophthalmol Clin North Am 2005;18:41–53.

17 Krishnakumar S, Sundaram A, Abhyankar D, et al. Major histocompatibilityantigens and antigen-processing molecules in retinoblastoma. Cancer2004;100:1059–69.

18 Mallikarjuna K, Pushparaj V, Biswas J, et al. Expression of epidermal growth factorreceptor, ezrin, hepatocyte growth factor, and c-Met in uveal melanoma: animmunohistochemical study. Curr Eye Res 2007;32:281–90.

19 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-timequantitative PCR and the 2(-Delta Delta C(T)). Method. Methods 2001;25:402–8.

20 Kakolyris S, Kaklamanis L, Engels K, et al. Human AP endonuclease 1 (HAP1)protein expression in breast cancer correlates with lymph node status andangiogenesis. Br J Cancer 1998;77:1169–73.

21 Di Maso V, Avellini C, Croce LS, et al. Subcellular localization of APE1/Ref-1 inhuman hepatocellular carcinoma: possible prognostic significance. Mol Med2007;13:89–96.

22 Moore DH, Michael H, Tritt R, et al. Alterations in the expression of the DNA repair/redox enzyme APE/ref-1 in epithelial ovarian cancers. Clin Cancer Res2000;6:602–9.

23 Wu HH, Cheng YW, Chang JT, et al. Subcellular localization of apurinicendonuclease 1 promotes lung tumor aggressiveness via NF-kappaB activation.Oncogene 2010;29:4330–40.

24 Wu HH, Chu YC, Wang L, et al. Cytoplasmic ape1 expression elevated by p53aberration may predict survival and relapse in resected non-small cell lung cancer.Ann Surg Oncol 2013;20:336–47.

25 Kashyap S, Sethi S, Meel R, et al. A histopathologic analysis of eyes primarilyenucleated for advanced intraocular retinoblastoma from a developing country. ArchPathol Lab Med 2012;136:190–3.

Sudhakar J, et al. Br J Ophthalmol 2014;98:402–407. doi:10.1136/bjophthalmol-2013-304166 407

Laboratory science

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from

doi: 10.1136/bjophthalmol-2013-3041662, 2014

2014 98: 402-407 originally published online JanuaryBr J Ophthalmol Job Sudhakar, Vikas Khetan, Srinivasan Madhusudan, et al. retinoblastoma(APE1) expression in advancedapurinic/apyrimidinic endonuclease 1 Dysregulation of human

http://bjo.bmj.com/content/98/3/402.full.htmlUpdated information and services can be found at:

These include:

References http://bjo.bmj.com/content/98/3/402.full.html#ref-list-1

This article cites 25 articles, 9 of which can be accessed free at:

serviceEmail alerting

the box at the top right corner of the online article.Receive free email alerts when new articles cite this article. Sign up in

CollectionsTopic

(624 articles)Eye (globe)   � Articles on similar topics can be found in the following collections

Notes

http://group.bmj.com/group/rights-licensing/permissionsTo request permissions go to:

http://journals.bmj.com/cgi/reprintformTo order reprints go to:

http://group.bmj.com/subscribe/To subscribe to BMJ go to:

group.bmj.com on September 29, 2014 - Published by bjo.bmj.comDownloaded from