Apurinic/apyrimidinic endonuclease is inversely associated with response to radiotherapy in pediatric ependymoma
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Apurinic/apyrimidinic endonuclease is inversely associatedwith response to radiotherapy in pediatric ependymoma
Michael S. Bobola1,2, Pawel P. Jankowski2,3, Mary E. Gross2, Jeffery Schwartz4, Laura S. Finn5,6,
A. Blank5, Richard G. Ellenbogen1,2 and John R. Silber1
1 Department of Neurological Surgery, University of Washington, Seattle, WA2 Division of Neurosurgery, Department of Surgery; Childrens Hospital and Regional Medical Center, Seattle, WA3 Division of Neurosurgery, University of California, San Diego, CA4 Department of Radiation Oncology, University of Washington, Seattle, WA5 Department of Pathology, University of Washington, Seattle, WA6 Department of Laboratories, Childrens Hospital and Regional Medical Center, Seattle, WA
Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells.
Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors
for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36
ependymomas and expression of Ape1/Ref-1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining.
Cox proportional hazards regression models were used to analyze the association of activity or expression with progression-
free survival or with overall survival. Activity varied 13-fold and was not associated with tumor or patient characteristics. In
univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a
factor of 2.18 for every 0.01 unit increase in activity (p 0.003) in 24 grade II ependymomas. Risk for death increased by afactor of 1.89 (p 0.02) in the same population. The fraction of Ape1/Ref-1 immunopositive cells varied widely withinindividual tumors and was not associated with either progression-free or with overall survival. Suppressing Ap endo activity in
pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with
radiation response reflected, at least in part, repair of radiation-induced DNA lesions. Our data indicate that Ap endo activity
is predictive of outcome following radiotherapy, and suggest that Ape1/Ref-1 promotes radiation resistance in pediatric
ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.
Ependymomas are the third most common pediatric centralnervous system tumor, showing a peak incidence in children56 years old.1 These generally slow growing tumors arisemost frequently in the posterior fossa, especially in youngerpatients, but also commonly occur in the cerebral hemi-spheres of older children, and rarely in the spine.2 Regardlessof anatomic location, the tumors are histologically similar,presumably reecting the transformation of a common pro-genitor. Emerging evidence indicates that ependymomas arise
from cells with functional and molecular features similar toradial glia, multipotent progenitors of normal neurons andglial cells.3 Recent work has revealed characteristic differencesin gene expression that distinguish ependymomas located inthe posterior fossa from those found in the cerebrum and thespine [e.g., as shown in Ref. 2,4]. Many of the differentiallyexpressed genes promote anatomic localization and matura-tion of radial glia, suggesting that ependymomas encompassdistinct tumor subgroups derived from a common progenitor.The underlying heterogeneity among ependymomas is high-lighted by the wide variability in clinical outcome observedamong tumors that are indistinguishable histologically.1
Despite advances in neuro-imaging, surgical techniquesand radiation treatment, the overall prognosis for pediatricependymomas remains unfavorable with 5-year survival ratesless than 50% in many studies.5,6 Gross total resection isassociated with longer progression-free survival, but anappreciable fraction of completely excised tumors recur, arisk for which there are no reliable clinical or biologicalmarkers.2,7 Moreover, in the case of posterior fossa tumors,gross total resection is frequently precluded by proximity tothe brain stem or involvement of cranial nerves. While clini-cal trials have documented the benet of radiation inprolonging survival, treatment of infants and very young chil-dren is restricted by a substantial risk of adverse sequelae,
Key words: Ape1/Ref-1, brain tumor, DNA repair, c-ray resistance
Abbreviations: Ap endo: apurinic/apyrimidinic endonuclease; ASO:
antisense oligonucleotide; CI: condence interval; HR: hazard ratio;
PFS: progression-free survival
Grant sponsor: The American Cancer Society; Grant number: RSG
01 9101 CCE; Grant sponsor: NIH; Grant numbers: CA82622,
History: Received 29 Jun 2010; Accepted 4 Nov 2010; Online 4 Jan
Correspondence to: John R. Silber, Department of Neurological
Surgery, University of Washington, Seattle, WA 98195-6470,
Tel.: 206-685-8642, Fax: 206-543-8315, E-mail: jrsilber@
Int. J. Cancer: 129, 23702379 (2011) VC 2011 UICC
International Journal of Cancer
including cognitive impairment.1 Effective chemotherapy regi-mens to be used either in lieu of radiation in newly operatedtumors or for tumors that recur after radiotherapy remain tobe unequivocally demonstrated.1,5 Because of these limitations,the contemporary standard of care for pediatric ependymomaincludes surgical excision to the greatest extent possible fol-lowed, when permissible, by conformal radiotherapy.1,5
In light of the poor prognosis that accompanies a diagno-sis of ependymoma, development of more effective post-ope-rative therapies is an urgent priority. Characterization ofmechanisms that promote radiation resistance will likely sug-gest novel approaches to improve treatment efcacy andprovide new molecular markers of clinical outcome. The tu-moricidal activity of radiation is mediated almost exclusivelyby double-strand breaks that are formed either directly8 or,more commonly, as a consequence of oxidative free radical-induced DNA lesions that impede DNA replication forkprogression.9 Abasic (apurinic/apyrmidinic) sites and single-strand breaks are the two most abundant replication-blockinglesions produced by radiation.10,11 Abasic sites result fromfree radical attack at deoxyribose or cleavage by DNA glyco-sylases at oxidized bases.12 Single-strand breaks are also pro-duced by radical attack at deoxyribose or by spontaneoushydrolysis of abasic sites. Radiation-induced single-strandbreaks frequently contain deoxyribose fragments that must beremoved to permit DNA repair synthesis and strand liga-tion.8,13 The multifunctional DNA repair protein Ape1/Ref-1is essential for radiation resistance in mammalian cells.Ape1/Ref-1 has a strong endonuclease activity that incisesDNA 50 to abasic sites, leaving 30-OH and 50-deoxyribosephosphate termini. The enzyme also excises oxidized deoxyri-bose fragments from single-strand breaks via a 30-phopho-diesterase activity.12 Importantly, suppression of Ape1/Ref-1is accompanied by hypersensitivity to radiation and oxidizingagents in human and other mammalian cells.12,14
We have reported evidence that the activity of Ape1/Ref-1mediates resistance to radiation in adult gliomas15 and toradiation followed by chemotherapy in pediatric medulloblas-tomas.16 In both instances, we observed a strong inverse corre-lation between tumor Ap endo activity and progression-freesurvival following adjuvant therapy, that is, the higher theactivity, the shorter the time to tumor recurrence. These nd-ings led us to assess the contribution of Ap endo activity toradiation resistance in pediatric ependymoma. We report herean initial retrospective study of 36 childhood ependymomas.Our results indicate that Ap endo activity is predictive of out-come following radiotherapy, and suggest that Ape1/Ref-1promotes radiation resistance in pediatric ependymomas.
Material and MethodsTissue
Tumors were obtained from patients operated with informedconsent at Childrens Hospital and Regional Medical Centerbetween 1994 and 2004. Diagnosis was obtained from thenal neuropathology report. Demographic and treatment out-
come information was obtained in accord with protocolsapproved by the Institutional Review Board at ChildrensHospital and Regional Medical Center. The precautions takento preserve tissue viability and enzymatic activity duringtransport of tissue to the laboratory, and the procedure fordetermining cell number are described elsewhere.17
Apurinic/apyrimidinic endonuclease activity
Activity was quantied in supernatants of intact tissuehomogenates using a highly sensitive assay that measuresconversion of acid-treated, super-coiled plasmid DNA torelaxed form caused by incision at an abasic site. The proto-col for extract preparation and activity determination,together with controls validating the assay, has beendescribed in extensive detail elsewhere.16
Immunoperoxidase staining was done on parafn blocks offormalin-xed tumors as described in Bobola et al., 2005.16
Briey, de-waxed 5 lm tissue sections were treated with 0.6%hydrogen peroxide/methanol to quench endogenous peroxi-dase. Following rehydration, trypsin digestion, and incubationwith blocking solution (10% goat serum and 3% bovine calfserum), the slides were serially incubated with a 1:5000 dilu-tion of mouse anti-Ape1/Ref-1 monoclonal antibody (NB100-116B1; Novus) and a 1:100 dilution of a biotin-conjugatedgoat anti-mouse IgG antibody (BA-9200, Vector). After incu-bation with strepavidin-conjugated horseradish peroxidase,antibody binding was visualized by incubation with 3,30-dia-minobenzidine tetrachloride. Slides were counterstained withmethyl green. Negative controls were treated identicallyexcept for omission of the primary antibody. Slides of Ape1/Ref-1-expressing medulloblastomas16 served as positive con-trols. Stained slides were scanned at low power to evaluateregional variation. Five hundred cells, 100 from each of veregions chosen at random, were counted and averaged to cal-culate the fraction of immunopositive cells.
Suppression of Ap endo activity
Ap endo activity in the pediatric ependymoma line Res19618
was suppressed by two cycles of transfection with eitherantisense oligonucleotides (ASOs) or siRNA targeting humanApe1/Ref-1, using the same oligomer sequences and proce-dures similar to those we have previously described.16 Inpreparation for transfection, subconuent cultures of Res196in 35 mm plates were incubated for 24 hr at 37C inhumidied 95%/5% air/CO2 in 3 mL of Opti-MEM contain-ing 5% iron supplemented bovine serum. Immediately priorto transfection cells were washed once with serum-freeOpti-Mem. Optimal oligonucleotide concentrations andtransfection procedures were established for Res196 asfollows.
For transfection with ASOs, separate 50 ll aliquots of unsup-plemented Opti-MEM (Life Technologies, Inc) were mixed with20 lL of 14.5 lM ASO (50-TTTCCCACGCTTCGGCATTCC-30
Bobola et al. 2371
Int. J. Cancer: 129, 23702379 (2011) VC 2011 UICC
complementary to the translation start site) or with 20 lL ofcationic lipid (Lipofectin; Life Technologies, Inc) and held atroom temperature for 30 min. The oligonucleotide and cationiclipid mixtures were gently combined and held overnight at 4C.The oligonucleotide mixture, diluted to 2.9 mL with unsupple-mented Opti-MEM (nal ASO concentration, 1 lM), was addedto washed cells and incubation continued for 18 hr. Three mLof Opti-MEM supplemented with 10% iron-supplemented bo-vine serum was then added and incubation continued for 30 hr;this cycle was repeated. Transfection with a random oligomer ofthe same nucleotide composition as the ASOs (i.e., a scrambledoligonucleotide) served as a control; the scrambled oligomeryielded no reduction in Ap endo activity relative to untrans-fected cells (0.16 6 0.09 vs 0.18 6 0.03 fmol abasic sitesincised/cell/min).
For transfection with siRNA, 8 lL of 20 lM siRNA (50-CUUCAGGAGCUGCCUGGACUC-30, 30-UUGAAGUCUCGACGGACCUG-50, corresponding to nucleotides 532-552 ofthe human Ape1/Ref-1 cDNA sequence), was mixed with42 lL of unsupplemented Opti-MEM; in a separate tube10 lL of cationic lipid was mixed with 40 lL Opti-MEM.After 20 min at room temperature, the two mixtures werecombined and incubated an additional 30 min. Washed cul-tures in 1.9 mL of Opti-MEM were incubated with thesiRNA mixture for 4 to 18 hr; nal siRNA concentration was80 nM. Two mL of Opti-MEM containing 10% iron supple-mented bovine serum was then added and incubation con-tinued. This cycle was repeated 48 hr after initiating siRNAtreatment. Transfection with an inactive siRNA duplex (50-AAUGUGGAUGGGCUUCGAGCC-30, 30-CCUUACACCUACCCGAAGCUC-50, corresponding to nucleotides 412432 ofthe human Ape1/Ref-1 cDNA sequence) served as a control;this sequence yielded no reduction in Ap endo activity rela-tive to untransfected cells (0.17 6 0.07 vs 0.18 6 0.03 fmolabasic sites incised/cell/min).
The effect of suppressing of Ap endo activity on the radiationsensitivity of Res196 was determined by clonogenic assayessentially as previously described in detail.19 Briey, 24 hrafter initiating the second cycle of transfection with ASOs orsiRNA, 50020,000 cells were sub-cultured into 35 mmplates. Twenty-four hour later cells were exposed to 137Cs-c-ray irradiation at 1 Gy/min under ambient conditions. Incu-bation was continued for approximately 14 days to allow for-mation of colonies 50 cells. Survival (mean 6 SD) is theratio of colony-forming ability of treated cells to that ofuntreated cells.
Survival curves were determined using the linear-quad-ratic model20 from which the following parameters werederived that quantitatively describe sensitivity: D1, the initialrate of cell killing, D0, the nal rate of killing, Dq, thequasi-threshold dose below which cells are insensitive toradiation killing, and D10, the dose required to reduce sur-viving fraction to 10%.
Statistical procedures were performed using Stata (Stata Cor-poration). Comparison of means was by Students t-testassuming unequal variances. Relationships between continu-ous variables were assessed by regression analysis. In theseanalyses, the outcome variables progression-free and overallsurvival are measures of time from initiation of radiotherapyto tumor progression assessed by radiologic imaging and dateof death, respectively. Tumor progression was dened as:appearance of tumor growth in the case of gross total resec-tion; increase in the largest dimension of residual tumor byat least 25% and/or tumor growth at a different site in thecase of sub-total resection. Observations were censored at thelast documented follow-up time if progression or death wasnot yet observed. Median survival was determined by themethod of Kaplan and Meier. The hazard ratio (HR) for tu-mor progression as a function of Ap endo...