Apurinic/Apyrimidinic Endonuclease Activity Is Associated With

CancerTherapy: Clinical

Apurinic/Apyrimidinic Endonuclease Activity Is Associated with Response to Radiation and Chemotherapy in Medulloblastoma and Primitive Neuroectodermal Tumors Michael S. Bobola,1,4 Laura S. Finn,3,5 Richard G. Ellenbogen,1,4 J. Russell Geyer,2,6 Mitchel S. Berger,7 Justin M. Braga,1Elizabeth H. Meade,1,4 Mary E. Gross,1andJohn R. Silber4

Abstract Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to radiation- and alkylator-induced cytotoxic abasic sites in human cells. We assayed apurinic/apyrimidinic endonuclease activity in medulloblastomas and primitive neuroectodermal tumors (PNET) to establish correlates with tumor and patient characteristics and with response to adjuvant radiation plus multiagent chemotherapy. Experimental Design: Ap endo activity was assayed in 52 medulloblastomas and 10 PNETs from patients 0.4 to 21years old. Ape1/Ref-1,the predominant human Ap endo activity, was measured in 42 medulloblastomas by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity with time to tumor progression (TTP). Results: Tumor Ap endo activity varied 180-fold and was significantly associated with age and gender.Tumor Ape1/Ref-1was detected almost exclusively in nuclei. In a multivariate model, with Ap endo activity entered as a continuous variable, the hazard ratio for progression after adjuvant treatment in 46medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P V 0.001) and was independent of age and gender. Suppressing Ap endo activity in a human medulloblastoma cell line significantly increased sensitivity to 1,3-bis(2-chlororethyl)-1-nitrosourea and temozolomide, suggesting that the association of tumor activity withTTP reflected, at least in part, abasic site repair. Conclusions: Our data (a) suggest that Ap endo activity promotes resistance to radiation plus chemotherapy in medulloblastomas/PNETs, (b) provide a potential marker of treatment outcome, and (c) suggest clinical use of Ap endo inhibitors to overcome resistance.

Medulloblastomas and primitive neuroectodermal tumors classification (5), are histologically similar, being composed (PNET) are highly malignant (i.e., WHO grade 4) cancers that of small, morphologically undifferentiated cells of uncertain account for 20% to 25% of the f2,200 pediatric primary histiogenesis that occasionally contain focal areas displaying brain tumors diagnosed annually in the United States (1, 2). morphologic and immunohistochemical features of mature The median age at diagnosis is about 9 years, with the neurons or glia (5, 6). Although emerging evidence suggests majority of cases (62%) occurring in males (3). Medulloblas- that medulloblastomas and PNETs harbor distinctive genetic tomas and PNETs also occur infrequently in younger adults aberrations (6), these tumors are primarily distinguished by between 20 and 40 years of age (4). These malignancies, their anatomic sites of occurrence: medulloblastomas arise in categorized as embryonal tumors in the current WHO the cerebellum, whereas PNETs occur almost exclusively in the cerebral hemispheres. Improvements in surgical technique and the development of effective postoperative treatments have dramatically in- Authors’Affiliations: 1Division of Neurosurgery, Department of Surgery; 2Division creased survival rates for medulloblastomas in the last 30 3 of Hematology/Oncology; and Department of Laboratories, Children’s Hospital years (1, 2). Better outcome has also been achieved by using and Regional Medical Center; Departments of 4Neurological Surgery, 5Pathology and 6Pediatrics, University of Washington, Seattle, Washington; and 7Department patient and tumor characteristics to stratify medulloblastomas of Neurological Surgery, University of California, San Francisco, California into average-risk and high-risk groups that differ in predicted Received 5/13/05; revised 7/12/05; accepted 7/21/05. response and likelihood of recurrence (6–8). The current Grant support: American Cancer Society grant RPG-97-019 CN and NIH grants standard of care is surgical excision to the greatest extent CA70790, CA82622, and CA104593. possible followed by adjuvant radiotherapy and chemother- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance apy. For patients receiving this care, overall 5-year survival with 18 U.S.C. Section 1734 solely to indicate this fact. rates range from 60% to 85% for average-risk medulloblasto- Requests for reprints: Michael S. Bobola, Division of Neurosurgery, Department mas to about 40% for high-risk medulloblastomas (6, 7, 9). of Surgery, Children’s Hospital and Regional Medical Center, Seattle, WA 98105. In contrast, PNETs are more aggressive and less responsive, Phone: 206-987-2046; Fax: 206-527-3925; E-mail: michael.bobola@ seattleschildren.org. with 5-year survival rates of <20% (10). Unfortunately, long- F 2005 American Association for Cancer Research. term survival is frequently accompanied by detrimental doi:10.1158/1078-0432.CCR-05-1068 physical and neuropsychological sequelae produced by

www.aacrjournals.org7405 Clin Cancer Res 2005;11(20) October 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. CancerTherapy: Clinical adjuvant radiotherapy and chemotherapy, especially in in- thology report. Tissue and demographic information was obtained in fants and young children (11). Moreover, there is no ther- accordance with protocols approved by the Institutional Review apy that produces long-term remission in recurrent disease Boards at Children’s Hospital and Regional Medical Research Center (2, 7, 10). and the University of Washington Medical Center. Immediately upon resection, tissue was placed in ice-cold DMEM/F12 supplemented with In light of the detrimental long-term effects of adjuvant 5% fetal bovine serum and transported to the laboratory. The therapy and the poor prognosis for the recurrent tumors that precautions taken to preserve tissue viability and enzymatic activity afflict ca. 50% of patients (2, 7, 10), development of more during transport of specimens and the procedure for determining cell effective approaches to treating medulloblastomas/PNETs is an number are described elsewhere (23). urgent priority. Little is known about the molecular mecha- Apurinic/apyrimidinic endonuclease activity. Ap endo activity was nisms that underlie adjuvant therapy failure. Delineating quantitated in whole cell extracts as we have previously described for resistance mechanisms will likely suggest new strategies to human brain tumors (22), brain tumor–derived cell lines (21), and improve the efficacy of existing therapies and provide new normal brain (22). The highly sensitive assay measures conversion of markers of clinical outcome. As a group, medulloblastomas/ acid-treated, supercoiled plasmid DNA to relaxed form caused by PNETs are relatively responsive to radiation and chemother- incision at an abasic site (24). Briefly, tissue was rinsed in PBS, weighed, and minced into small pieces with scalpel blades. Tissue apeutic agents that damage DNA (2), suggesting that DNA fragments were resuspended in PBS and passed serially through 16-, repair may be a determinant of tumor response. Ionizing 18-, and 20-gauge needles to produce a single cell suspension. Cells radiation and the nitrosourea- and nitrogen mustard–based were pelleted by centrifugation at 2,000 Â g for 5 minutes at 4jC and alkylating agents used to treat medulloblastomas/PNETs suspended at f1 Â 107 cells/mL in 50 mmol/L Tris-HCl (pH 7.5), produce cytotoxic abasic sites (apurinic/apyrimidinic sites) in 1 mmol/L EDTA, 100 mmol/L NaCl, 0.1 mmol/L phenylmethylsulfonyl DNA (12–14). Abasic sites, which constitute a major lethal fluoride, and 1 Ag/mL each of aprotinin, leupeptin, and pepstatin. Cell lesion, block progression of replicative DNA polymerases and suspensions were sonicated on ice for four 15-second intervals, and are believed to cause replication forks to stall and/or collapse debris was pelleted by centrifugation at 10,000 Â g for 5 minutes at 4jC. (15–17). The vast majority of abasic site repair in mammalian In some instances, extraction was done in 1.5Â Ap endo reaction buffer cells is catalyzed by Ape1/Ref-1, an abundant enzyme [75 mmol/L HEPES (pH 7.5), 225 mmol/L KCl, 7.5 mmol/L MgCl2, A possessing a strong apurinic/apyrimidinic endonuclease 0.75 mmol/L CoCl2, and 150 g/mL bovine serum albumin]. Experi- ments with pediatric brain tumor cell lines revealed identical (Ap endo) activity that hydrolyzes the phosphodiester bond Ap endo activities for extracts prepared in both extraction buffers. V 5 to baseless sites (12, 17–19). Assay mixtures (30 AL) contained 0.033 Ag/mL depurinated pKT100 Our recent evidence indicates that the Ap endo activity of plasmid DNA (3.5 kb), containing, on average, 1.5 abasic sites per Ape1/Ref-1 mediates resistance to adjuvant radiation and molecule, 50 mmol/L HEPES (pH 7.5), 150 mmol/L KCl, 5 mmol/L alkylating agent–based chemotherapy in adult gliomas (20– MgCl2, 0.5 mmol/L CoCl2, 100 Ag/mL bovine serum albumin, and 22). We have observed a strong inverse correlation between extract equivalent to 10 to 104 cells. Preparation of the DNA substrate is glioma Ap endo activity and time to tumor progression (TTP) described in ref. 22. After incubation for 10 minutes at 37jC, reaction following either radiation or alkylator therapy (20); that is, products were resolved on an 0.8% agarose gel in 40 mmol/L the higher the activity, the shorter the time to recurrence. We Tris-acetate, 2 mmol/L EDTA. The gel was stained with ethidium have also found that suppressing Ap endo activity, and Ape1/ bromide to visualize supercoiled and nicked, relaxed plasmid DNA and was photographed with a Kodak DC290 digital camera. Band density Ref-1 content, in a human adult glioma cell line reduces was quantitated using Kodak 1D version 3.5.4 image processing resistance to the clinical alkylators 1,3-bis(2-chloroethyl)-1- software, with HindIII-linearized pKT100 as a standard. Activity (fmol nitrosourea (BCNU) and temozolomide (21). A concurrent abasic sites incised/cell/min, abbreviated to fmol/cell/min) is the mean increase in abasic sites supports the conclusion that abasic site of at least three separate determinations that differed, in general, by repair promotes resistance and shows a functional basis for <15%, and in all cases, by <30%. Each determination comprised assay the in vivo findings. Lastly, Ap endo activity in human of increasing amounts of sample and yielded activity calculated by gliomas is positively correlated with tumor characteristics regression analysis of points on the linear portion of the curve. associated with greater malignancy and poor response (22). Normalizing activity to cell number affords a uniform basis for These findings in adult gliomas led us to assess the comparing tissues of different histology, avoiding potential problems attributable to tissue- and tumor-specific differences in the amount and contribution of Ap endo activity to the resistance of pediatric composition of intracellular and extracellular proteins (e.g., ref. 25). brain tumors to adjuvant therapy. Here, we report an initial Immunohistochemistry. Immunoperoxidase staining was done on study of 52 medulloblastomas and 10 PNETs in children and paraffin blocks of formalin-fixed tumors. Briefly, 7-Am tissue sections young adults. Our results suggest that Ap endo activity were mounted on 3-aminopropyltriethoxysilane-treated slides, de- promotes resistance to combined radiation and chemotherapy waxed, and rinsed twice in 100% ethanol followed by treatment with containing alkylating and/or platinating agents and is 0.6% hydrogen peroxide/methanol to quench endogenous peroxidase. predictive of outcome following this adjuvant therapy in Slides were rehydrated by serial rinses in 95% ethanol, 80% ethanol medulloblastomas/PNETs. then distilled water followed by a 5-minute incubation in TBS (50 mmol/L Tris pH 7.5, 150 mmol/L NaCl and 12.5 mmol/L KCl). Sections were incubated for 5 minutes at room temperature in 0.1% Materials and Methods trypsin in TBS followed by 3 Â 5-minute washes with TBS. Slides were incubated with 10% goat serum and 3% bovine calf serum in TBS for 1 Tissue. Tumors were obtained with informed consent from patients hour at room temperature and then incubated overnight at 4jC with a operated at the Children’s Hospital and Regional Medical Research 1:5,000 dilution of a mouse monoclonal anti-Ape1/Ref-1 antibody Center and the University of Washington Medical Center. Care was (NB100-116B1, Novus, Littleton, CO) in TBS containing 10% goat taken to ensure that tumor specimens were distant from the gross serum. Slides were washed in TBS and incubated with a 1:100 dilution interface with normal tissue. All tissues were reviewed by a of a biotin-conjugated goat anti-mouse IgG antibody (BA-9200, Vector, neuropathologist and diagnosis was obtained from the final neuropa- Burlingame, CA) for 1 hour at room temperature. After washing with

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TBS, the slides are incubated for 30 minutes at room temperature with fected with antisense oligonucleotides or siRNA. The trays were strepavidin-conjugated horseradish peroxidase and washed again with incubated for 6 to 8 hours to allow cells to attach and resume TBS. Primary antibody binding is visualized by incubation with 3,3V- proliferation before further incubation with alkylating agent for diaminobenzidine tetrachloride until a satisfactory signal develops. 1 hour. In each experiment, single or duplicate wells of cells were Slides were counterstained with methyl green or Gill’s hematoxylin. exposed to at least 10 drug doses that serially increased by 10% to Negative controls were treated identically except for omission of the 20% increments. Alkylator sensitivity was assayed in two and three primary antibody. Stained slides were scanned at low power to evaluate independent experiments for temozolomide and BCNU, respectively; regional variation. One hundred cells from five regions were counted each experiment employing different drug doses. The cells were and the average percent of positive cells for each tumor was calculated. washed free of residual alkylator and incubated in fresh, supple- Both nuclear and cytoplasmic staining were considered positive. mented medium for 5 to 7 days to allow formation of colonies. The Staining in any fragments of normal brain was also assessed. colonies were stained with 0.5% methylene blue in 1:1 methanol/ Suppression of apurinic/apyrimidinic endonuclease activity and H2O (v/v) to aid visualization during counting by light microscopy. alkylating agent sensitivity. Ap endo activity in the human medullo- Colonies containing z50 cells were counted. The colony-forming blastoma-derived cell line UW228-2 (26) was suppressed by transfection efficiency of untreated UW228-2 was f25%. with either antisense oligonucleotides or siRNA targeting Ape1/Ref-1. Drug sensitivity was determined by analysis of survival curves (log For transfection with antisense oligonucleotides, separate 50-AL aliquots surviving fraction versus dose) using standard methods, as we have of unsupplemented Opti-MEM (Gibco, Invitrogen, Carlsbad, CA) were previously described in detail (e.g., Fig. 1 of ref. 28). The survival mixed with either 2.9 nmol of an antisense oligonucleotide comple- variables LD10 (the dose required to reduce overall survival to 10%), mentary to the translation start site (5V-TTTCCCACGCTTCGGCATTCC- DT (the threshold dose below which cells are insensitive to alkylator 3V) or with 15 AL of cationic lipid (Lipofectin, Life Technologies) cytotoxicity), and D37 (the rate of killing as indicated by the slope of and held at room temperature for 30 minutes. The antisense the linear portion of the survival curve) were derived by regression oligonucleotide and cationic lipid mixtures were gently combined and analysis of the linear portion of a composite kill curve derived from held overnight at 4jC. Subconfluent cultures of UW228-2 incubated at the replicate assays. The three survival variables are presented as mean F 37jC in humidified 95%/5% air/CO2 overnight in 3 mL of Opti-MEM SE and provide a complete description of alkylator sensitivity. containing 5% iron-supplemented bovine serum in a 35-mm dish were Statistical analysis. Data analysis and statistical procedures were washed once with unsupplemented Opti-MEM before adding the done using Microsoft Excel and the statistics program Intercooled Stata antisense oligonucleotide mixture. Cells were incubated with antisense 8 (Stata Corp., College Station, TX). Comparison of means was by oligonucleotides for 18 hours before adding 2 mL of Opti-MEM Student’s t test assuming unequal variances. Relationships between containing with 10% iron-supplemented bovine serum and continuing continuous variables were assessed by regression analysis. The incubation. Final antisense oligonucleotide concentration was 1 Amol/L. association of Ap endo activity with TTP was assessed by standard Seventy-two hours after initiating transfection, cells were harvested by methods. In these analyses, the outcome variable TTP was assessed by trypsinization for determination of alkylator sensitivity and assay of Ap radiologic imaging. Tumor progression was defined as appearance of endo activity. Transfection with a random oligomer composed of the tumor growth in the case of gross total resection: increase in the largest same nucleotide composition as the antisense oligonucleotide served as dimension of residual tumor by at least 25% and/or tumor growth at a a control. different site. Observations were censored at the last documented A protocol similar to that described by Wang et al. (27) was used for follow-up time if progression was not yet observed. Mean TTP was transfection with siRNA. In separate tubes, 1.6 nmol of the siRNA determined by the method of Kaplan and Meier. The hazard ratio (HR) (5V-CUUCAGGAGCUGCCUGGACUC-3V,3V-UUGAAGUCCUCGACG- for tumor progression as a function of Ap endo activity was calculated GACCUG-5V, corresponding to nucleotides 532-552 of the human by using Cox regression analysis. Ap endo was entered into the Ape1/Ref-1 cDNA sequence) was mixed with 0.1 mL of unsupple- regression models as a continuous variable and scaled (i.e., multiplied mented Opti-MEM, and 15 AL of cationic lipid was mixed with 0.4 mL by 100) so that the tabulated HR represents the relative change in Opti-MEM. After 10 minutes at room temperature, the two mixtures hazard for a 0.01 unit change in measured Ap endo activity. Possible were combined and added to 35-mm dishes of UW228-2 grown as confounding by age and gender was examined by using multivariate described above. Cells were incubated with siRNA for 4 to 18 hours Cox regression analysis. Statistically significant relationships were before adding 2 mL of Opti-MEM containing 10% iron-supplemented determined at the 95% confidence level (95% CI). bovine serum and continuing incubation; suppression of Ap endo activity or effect on alkylator sensitivity was unaffected by length of Results incubation with siRNA. Final siRNA concentration was 320 nmol/L. At 72 hours after initiating transfection, cells were harvested by Patient and tumor characteristics. Fifty-two medulloblasto- trypsinization for determination of alkylator sensitivity and assay of mas and 10 cerebral PNETs were obtained from patients Ap endo activity. Transfection with an inactive siRNA duplex F F (5V-AAUGUGGAUGGGCUUCGAGCC-3V,3V-CCUUACACCUACCC- ranging in age from 4 days to 21 years (mean SD, 8.4 5.0 GAAGCUC-5V, corresponding to nucleotides 412-432 of the human years). The majority of tumors occurred in males (61%), in Ape1/Ref-1 cDNA sequence) served as a control. accord with previous observations of gender bias (3). Fifty-five Alkylating agent survival. BCNU and temozolomide were obtained tumors were newly diagnosed, two were recurrent after from the pharmacies of the University of Washington Medical Center surgery, one after surgery and chemotherapy, and four after and Children’s Hospital and Regional Medical Center, respectively. surgery, radiation, and chemotherapy. BCNU was dissolved in absolute ethanol at a concentration of Association of apurinic/apyrimidinic endonuclease activity with 1 mol/L; temozolomide was dissolved in DMSO at a concentration tumor and patient characteristics. Ap endo activity in the 62 of 0.15 mol/L. Both drugs were stored as single-use aliquots at tumors ranged 180-fold from 0.0028 to 0.50 fmol/cell/min À80jC. All drugs were diluted in the appropriate solvent immedi- (Table 1). The mean was 0.12 F 0.12 fmol/cell/min and did ately before use so that a constant volume was added for all doses. F No-drug controls received an equivalent volume of solvent. The final not differ between medulloblastomas and PNETs (0.11 0.13 F concentration of DMSO or ethanol was 1%. versus 0.13 0.13 fmol/cell/min), indicating that anatomic Alkylating agent survival was assayed as previously described in location (i.e., cerebellum versus cerebrum) did not affect detail (e.g., ref. 28). Six-well (35 mm) trays were inoculated with 2 mL activity. Newly operated and previously treated tumors (see of supplemented DMEM/F12 containing 1,000 to 2,000 cells trans- above) did not differ significantly in activity (0.11 F 0.12

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0.089 F 0.11 fmol/cell/min, P V 0.040). However, the female sample population had a greater proportion of infants (33% versus 5%) than the male, suggesting that the gender difference could reflect the effect of age on activity. To address this possibility, the relationship among Ap endo activity, age, and gender was further analyzed by using a multivariate model. Controlling for variation in age, activity remained 0.060 fmol/cell/min higher in females. Controlling for gender, the age-related decrease in activity was 0.0054 fmol/cell/min per year. The consistency of the age and gender relationships with activity when controlling for one another indicates that these relationships are independent. Ape1/Ref-1 is localized in tumor cell nuclei. Intracellular localization of APE1/Ref-1, the predominant Ap endo activity in human cells (18), was examined by immunohistochemistry in 42 medulloblastomas. As is characteristic of medulloblastomas, all tumors were highly cellular, composed of small, poorly differentiated, round cells that displaced rather than infiltrated surrounding normal parenchyma. Immunostaining was detected in all tumors examined, and as illustrated in

Fig. 1. Apurinic/apyrimidinic endonuclease (Ap endo) activity as function of age. Fig. 2, seemed almost exclusively nuclear in tumor cells. The regressionline shows decreased activity with age in pediatric medulloblastomas/ Between specimens, the average fraction of immunopositive PNETs from female (.)andmale(Â) patients. Also illustrated are the wide range of cells varied from f8% to 73% and differences in the relative Ap endo activity and the greater activity in tumor from females. intensity of staining were clearly discernible (e.g., Fig. 2A-F). However, within individual tumors, the distribution of stained cells and the intensity of staining displayed little variability versus 0.19 F 0.18 fmol/cell/min), suggesting that prior when different areas of the tumor were examined; that is, there therapy had no lasting effect on Ap endo activity. seemed no focal areas that differed in stained fraction or Regression analysis revealed a significant inverse associa- intensity. Moreover, the fraction of stained cells was strongly tion between activity and age (r = 0.262, P V 0.040), with correlated with relative staining intensity, estimated visually as activity decreasing 0.0070 fmol/cell/min per year (Fig. 1). +1 to +3 (P V 0.003). These findings suggest that the pattern of This decline with age, illustrated by the 2-fold higher activity expression of Ap endo activity may be uniform within a in infants compared with adolescents (Table 1), was observed tumor, a conclusion consistent with a near-significant associ- in medulloblastomas and PNETs separately, and in males ation between activity and the fraction of immunopositive and females (data not shown). Ap endo activity also differed cells (r = 0.314, P < 0.06). Immunoreactivity was largely significantly between genders (Table 1) as evidenced by an absent in adjacent normal cerebellum (Fig. 2G) with the f2-fold greater activity in females (0.16 F 0.14 versus exception of cytoplasmic staining of occasional Purkinje cells

Ta b l e 1. Ap endo activity by tumor and patient characteristics

Tu m o r s n Activity* Mean F SDRange Diagnosis All 62 0.12 F 0.12 0.0028-0.50 Medulloblastoma 52 0.11 F0.13 0.0028-0.50 PNET 10 0.13 F 0.13 0.0042-0.34 Treatment Newly operated 55 0.11 F0.12 0.0028-0.50 Previously treated 7 0.19 F 0.18 0.023-0.43 Gender Female 24 0.16 F 0.14 0.0042-0.45 Male 38 0.089 F 0.11 0.0028-0.50 Age Infants (<3.0 y) 10 0.17 F 0.14 0.0076-0.40 Children (z3.0, <12 .0 y) 37 0.12 F 0.13 0.0091-0.50 Adolescents (>12 y) 15 0.078 F 0.11 0.0028-0.34

*fmol abasic sites incised/cell/min.

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Fig. 2. Nuclear staining of Ape1/Ref-1in medulloblastoma cells. A-F, relative intensity of nuclear immunopositivity is compared at Â10 and Â40 power (original magnification) for weak (+1; A-B), moderate (+2; C-D ), and strong (+3; E-F) staining.Tumor nuclei expressing detectable Ape1/Ref-1are brown; negative nuclei are green (stained with 3,3V-diaminobenzidine tetrachloride and methyl green). G, Ape1/Ref-1immunostaining in a medulloblastoma and adjacent normal cerebellum. Low-power view shows a marked contrast between a fragment of tumor (left) adjacent to cerebellum (right), with diffuse staining of tumor cells at +3 intensity compared with nearly complete absence of staining in the normal cerebellum. H, cytoplasm of large Purkinje cells has intermediate staining, whereas rare cells of the inner granular layer (arrowhead) are positive. and, rarely, of cells of the inner granular layer (Fig. 2H). In 1.117;P < 0.0005). These data suggest that Ap endo activity accord, activity in two samples of normal cerebellum was 5.9- may be a useful predictor of progression following postoper- and 29-fold lower than that in adjacent medulloblastoma ative radiotherapy and/or chemotherapy in medulloblastoma/ (data not shown). PNETs and are consistent with the proposal that Ap endo Apurinic/apyrimidinic endonuclease activity is inversely activity mediates resistance to adjuvant therapy. associated with time to tumor progression following adjuvant Ape1/Ref-1 immunopositivity and time to tumor progression. therapy. To examine the association of Ap endo activity with Previous studies have suggested that the fraction of Ape1/Ref-1 response to postoperative treatment, we analyzed the associ- immunopositive cells is associated with treatment outcome or ation of activity with TTP after initiation of adjuvant therapy overall survival in diverse pediatric and adult (reviewed in in 46 medulloblastomas and four PNETs. All patients received craniospinal radiation (23.4-36 Gy) with additional focal irradiation to the tumor bed for a total dose of 54.4 to 58.8 Gy. Patients also received vincristine during radiation and Ta b l e 2 . HR estimates for the association of Ap endo during subsequent multiagent chemotherapy that included an activity with TTP following combined radiotherapy alkylating agent (1-(2-chloroethyl)-3-cyclohexyl-L-nitrosourea and chemotherapy and/or cyclophosphamide) and a platinating agent (cisplatin or carboplatin). Fourteen tumors progressed and 36 were Variable HR (95 % CI) P censored: median time at risk was 47.8 months. In a uni- Univariate analysis variate Cox proportional hazards regression model (Table 2) Apurinic/apyrimidinic 1.0 67 (1.0 2 9 -1.10 6) 0.0 01 with Ap endo activity entered as a continuous variable, the HR endonuclease* increased by a factor of 1.067 for every increase of 0.01 in Agec 0.972 (0.863-3.318) 0.80 activity (95% CI, 1.029-1.106; P < 0.0001). Thus, greater Male versus femaleb 1.218 (0.407-3.642) 0.72 apurinic/apyrimidinic endonuclease activity was associated, on Multivariate analysis average, with an increasing risk for progression (i.e., shorter Apurinic/apyrimidinic 1.073 (1.030-1.117) 0.001 TTP). For example, in our sample population, the tumor with endonuclease* f (0.501-0.0028)/0.01 the highest activity is 25-times [i.e., 1.067 ] Agec 1.048 (0.919-1.196) 0.49 more likely to progress than the lowest-activity tumor. Male versus femaleb 1.041 (0.342-3.166) 0.94 Adjusting for age and gender, variables that have been associated with treatment outcome and/or overall survival in *Per 0.01increase in activity. both pediatric and adult primary brain tumors (6–8), in a cPer1-y increase. multivariate analysis had no effect on the relationship bMale = 0, female = 1. between activity and TTP (HR, 1.073; 95% CI, 1.030-

www.aacrjournals.org 7409 Clin Cancer Res 2005;11(20) October 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. CancerTherapy: Clinical refs. 18, 19, 29) tumors. TTP following adjuvant radiotherapy temozolomide. Antisense oligonucleotide–mediated reduc- and chemotherapy was available for 37 of the 42 medullo- tion of Ap endo activity decreased LD10 for temozolomide blastomas (13 progressed) examined for Ape1/Ref-1 by immu- by 1.9-fold (Fig. 3D; Table 2). The enhanced sensitivity to nohistochemistry. Univariate analysis revealed no relationship temozolomide reflected an increased rate of killing (i.e., between TTP and the fraction of immunopositive cells (HR, lower D37), whereas DT was unchanged. 0.992; P < 0.61), the intensity of staining (HR, 0.998; P < 0.72), or the product of stained fraction and intensity Discussion (HR, 0.998; P < 0.68). Apurinic/apyrimidinic endonuclease activity is associated with Improvements in adjuvant radiotherapy and chemotherapy time to tumor progression following adjuvant therapy within have greatly increased survival for pediatric medulloblasto- both high-risk and average-risk medulloblastomas. Newly mas/PNETs (1, 2). However, the benefit is not equally diagnosed medulloblastomas can be stratified into high risk afforded to all patients, and postoperative radiotherapy is and average risk for treatment failure and recurrence based on frequently accompanied by physical and neuropsychological patient age, extent of tumor resection, and dissemination to detriments (11). Although clinical experience has identified other sites in the central nervous system (6, 7, 8). In our patient and tumor features that are associated with overall study, high-risk tumors were defined using the Children’s survival, there are few molecular markers that predict Cancer Study Group criteria; that is, high-risk patients are response to the combined radiotherapy and chemotherapy those with >1.5 cm3 residual tumor after surgery, are younger used to treat medulloblastomas/PNETs. The cytotoxicity than 3 years, and have evidence of tumor dissemination to of unrepaired abasic sites induced by radiation and alkylators other sites (7). In accord with the increasing hazard for (12, 13), and the evidence that Ap endo activity mediates progression associated with increasing Ap endo activity, the resistance of human gliomas to treatment with these agents mean Ap endo activity in 20 high-risk tumors in our sample (20–22), led us to hypothesize that Ap endo activity is was twice that of 29 average-risk tumors (0.18 F 0.18 versus a mechanistically relevant predictor of response to adjuvant 0.083 F 0.14 fmol/cell/min; P V 0.05). Univariate analysis therapy in medulloblastomas/PNETs. The work reported here revealed a significant inverse relationship between activity and substantiates our hypothesis, shows a functional basis for TTP in both high-risk tumors (HR, 1.149; 95% CI, 1.016- the association of Ap endo activity with recurrence, and 1.300; P = 0.027) and average-risk tumors (HR, 1.076; 95% suggests that apurinic/apyrimidinic endonuclease activity may CI, 1.013-1.144; P = 0.018), indicating the use of Ap endo in be useful as a predictor of treatment outcome and as a target predicting treatment outcome within each risk group. for antiresistance therapies. Suppression of apurinic/apyrimidinic endonuclease activity Support for our hypothesis is provided by the strong, increases alkylator killing of medulloblastoma cells. The inverse association between Ap endo activity and TTP association between Ap endo activity and TTP following following combined radiotherapy and chemotherapy that postoperative therapy suggests that repair of abasic sites included an alkylating and/or a platinating agent (Table 2). promotes medulloblastoma/PNET resistance to radiation and/ Based on biochemical, genetic and cell/molecular biological or alkylating agents in situ. To address this hypothesis, studies, particularly in human glioma cells (20–22), the alkylator sensitivity was determined by clonogenic colony- relationship between activity and TTP likely reflects repair of forming assay following suppression of Ap endo activity in abasic sites, at least in part. Both radiation and alkylating the human medulloblastoma–derived cell line UW228-2. agents induce baseless sites via DNA glycosylase–mediated The effect of transfection with either antisense oligonucleo- excision of oxidized and alkylated purines and pyrimidines tides or siRNA targeting Ape1/Ref-1 on Ap endo activity, (12, 13). In addition, radiation-induced oxidative free Ape1/Ref-1 content, and alkylator sensitivity is illustrated in radicals can induce abasic sites by direct attack on deoxy- Fig. 3. Antisense oligonucleotide treatment reduced activity ribose (12). Unrepaired abasic sites are lethal, most likely a 3.8-fold (0.23 F 0.044 to 0.061 F 0.022 fmol/cell/min) in consequence of their ability to strongly impede the progress three separate experiments, whereas transfection with siRNA of DNA replication (15–17). The contribution of abasic sites reduced activity 5.4-fold (0.21 F 0.048 to 0.039 F 0.017 to the cytotoxicity of radiation and alkylating agents is fmol/cell/min) in four separate experiments. In accord, evidenced by the hypersensitivity of bacterial, yeast, and Western analysis (Fig. 3B) revealed reduced abundance of mammalian cells that are deficient in repair of this lesion Ape1/Ref-1, evident within 30 hours after transfection with (reviewed in refs. 12, 17–19). Moreover, suppression of antisense oligonucleotides or siRNA. Suppression of Ap endo Ape1/Ref-1, the major human Ap endo activity (18), results activity was accompanied by significantly greater sensitivity to in hypersensitivity to oxidizing and alkylating agents in both the clinical chloroethylating agent BCNU, as illustrated in rat (30) and human adult glioma cells (21)8 Similar findings Fig. 3C for siRNA. As summarized in Table 3, treatment with have been reported for a variety of additional mammalian antisense oligonucleotides or siRNA yielded 2.4- and 1.8-fold cell lines (e.g., refs. 27, 31, 32). The association between Ap reductions, respectively, in LD10 for BCNU, relative to cells endo activity and TTP may also reflect mechanisms in transfected with control sequences. Greater sensitivity addition to, or other than, excision of abasic sites. Ape1/ reflected (a) 2.3- and 1.3-fold reductions in DT, respectively, Ref-1 has multiple catalytic activities in vitro, including indicating that apurinic/apyrimidinic endonuclease contrib- 3V-phosphodiesterase, 3V-phosphatase, and 3V-exonuclease utes to insensitivity to low doses of BCNU and (b) increased (18, 19) that may promote resistance to radiation damage rates of killing, as evidenced by 2.2- and 1.9-fold lower D37 values, respectively. Suppression of Ap endo activity also yielded enhanced sensitivity to the clinical methylator 8 J.R. Silber and M.S. Bobola, unpublished results.

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Fig. 3. Antisense oligonucleotides (ASO) or siRNA suppression of apurinic/ apyrimidinic endonuclease (Ap endo) activity and Ape1/Ref-1content produces greater alkylator sensitivity in the medulloblastoma cell line UW228-2. A, subconfluent cells were transfected with antisense oligonucleotides (Â), siRNA (5), or control oligomers (.) as described in Materials and Methods. After incubation with oligomers for 72 hours, cells were harvested and Ap endo activity was determined in a plasmid-nicking assay that monitors conversion from supercoiled to relaxed DNA. B, Ape1/Ref-1protein content at 30, 48, and 72 hours after transfection with antisense oligonucleotides (top) or siRNA (bottom) assessed by immunoblotting with chemiluminescent detection. Abbreviations: Con, cells transfected with control oligomers. Mr, molecular weight standards. C, BCNU sensitivity determined by clonogenic assay 72 hours after transfection with siRNA (Â) or control oligomer (o). Each point is a single determination at each drug dose. D, temozolomide (TMZ ) sensitivity determined by clonogenic assay 72 hours after transfection with antisense oligonucleotides (Â) and control oligomer (o). Each point is a single determination at each drug dose. Panels A-D show representative results from different single experiments.

by excising fragmented deoxyribose phosphate moieties at proapoptotic activities of p53 (33). Thus, it is possible that single-strand breaks (12). In addition, the reduction-oxida- multiple redox-sensitive proteins that are mechanistically tion activity Ref-1, located at the NH2 terminus of Ape1/Ref-1 associated with Ref-1 contribute to the associations with (18), mediates signal transduction in response to radiation outcome that we observed here. Whatever the case, involve- and other genotoxic stress (18, 19). For example, Ref-1 has ment of the DNA repair activity of Ape1/Ref-1 is supported been implicated in regulating the transactivation and by the finding that a chimeric protein containing Ap endo

Ta b l e 3 . Suppression of Ape1/Ref-1-catalyzed Ap endo activity increases alkylator cytotoxicity in human medulloblastoma cells

Antisense siRNA Control (Mmol/L) ASO (Mmol/L) Con/ASO P Control (Mmol/L) siRNA (Mmol/L) Con/siRNA P

BCNU

LD10 85 F 2.9 36 F 1. 9 2 . 4 V0.001 79 F 2.3 44 F 1. 3 1. 8 V0.001

DT 27 F 2.0 12 F 2.5 2.3 V0.03 20 F 2.4 15 F 2.0 1.3 V0.1

D37 22 F 2.8 10 F 1.8 2.2 V0.001 25 F 1. 613 F 1. 4 1. 9 V0.001 Te m o z o l o m i d e

LD10 460 F 9.8 242 F 9.0 1.9 0.001

DT 12 9 F 10 130 F 14 1 0.77

D37 143 F 7.3 45 F 10 3.2 0.001

Abbreviation: ASO, antisense oligonucleotides.

www.aacrjournals.org 7411 Clin Cancer Res 2005;11(20) October 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. CancerTherapy: Clinical and O6-methylguanine-DNA methyltransferase activities, but high risk versus low risk in medulloblastomas) is associated no Ref-1, conferred greater alkylation resistance to human with significantly higher mean activity. Correlation with degree cells than O6-methylguanine-DNA methyltransferase activity of malignancy may reflect elevated oxidative stress accompa- alone (34). These survival-promoting functions of Ref-1 may nying increased rates of proliferation and/or tumor cell be especially important in promoting resistance to multiagent migration. As also observed for an adult glioma line (21), chemotherapy protocols such as those used to treat medul- suppression of Ape1/Ref-1 expression in the medulloblastoma loblastomas/PNETs that include DNA-damaging agents that line UW228-2 increased killing by the clinical alkylators BCNU do not produce abasic sites or oxidative lesions (e.g., and temozolomide. Finally, in both medulloblastomas/PNETs cisplatin) and cytotoxic drugs that do not damage DNA and adult gliomas, there is a strong significant inverse (e.g., vincristine). association between Ap endo activity and TTP following Additional evidence associating Ap endo activity with adjuvant therapy with radiation and/or alkylating agent–based clinical outcome following postoperative treatment in medul- chemotherapy (both modalities combined in the present study, loblastoma is the difference in mean activity between tumors and each modality separately in gliomas). It is important to that are average risk and high risk for recurrence. One note that, in our sample populations, the hazard for criterion of higher risk, age of <3 years (i.e., infants), is progression differed by 25-fold within the range of Ap endo associated with significantly greater Ap endo activity com- activities observed in 52 medulloblastomas/PNETs and by 13- pared with older children and adolescents (Table 1). The to 14-fold within the range of activities observed in 30 and 44 worse outcome observed for infants has been attributed to adult gliomas treated with either alkylators or radiotherapy, restriction of adjuvant treatment to chemotherapy to avoid respectively. Taken together, our results suggest that Ap endo the severe morbidity caused by radiotherapy in the very may contribute to resistance to postoperative radio/chemo- young (2). Our data suggest that greater Ap endo activity/ therapy and provide a predictor of outcome in a variety of Ape1/Ref-1 content could also contribute to the poor prog- primary brain tumors. nosis in infants by promoting chemoresistance. In children We observed extensive variation between medulloblastomas older than 3 years and adolescents, Ap endo activity is also in the fraction of cells that were immunopositive for Ape1/ greater in high-risk medulloblastoma (0.17 F 0.17 fmol/cell/ Ref-1 (f8-73%) and in the intensity of staining scored on a min) versus average-risk medulloblastoma (0.083 F 0.14 scale of +1 to +3 (e.g., Fig. 2). This variability is consistent fmol/cell/min). In these patients, high risk is defined by with the 180-fold range in Ap endo activity measured in our tumor invasion of surrounding brain that prevents gross total biochemical assay. The staining pattern seemed uniform within resection or dissemination of tumor elsewhere in the neuro- each of the 42 tumors examined, and there was a strong axis (7). Our data indicate that elevation of Ap endo activity correlation between fraction of stained cells and staining accompanies and/or promotes acquisition of these aggressive intensity, as well as a nearly significant correlation between characteristics. fraction of stained cells and activity. Ape1/Ref-1 was detectable We observed that Ap endo activity is associated with age and only in nuclei, as has been observed for some, but not all tumors gender in medulloblastomas/PNETs. The inverse correlation (18, 19). Lack of cytoplasmic staining may reflect the small, between activity and age (Fig. 1) may reflect processes round cell morphology and large nuclear volume that are associated with physical and functional maturation of the histologic hallmarks of medulloblastomas. More intriguing are brain that are intrinsic to the progenitor cells of medulloblas- the mechanisms that may account for the variability in stained tomas/PNETs. Alternatively, declining tumor Ap endo activity cell fraction and intensity. Numerous studies have implicated with age may reflect reduction in exogenous stress in the oxidative stress as a modulator of Ap endo activity and Ape/ surrounding brain associated with cessation of proliferation of Ref-1 content in cell lines and tissue (19). It is possible that the neural cell progenitors and neuronal maturation. Notably, intertumor heterogeneity in immunopositivity reflects oxidative reduction of Ape1/Ref-1 mRNA detected by in situ hybridiza- stress associated either with cell proliferation or episodes of tion accompanies the maturation of developing brain into transient hypoxia due to inadequate/impaired vasculature. An adulthood in rodents (35, 36). Possible mechanisms underly- association with Ape1/Ref-1 content, assessed by immunohis- ing the 2-fold higher activity in females are less evident. Several tochemistry, with treatment outcome has been reported for studies have shown hormonal and trophic factor regulation of some human tumors, including head and neck cancer, germ cell Ape1/Ref-1 mRNA and protein expression in human and tumors, cervical cancer, ovarian carcinoma, and osteosarcomas rodent cell lines (37–39), suggesting that estrogenic hormones (reviewed in refs. 18, 19, 27, 40). In contrast to these studies, a responsible for sexual differentiation in the brain may stim- Cox univariate model did not reveal a significant relationship ulate Ape1/Ref-1 expression. between either the fraction of Ape1/Ref-1-immunopositive cells, It is of interest to relate our findings for medulloblastomas/ the intensity of staining, or their product, and TTP following PNETs to those for adult gliomas (20–22). Medulloblastomas/ adjuvant treatment for medulloblastomas. However, this lack of PNETs differ from adult gliomas in biological and clinical association may reflect the relatively small number of tumors properties, particularly in their greater responsiveness to examined, the low proportion that had progressed (35%), and/ radiation and chemotherapy. Despite these differences, we or the relative insensitivity of a discontinuous variable (staining found that mean Ap endo activity for medulloblastomas/ intensity) versus a continuous variable (activity). Nonetheless, PNETs (0.12 F 0.12 fmol/cell/min) was comparable with that Ap endo activity permitted detection of an association for a for high-grade gliomas (0.090 F 0.11 fmol/cell/min; ref. 22). similar sample size and fraction of treatment failures, suggesting However, we observed no association of activity with age or that activity may be a more sensitive predictor of clinical gender in gliomas. In both groups of tumors, increasing outcome. Increased nuclear Ape1/Ref-1 content and localization aggressiveness (high grade versus low grade in gliomas and has been observed to accompany malignant progression in

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ovarian carcinomas (40), suggesting that the nuclear localiza- significant increases in alkylator sensitivity in vitro (Fig. 3; tion of Ape1/Ref-1 may reflect the malignant character of ref. 20), in contrast to the complete and prolonged ablation of medulloblastomas. MGMT required to produce enhanced alkylator cytotoxicity Our results indicate that Ap endo activity may have utility in (41). A number of low molecular weight compounds have guiding postoperative radiation and chemotherapy in medul- been described that inhibit the excision of abasic sites (14, 42). loblastoma/PNET. High-activity tumors may be candidates for For example, lucanthone, a clinically used antischistosome, novel, potentially more effective, treatments (e.g., immuno- inhibits the Ap endo activity of human Ape1/Ref-1 in vitro and therapy) that spare patients the systemic toxicity caused by increases the abundance of abasic sites in HeLa cells (42). radiation and DNA-damaging agents. Alternatively, high Lucanthone also sensitizes breast cancer cells to methylating activity may identify tumors that are amenable to anti- agents (43) and adult glioma cells8 to both alkylating and resistance therapies targeting Ape1/Ref-1. Numerous studies oxidizing agents. More importantly, lucanthone has been have shown hypersensitivity to oxidizing and alkylating agents observed to accelerate the regression of brain metastases treated accompanying suppression of Ap endo activity and/or Ape/ with radiation, a result attributed to decreased repair of abasic Ref-1 protein level in a variety of tumor cell lines (e.g., refs. 21, sites (44). These findings suggest that antiresistance therapies 27, 30, 31). The potentiation of alkylator cytotoxicity effected targeting Ap endo activity are pharmacologically tractable and by antisense- and/or siRNA-mediated suppression of Ap endo potentially efficacious. activity and Ape1/Ref-1 protein content that we observed here in the medulloblastoma line UW228-2 and earlier in an the adult glioma line (21) provide proof of principal of the Acknowledgments

efficacy and clinical potential of strategies to circumvent We thank Dr. A. Blank for critical reading of the article, Dr. Kristy Seidel for guid- radiotherapy and chemoresistance in primary brain tumors. ance with statistical analysis, and Bobby Stevens and Douglas Kolstoe for technical Importantly, partial elimination of Ap endo activity results in assistance.

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Michael S. Bobola, Laura S. Finn, Richard G. Ellenbogen, et al.

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