Clinical Cancer Research Targets Advances in Breast Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bobola, M. S.
Right arrow Articles by Silber, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bobola, M. S.
Right arrow Articles by Silber, J. R.
Clinical Cancer Research Vol. 11, 7405-7414, October 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Clinical

Apurinic/Apyrimidinic Endonuclease Activity Is Associated with Response to Radiation and Chemotherapy in Medulloblastoma and Primitive Neuroectodermal Tumors

Michael S. Bobola1,4, Laura S. Finn3,5, Richard G. Ellenbogen1,4, J. Russell Geyer2,6, Mitchel S. Berger7, Justin M. Braga1, Elizabeth H. Meade1,4, Mary E. Gross1 and John R. Silber4

Authors' Affiliations: 1 Division of Neurosurgery, Department of Surgery; 2 Division of Hematology/Oncology; and 3 Department of Laboratories, Children's Hospital and Regional Medical Center; Departments of 4 Neurological Surgery, 5 Pathology and 6 Pediatrics, University of Washington, Seattle, Washington; and 7 Department of Neurological Surgery, University of California, San Francisco, California

Requests for reprints: Michael S. Bobola, Division of Neurosurgery, Department of Surgery, Children's Hospital and Regional Medical Center, Seattle, WA 98105. Phone: 206-987-2046; Fax: 206-527-3925; E-mail: michael.bobola{at}seattleschildren.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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 21 years 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-1 was 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 46 medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P ≤ 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 with TTP 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 (PNET) are highly malignant (i.e., WHO grade 4) cancers that account for 20% to 25% of the ~2,200 pediatric primary brain tumors diagnosed annually in the United States (1, 2). The median age at diagnosis is about 9 years, with the majority of cases (62%) occurring in males (3). Medulloblastomas and PNETs also occur infrequently in younger adults between 20 and 40 years of age (4). These malignancies, categorized as embryonal tumors in the current WHO classification (5), are histologically similar, being composed of small, morphologically undifferentiated cells of uncertain histiogenesis that occasionally contain focal areas displaying morphologic and immunohistochemical features of mature neurons or glia (5, 6). Although emerging evidence suggests that medulloblastomas and PNETs harbor distinctive genetic aberrations (6), these tumors are primarily distinguished by their anatomic sites of occurrence: medulloblastomas arise in the cerebellum, whereas PNETs occur almost exclusively in the cerebral hemispheres.

Improvements in surgical technique and the development of effective postoperative treatments have dramatically increased survival rates for medulloblastomas in the last 30 years (1, 2). Better outcome has also been achieved by using patient and tumor characteristics to stratify medulloblastomas into average-risk and high-risk groups that differ in predicted response and likelihood of recurrence (6–8). The current standard of care is surgical excision to the greatest extent possible followed by adjuvant radiotherapy and chemotherapy. For patients receiving this care, overall 5-year survival rates range from 60% to 85% for average-risk medulloblastomas to about 40% for high-risk medulloblastomas (6, 7, 9). In contrast, PNETs are more aggressive and less responsive, with 5-year survival rates of <20% (10). Unfortunately, long-term survival is frequently accompanied by detrimental physical and neuropsychological sequelae produced by adjuvant radiotherapy and chemotherapy, especially in infants and young children (11). Moreover, there is no therapy that produces long-term remission in recurrent disease (2, 7, 10).

In light of the detrimental long-term effects of adjuvant therapy and the poor prognosis for the recurrent tumors that afflict ca. 50% of patients (2, 7, 10), development of more effective approaches to treating medulloblastomas/PNETs is an urgent priority. Little is known about the molecular mechanisms that underlie adjuvant therapy failure. Delineating resistance mechanisms will likely suggest new strategies to improve the efficacy of existing therapies and provide new markers of clinical outcome. As a group, medulloblastomas/PNETs are relatively responsive to radiation and chemotherapeutic agents that damage DNA (2), suggesting that DNA repair may be a determinant of tumor response. Ionizing radiation and the nitrosourea- and nitrogen mustard–based alkylating agents used to treat medulloblastomas/PNETs produce cytotoxic abasic sites (apurinic/apyrimidinic sites) in DNA (12–14). Abasic sites, which constitute a major lethal lesion, block progression of replicative DNA polymerases and are believed to cause replication forks to stall and/or collapse (15–17). The vast majority of abasic site repair in mammalian cells is catalyzed by Ape1/Ref-1, an abundant enzyme possessing a strong apurinic/apyrimidinic endonuclease (Ap endo) activity that hydrolyzes the phosphodiester bond 5' to baseless sites (12, 17–19).

Our recent evidence indicates that the Ap endo activity of Ape1/Ref-1 mediates resistance to adjuvant radiation and alkylating agent–based chemotherapy in adult gliomas (20–22). We have observed a strong inverse correlation between glioma Ap endo activity and time to tumor progression (TTP) following either radiation or alkylator therapy (20); that is, the higher the activity, the shorter the time to recurrence. We have also found that suppressing Ap endo activity, and Ape1/Ref-1 content, in a human adult glioma cell line reduces resistance to the clinical alkylators 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (21). A concurrent increase in abasic sites supports the conclusion that abasic site repair promotes resistance and shows a functional basis for the in vivo findings. Lastly, Ap endo activity in human gliomas is positively correlated with tumor characteristics associated with greater malignancy and poor response (22). These findings in adult gliomas led us to assess the contribution of Ap endo activity to the resistance of pediatric brain tumors to adjuvant therapy. Here, we report an initial study of 52 medulloblastomas and 10 PNETs in children and young adults. Our results suggest that Ap endo activity promotes resistance to combined radiation and chemotherapy containing alkylating and/or platinating agents and is predictive of outcome following this adjuvant therapy in medulloblastomas/PNETs.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue. Tumors were obtained with informed consent from patients operated at the Children's Hospital and Regional Medical Research Center and the University of Washington Medical Center. Care was taken to ensure that tumor specimens were distant from the gross interface with normal tissue. All tissues were reviewed by a neuropathologist and diagnosis was obtained from the final neuropathology report. Tissue and demographic information was obtained in accordance with protocols approved by the Institutional Review Boards at Children's Hospital and Regional Medical Research Center and the University of Washington Medical Center. Immediately upon resection, tissue was placed in ice-cold DMEM/F12 supplemented with 5% fetal bovine serum and transported to the laboratory. The precautions taken to preserve tissue viability and enzymatic activity during transport of specimens and the procedure for determining cell number are described elsewhere (23).

Apurinic/apyrimidinic endonuclease activity. Ap endo activity was quantitated in whole cell extracts as we have previously described for human brain tumors (22), brain tumor–derived cell lines (21), and normal brain (22). The highly sensitive assay measures conversion of acid-treated, supercoiled plasmid DNA to relaxed form caused by incision at an abasic site (24). Briefly, tissue was rinsed in PBS, weighed, and minced into small pieces with scalpel blades. Tissue fragments were resuspended in PBS and passed serially through 16-, 18-, and 20-gauge needles to produce a single cell suspension. Cells were pelleted by centrifugation at 2,000 x g for 5 minutes at 4°C and suspended at ~1 x 107 cells/mL in 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 100 mmol/L NaCl, 0.1 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL each of aprotinin, leupeptin, and pepstatin. Cell suspensions were sonicated on ice for four 15-second intervals, and debris was pelleted by centrifugation at 10,000 x g for 5 minutes at 4°C. In some instances, extraction was done in 1.5x Ap endo reaction buffer [75 mmol/L HEPES (pH 7.5), 225 mmol/L KCl, 7.5 mmol/L MgCl2, 0.75 mmol/L CoCl2, and 150 µg/mL bovine serum albumin]. Experiments with pediatric brain tumor cell lines revealed identical Ap endo activities for extracts prepared in both extraction buffers.

Assay mixtures (30 µL) contained 0.033 µg/mL depurinated pKT100 plasmid DNA (3.5 kb), containing, on average, 1.5 abasic sites per molecule, 50 mmol/L HEPES (pH 7.5), 150 mmol/L KCl, 5 mmol/L MgCl2, 0.5 mmol/L CoCl2, 100 µg/mL bovine serum albumin, and extract equivalent to 10 to 104 cells. Preparation of the DNA substrate is described in ref. 22. After incubation for 10 minutes at 37°C, reaction products were resolved on an 0.8% agarose gel in 40 mmol/L Tris-acetate, 2 mmol/L EDTA. The gel was stained with ethidium bromide to visualize supercoiled and nicked, relaxed plasmid DNA and was photographed with a Kodak DC290 digital camera. Band density was quantitated using Kodak 1D version 3.5.4 image processing software, with HindIII-linearized pKT100 as a standard. Activity (fmol abasic sites incised/cell/min, abbreviated to fmol/cell/min) is the mean of at least three separate determinations that differed, in general, by <15%, and in all cases, by <30%. Each determination comprised assay of increasing amounts of sample and yielded activity calculated by regression analysis of points on the linear portion of the curve. Normalizing activity to cell number affords a uniform basis for comparing tissues of different histology, avoiding potential problems attributable to tissue- and tumor-specific differences in the amount and composition of intracellular and extracellular proteins (e.g., ref. 25).

Immunohistochemistry. Immunoperoxidase staining was done on paraffin blocks of formalin-fixed tumors. Briefly, 7-µm tissue sections were mounted on 3-aminopropyltriethoxysilane-treated slides, dewaxed, and rinsed twice in 100% ethanol followed by treatment with 0.6% hydrogen peroxide/methanol to quench endogenous peroxidase. Slides were rehydrated by serial rinses in 95% ethanol, 80% ethanol 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% trypsin in TBS followed by 3 x 5-minute washes with TBS. Slides were incubated with 10% goat serum and 3% bovine calf serum in TBS for 1 hour at room temperature and then incubated overnight at 4°C with a 1:5,000 dilution of a mouse monoclonal anti-Ape1/Ref-1 antibody (NB100-116B1, Novus, Littleton, CO) in TBS containing 10% goat serum. Slides were washed in TBS and incubated with a 1:100 dilution of a biotin-conjugated goat anti-mouse IgG antibody (BA-9200, Vector, Burlingame, CA) for 1 hour at room temperature. After washing with TBS, the slides are incubated for 30 minutes at room temperature with strepavidin-conjugated horseradish peroxidase and washed again with TBS. Primary antibody binding is visualized by incubation with 3,3'-diaminobenzidine tetrachloride until a satisfactory signal develops. Slides were counterstained with methyl green or Gill's hematoxylin. Negative controls were treated identically except for omission of the primary antibody. Stained slides were scanned at low power to evaluate regional variation. One hundred cells from five regions were counted and the average percent of positive cells for each tumor was calculated. Both nuclear and cytoplasmic staining were considered positive. Staining in any fragments of normal brain was also assessed.

Suppression of apurinic/apyrimidinic endonuclease activity and alkylating agent sensitivity. Ap endo activity in the human medulloblastoma-derived cell line UW228-2 (26) was suppressed by transfection with either antisense oligonucleotides or siRNA targeting Ape1/Ref-1. For transfection with antisense oligonucleotides, separate 50-µL aliquots of unsupplemented Opti-MEM (Gibco, Invitrogen, Carlsbad, CA) were mixed with either 2.9 nmol of an antisense oligonucleotide complementary to the translation start site (5'-TTTCCCACGCTTCGGCATTCC-3') or with 15 µL of cationic lipid (Lipofectin, Life Technologies) and held at room temperature for 30 minutes. The antisense oligonucleotide and cationic lipid mixtures were gently combined and held overnight at 4°C. Subconfluent cultures of UW228-2 incubated at 37°C in humidified 95%/5% air/CO2 overnight in 3 mL of Opti-MEM containing 5% iron-supplemented bovine serum in a 35-mm dish were washed once with unsupplemented Opti-MEM before adding the antisense oligonucleotide mixture. Cells were incubated with antisense oligonucleotides for 18 hours before adding 2 mL of Opti-MEM containing with 10% iron-supplemented bovine serum and continuing incubation. Final antisense oligonucleotide concentration was 1 µmol/L. Seventy-two hours after initiating transfection, cells were harvested by trypsinization for determination of alkylator sensitivity and assay of Ap endo activity. Transfection with a random oligomer composed of the same nucleotide composition as the antisense oligonucleotide served as a control.

A protocol similar to that described by Wang et al. (27) was used for transfection with siRNA. In separate tubes, 1.6 nmol of the siRNA (5'-CUUCAGGAGCUGCCUGGACUC-3', 3'-UUGAAGUCCUCGACGGACCUG-5', corresponding to nucleotides 532-552 of the human Ape1/Ref-1 cDNA sequence) was mixed with 0.1 mL of unsupplemented Opti-MEM, and 15 µL of cationic lipid was mixed with 0.4 mL Opti-MEM. After 10 minutes at room temperature, the two mixtures were combined and added to 35-mm dishes of UW228-2 grown as described above. Cells were incubated with siRNA for 4 to 18 hours before adding 2 mL of Opti-MEM containing 10% iron-supplemented bovine serum and continuing incubation; suppression of Ap endo activity or effect on alkylator sensitivity was unaffected by length of incubation with siRNA. Final siRNA concentration was 320 nmol/L. At 72 hours after initiating transfection, cells were harvested by trypsinization for determination of alkylator sensitivity and assay of Ap endo activity. Transfection with an inactive siRNA duplex (5'-AAUGUGGAUGGGCUUCGAGCC-3', 3'-CCUUACACCUACCCGAAGCUC-5', corresponding to nucleotides 412-432 of the human Ape1/Ref-1 cDNA sequence) served as a control.

Alkylating agent survival. BCNU and temozolomide were obtained from the pharmacies of the University of Washington Medical Center and Children's Hospital and Regional Medical Center, respectively. BCNU was dissolved in absolute ethanol at a concentration of 1 mol/L; temozolomide was dissolved in DMSO at a concentration of 0.15 mol/L. Both drugs were stored as single-use aliquots at –80°C. All drugs were diluted in the appropriate solvent immediately before use so that a constant volume was added for all doses. No-drug controls received an equivalent volume of solvent. The final concentration of DMSO or ethanol was 1%.

Alkylating agent survival was assayed as previously described in detail (e.g., ref. 28). Six-well (35 mm) trays were inoculated with 2 mL of supplemented DMEM/F12 containing 1,000 to 2,000 cells transfected with antisense oligonucleotides or siRNA. The trays were incubated for 6 to 8 hours to allow cells to attach and resume proliferation before further incubation with alkylating agent for 1 hour. In each experiment, single or duplicate wells of cells were exposed to at least 10 drug doses that serially increased by 10% to 20% increments. Alkylator sensitivity was assayed in two and three independent experiments for temozolomide and BCNU, respectively; each experiment employing different drug doses. The cells were washed free of residual alkylator and incubated in fresh, supplemented medium for 5 to 7 days to allow formation of colonies. The colonies were stained with 0.5% methylene blue in 1:1 methanol/H2O (v/v) to aid visualization during counting by light microscopy. Colonies containing ≥50 cells were counted. The colony-forming efficiency of untreated UW228-2 was ~25%.

Drug sensitivity was determined by analysis of survival curves (log surviving fraction versus dose) using standard methods, as we have previously described in detail (e.g., Fig. 1 of ref. 28). The survival variables LD10 (the dose required to reduce overall survival to 10%), DT (the threshold dose below which cells are insensitive to alkylator cytotoxicity), and D37 (the rate of killing as indicated by the slope of the linear portion of the survival curve) were derived by regression analysis of the linear portion of a composite kill curve derived from the replicate assays. The three survival variables are presented as mean ± SE and provide a complete description of alkylator sensitivity.



View larger version (13K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Apurinic/apyrimidinic endonuclease (Ap endo) activity as function of age. The regression line shows decreased activity with age in pediatric medulloblastomas/PNETs from female ({bullet}) and male (x) patients. Also illustrated are the wide range of Ap endo activity and the greater activity in tumor from females.

 
Statistical analysis. Data analysis and statistical procedures were done using Microsoft Excel and the statistics program Intercooled Stata 8 (Stata Corp., College Station, TX). Comparison of means was by Student's t test assuming unequal variances. Relationships between continuous variables were assessed by regression analysis. The association of Ap endo activity with TTP was assessed by standard methods. In these analyses, the outcome variable TTP was assessed by radiologic imaging. Tumor progression was defined as appearance of tumor growth in the case of gross total resection: increase in the largest dimension of residual tumor by at least 25% and/or tumor growth at a different site. Observations were censored at the last documented follow-up time if progression was not yet observed. Mean TTP was determined by the method of Kaplan and Meier. The hazard ratio (HR) for tumor progression as a function of Ap endo activity was calculated by using Cox regression analysis. Ap endo was entered into the regression models as a continuous variable and scaled (i.e., multiplied by 100) so that the tabulated HR represents the relative change in hazard for a 0.01 unit change in measured Ap endo activity. Possible confounding by age and gender was examined by using multivariate Cox regression analysis. Statistically significant relationships were determined at the 95% confidence level (95% CI).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Patient and tumor characteristics. Fifty-two medulloblastomas and 10 cerebral PNETs were obtained from patients ranging in age from 4 days to 21 years (mean ± SD, 8.4 ± 5.0 years). The majority of tumors occurred in males (61%), in accord with previous observations of gender bias (3). Fifty-five tumors were newly diagnosed, two were recurrent after surgery, one after surgery and chemotherapy, and four after surgery, radiation, and chemotherapy.

Association of apurinic/apyrimidinic endonuclease activity with tumor and patient characteristics. Ap endo activity in the 62 tumors ranged 180-fold from 0.0028 to 0.50 fmol/cell/min (Table 1). The mean was 0.12 ± 0.12 fmol/cell/min and did not differ between medulloblastomas and PNETs (0.11 ± 0.13 versus 0.13 ± 0.13 fmol/cell/min), indicating that anatomic location (i.e., cerebellum versus cerebrum) did not affect activity. Newly operated and previously treated tumors (see above) did not differ significantly in activity (0.11 ± 0.12 versus 0.19 ± 0.18 fmol/cell/min), suggesting that prior therapy had no lasting effect on Ap endo activity.


View this table:
[in this window]
[in a new window]

  		Table 1. Ap endo activity by tumor and patient characteristics

 
Regression analysis revealed a significant inverse association between activity and age (r = 0.262, P ≤ 0.040), with activity decreasing 0.0070 fmol/cell/min per year (Fig. 1). This decline with age, illustrated by the 2-fold higher activity in infants compared with adolescents (Table 1), was observed in medulloblastomas and PNETs separately, and in males and females (data not shown). Ap endo activity also differed significantly between genders (Table 1) as evidenced by an ~2-fold greater activity in females (0.16 ± 0.14 versus 0.089 ± 0.11 fmol/cell/min, P ≤ 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. 2, seemed almost exclusively nuclear in tumor cells. Between specimens, the average fraction of immunopositive cells varied from ~8% to 73% and differences in the relative 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 when different areas of the tumor were examined; that is, there seemed no focal areas that differed in stained fraction or intensity. Moreover, the fraction of stained cells was strongly correlated with relative staining intensity, estimated visually as +1 to +3 (P ≤ 0.003). These findings suggest that the pattern of expression of Ap endo activity may be uniform within a tumor, a conclusion consistent with a near-significant association between activity and the fraction of immunopositive cells (r = 0.314, P < 0.06). Immunoreactivity was largely absent in adjacent normal cerebellum (Fig. 2G) with the exception of cytoplasmic staining of occasional Purkinje cells and, rarely, of cells of the inner granular layer (Fig. 2H). In accord, activity in two samples of normal cerebellum was 5.9- and 29-fold lower than that in adjacent medulloblastoma (data not shown).



View larger version (76K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Nuclear staining of Ape1/Ref-1 in medulloblastoma cells. A-F, relative intensity of nuclear immunopositivity is compared at x10 and x40 power (original magnification) for weak (+1; A-B), moderate (+2; C-D), and strong (+3; E-F) staining. Tumor nuclei expressing detectable Ape1/Ref-1 are brown; negative nuclei are green (stained with 3,3'-diaminobenzidine tetrachloride and methyl green). G, Ape1/Ref-1 immunostaining 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.

 
Apurinic/apyrimidinic endonuclease activity is inversely associated with time to tumor progression following adjuvant therapy. To examine the association of Ap endo activity with response to postoperative treatment, we analyzed the association of activity with TTP after initiation of adjuvant therapy 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 during subsequent multiagent chemotherapy that included an alkylating agent (1-(2-chloroethyl)-3-cyclohexyl-L-nitrosourea and/or cyclophosphamide) and a platinating agent (cisplatin or carboplatin). Fourteen tumors progressed and 36 were censored: median time at risk was 47.8 months. In a univariate Cox proportional hazards regression model (Table 2) with Ap endo activity entered as a continuous variable, the HR increased by a factor of 1.067 for every increase of 0.01 in activity (95% CI, 1.029-1.106; P < 0.0001). Thus, greater apurinic/apyrimidinic endonuclease activity was associated, on average, with an increasing risk for progression (i.e., shorter TTP). For example, in our sample population, the tumor with the highest activity is ~25-times [i.e., 1.067(0.501-0.0028)/0.01] more likely to progress than the lowest-activity tumor. Adjusting for age and gender, variables that have been associated with treatment outcome and/or overall survival in both pediatric and adult primary brain tumors (6–8), in a multivariate analysis had no effect on the relationship between activity and TTP (HR, 1.073; 95% CI, 1.030-1.117;P < 0.0005). These data suggest that Ap endo activity may be a useful predictor of progression following postoperative radiotherapy and/or chemotherapy in medulloblastoma/PNETs and are consistent with the proposal that Ap endo activity mediates resistance to adjuvant therapy.


View this table:
[in this window]
[in a new window]

  		Table 2. HR estimates for the association of Ap endo activity with TTP following combined radiotherapy and chemotherapy

 
Ape1/Ref-1 immunopositivity and time to tumor progression. Previous studies have suggested that the fraction of Ape1/Ref-1 immunopositive cells is associated with treatment outcome or overall survival in diverse pediatric and adult (reviewed in refs. 18, 19, 29) tumors. TTP following adjuvant radiotherapy and chemotherapy was available for 37 of the 42 medulloblastomas (13 progressed) examined for Ape1/Ref-1 by immunohistochemistry. Univariate analysis revealed no relationship between TTP and the fraction of immunopositive cells (HR, 0.992; P < 0.61), the intensity of staining (HR, 0.998; P < 0.72), or the product of stained fraction and intensity (HR, 0.998; P < 0.68).

Apurinic/apyrimidinic endonuclease activity is associated with time to tumor progression following adjuvant therapy within both high-risk and average-risk medulloblastomas. Newly diagnosed medulloblastomas can be stratified into high risk and average risk for treatment failure and recurrence based on patient age, extent of tumor resection, and dissemination to other sites in the central nervous system (6, 7, 8). In our study, high-risk tumors were defined using the Children's Cancer Study Group criteria; that is, high-risk patients are those with >1.5 cm3 residual tumor after surgery, are younger than 3 years, and have evidence of tumor dissemination to other sites (7). In accord with the increasing hazard for progression associated with increasing Ap endo activity, the mean Ap endo activity in 20 high-risk tumors in our sample was twice that of 29 average-risk tumors (0.18 ± 0.18 versus 0.083 ± 0.14 fmol/cell/min; P ≤ 0.05). Univariate analysis revealed a significant inverse relationship between activity and TTP in both high-risk tumors (HR, 1.149; 95% CI, 1.016-1.300; P = 0.027) and average-risk tumors (HR, 1.076; 95% CI, 1.013-1.144; P = 0.018), indicating the use of Ap endo in predicting treatment outcome within each risk group.

Suppression of apurinic/apyrimidinic endonuclease activity increases alkylator killing of medulloblastoma cells. The association between Ap endo activity and TTP following postoperative therapy suggests that repair of abasic sites promotes medulloblastoma/PNET resistance to radiation and/or alkylating agents in situ. To address this hypothesis, alkylator sensitivity was determined by clonogenic colony-forming assay following suppression of Ap endo activity in the human medulloblastoma–derived cell line UW228-2. The effect of transfection with either antisense oligonucleotides or siRNA targeting Ape1/Ref-1 on Ap endo activity, Ape1/Ref-1 content, and alkylator sensitivity is illustrated in Fig. 3. Antisense oligonucleotide treatment reduced activity 3.8-fold (0.23 ± 0.044 to 0.061 ± 0.022 fmol/cell/min) in three separate experiments, whereas transfection with siRNA reduced activity 5.4-fold (0.21 ± 0.048 to 0.039 ± 0.017 fmol/cell/min) in four separate experiments. In accord, Western analysis (Fig. 3B) revealed reduced abundance of Ape1/Ref-1, evident within 30 hours after transfection with antisense oligonucleotides or siRNA. Suppression of Ap endo activity was accompanied by significantly greater sensitivity to the clinical chloroethylating agent BCNU, as illustrated in Fig. 3C for siRNA. As summarized in Table 3, treatment with antisense oligonucleotides or siRNA yielded 2.4- and 1.8-fold reductions, respectively, in LD10 for BCNU, relative to cells transfected with control sequences. Greater sensitivity reflected (a) 2.3- and 1.3-fold reductions in DT, respectively, indicating that apurinic/apyrimidinic endonuclease contributes to insensitivity to low doses of BCNU and (b) increased 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 temozolomide. Antisense oligonucleotide–mediated reduction of Ap endo activity decreased LD10 for temozolomide by 1.9-fold (Fig. 3D; Table 2). The enhanced sensitivity to temozolomide reflected an increased rate of killing (i.e., lower D37), whereas DT was unchanged.



View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Antisense oligonucleotides (ASO) or siRNA suppression of apurinic/apyrimidinic endonuclease (Ap endo) activity and Ape1/Ref-1 content produces greater alkylator sensitivity in the medulloblastoma cell line UW228-2. A, subconfluent cells were transfected with antisense oligonucleotides (x), siRNA ({square}), or control oligomers ({bullet}) 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-1 protein 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 (x) or control oligomer ({circ}). 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 (x) and control oligomer ({circ}). Each point is a single determination at each drug dose. Panels A-D show representative results from different single experiments.

 

View this table:
[in this window]
[in a new window]

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

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Improvements in adjuvant radiotherapy and chemotherapy have greatly increased survival for pediatric medulloblastomas/PNETs (1, 2). However, the benefit is not equally afforded to all patients, and postoperative radiotherapy is frequently accompanied by physical and neuropsychological detriments (11). Although clinical experience has identified patient and tumor features that are associated with overall survival, there are few molecular markers that predict response to the combined radiotherapy and chemotherapy used to treat medulloblastomas/PNETs. The cytotoxicity of unrepaired abasic sites induced by radiation and alkylators (12, 13), and the evidence that Ap endo activity mediates resistance of human gliomas to treatment with these agents (20–22), led us to hypothesize that Ap endo activity is a mechanistically relevant predictor of response to adjuvant therapy in medulloblastomas/PNETs. The work reported here substantiates our hypothesis, shows a functional basis for the association of Ap endo activity with recurrence, and suggests that apurinic/apyrimidinic endonuclease activity may be useful as a predictor of treatment outcome and as a target for antiresistance therapies.

Support for our hypothesis is provided by the strong, inverse association between Ap endo activity and TTP following combined radiotherapy and chemotherapy that included an alkylating and/or a platinating agent (Table 2). Based on biochemical, genetic and cell/molecular biological studies, particularly in human glioma cells (20–22), the relationship between activity and TTP likely reflects repair of abasic sites, at least in part. Both radiation and alkylating agents induce baseless sites via DNA glycosylase–mediated excision of oxidized and alkylated purines and pyrimidines (12, 13). In addition, radiation-induced oxidative free radicals can induce abasic sites by direct attack on deoxyribose (12). Unrepaired abasic sites are lethal, most likely a consequence of their ability to strongly impede the progress of DNA replication (15–17). The contribution of abasic sites to the cytotoxicity of radiation and alkylating agents is evidenced by the hypersensitivity of bacterial, yeast, and mammalian cells that are deficient in repair of this lesion (reviewed in refs. 12, 17–19). Moreover, suppression of Ape1/Ref-1, the major human Ap endo activity (18), results in hypersensitivity to oxidizing and alkylating agents in both rat (30) and human adult glioma cells (21)8 Similar findings have been reported for a variety of additional mammalian cell lines (e.g., refs. 27, 31, 32). The association between Ap endo activity and TTP may also reflect mechanisms in addition to, or other than, excision of abasic sites. Ape1/Ref-1 has multiple catalytic activities in vitro, including 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease (18, 19) that may promote resistance to radiation damage by excising fragmented deoxyribose phosphate moieties at single-strand breaks (12). In addition, the reduction-oxidation activity Ref-1, located at the NH2 terminus of Ape1/Ref-1 (18), mediates signal transduction in response to radiation and other genotoxic stress (18, 19). For example, Ref-1 has been implicated in regulating the transactivation and proapoptotic activities of p53 (33). Thus, it is possible that multiple redox-sensitive proteins that are mechanistically associated with Ref-1 contribute to the associations with outcome that we observed here. Whatever the case, involvement of the DNA repair activity of Ape1/Ref-1 is supported by the finding that a chimeric protein containing Ap endo and O6-methylguanine-DNA methyltransferase activities, but no Ref-1, conferred greater alkylation resistance to human cells than O6-methylguanine-DNA methyltransferase activity alone (34). These survival-promoting functions of Ref-1 may be especially important in promoting resistance to multiagent chemotherapy protocols such as those used to treat medulloblastomas/PNETs that include DNA-damaging agents that do not produce abasic sites or oxidative lesions (e.g., cisplatin) and cytotoxic drugs that do not damage DNA (e.g., vincristine).

Additional evidence associating Ap endo activity with clinical outcome following postoperative treatment in medulloblastoma is the difference in mean activity between tumors that are average risk and high risk for recurrence. One criterion of higher risk, age of <3 years (i.e., infants), is associated with significantly greater Ap endo activity compared with older children and adolescents (Table 1). The worse outcome observed for infants has been attributed to restriction of adjuvant treatment to chemotherapy to avoid the severe morbidity caused by radiotherapy in the very young (2). Our data suggest that greater Ap endo activity/Ape1/Ref-1 content could also contribute to the poor prognosis in infants by promoting chemoresistance. In children older than 3 years and adolescents, Ap endo activity is also greater in high-risk medulloblastoma (0.17 ± 0.17 fmol/cell/min) versus average-risk medulloblastoma (0.083 ± 0.14 fmol/cell/min). In these patients, high risk is defined by tumor invasion of surrounding brain that prevents gross total resection or dissemination of tumor elsewhere in the neuroaxis (7). Our data indicate that elevation of Ap endo activity accompanies and/or promotes acquisition of these aggressive characteristics.

We observed that Ap endo activity is associated with age and gender in medulloblastomas/PNETs. The inverse correlation between activity and age (Fig. 1) may reflect processes associated with physical and functional maturation of the brain that are intrinsic to the progenitor cells of medulloblastomas/PNETs. Alternatively, declining tumor Ap endo activity with age may reflect reduction in exogenous stress in the surrounding brain associated with cessation of proliferation of neural cell progenitors and neuronal maturation. Notably, reduction of Ape1/Ref-1 mRNA detected by in situ hybridization accompanies the maturation of developing brain into adulthood in rodents (35, 36). Possible mechanisms underlying the 2-fold higher activity in females are less evident. Several studies have shown hormonal and trophic factor regulation of Ape1/Ref-1 mRNA and protein expression in human and rodent cell lines (37–39), suggesting that estrogenic hormones responsible for sexual differentiation in the brain may stimulate Ape1/Ref-1 expression.

It is of interest to relate our findings for medulloblastomas/PNETs to those for adult gliomas (20–22). Medulloblastomas/PNETs differ from adult gliomas in biological and clinical properties, particularly in their greater responsiveness to radiation and chemotherapy. Despite these differences, we found that mean Ap endo activity for medulloblastomas/PNETs (0.12 ± 0.12 fmol/cell/min) was comparable with that for high-grade gliomas (0.090 ± 0.11 fmol/cell/min; ref. 22). However, we observed no association of activity with age or gender in gliomas. In both groups of tumors, increasing aggressiveness (high grade versus low grade in gliomas and high risk versus low risk in medulloblastomas) is associated with significantly higher mean activity. Correlation with degree of malignancy may reflect elevated oxidative stress accompanying increased rates of proliferation and/or tumor cell migration. As also observed for an adult glioma line (21), suppression of Ape1/Ref-1 expression in the medulloblastoma line UW228-2 increased killing by the clinical alkylators BCNU and temozolomide. Finally, in both medulloblastomas/PNETs and adult gliomas, there is a strong significant inverse association between Ap endo activity and TTP following adjuvant therapy with radiation and/or alkylating agent–based chemotherapy (both modalities combined in the present study, and each modality separately in gliomas). It is important to note that, in our sample populations, the hazard for progression differed by 25-fold within the range of Ap endo activities observed in 52 medulloblastomas/PNETs and by 13- to 14-fold within the range of activities observed in 30 and 44 adult gliomas treated with either alkylators or radiotherapy, respectively. Taken together, our results suggest that Ap endo may contribute to resistance to postoperative radio/chemotherapy and provide a predictor of outcome in a variety of primary brain tumors.

We observed extensive variation between medulloblastomas in the fraction of cells that were immunopositive for Ape1/Ref-1 (~8-73%) and in the intensity of staining scored on a scale of +1 to +3 (e.g., Fig. 2). This variability is consistent with the 180-fold range in Ap endo activity measured in our biochemical assay. The staining pattern seemed uniform within each of the 42 tumors examined, and there was a strong correlation between fraction of stained cells and staining intensity, as well as a nearly significant correlation between fraction of stained cells and activity. Ape1/Ref-1 was detectable only in nuclei, as has been observed for some, but not all tumors (18, 19). Lack of cytoplasmic staining may reflect the small, round cell morphology and large nuclear volume that are histologic hallmarks of medulloblastomas. More intriguing are the mechanisms that may account for the variability in stained cell fraction and intensity. Numerous studies have implicated oxidative stress as a modulator of Ap endo activity and Ape/Ref-1 content in cell lines and tissue (19). It is possible that the intertumor heterogeneity in immunopositivity reflects oxidative stress associated either with cell proliferation or episodes of transient hypoxia due to inadequate/impaired vasculature. An association with Ape1/Ref-1 content, assessed by immunohistochemistry, with treatment outcome has been reported for some human tumors, including head and neck cancer, germ cell tumors, cervical cancer, ovarian carcinoma, and osteosarcomas (reviewed in refs. 18, 19, 27, 40). In contrast to these studies, a Cox univariate model did not reveal a significant relationship between either the fraction of Ape1/Ref-1-immunopositive cells, the intensity of staining, or their product, and TTP following adjuvant treatment for medulloblastomas. However, this lack of association may reflect the relatively small number of tumors examined, the low proportion that had progressed (35%), and/or the relative insensitivity of a discontinuous variable (staining intensity) versus a continuous variable (activity). Nonetheless, Ap endo activity permitted detection of an association for a similar sample size and fraction of treatment failures, suggesting that activity may be a more sensitive predictor of clinical outcome. Increased nuclear Ape1/Ref-1 content and localization has been observed to accompany malignant progression in ovarian carcinomas (40), suggesting that the nuclear localization of Ape1/Ref-1 may reflect the malignant character of medulloblastomas.

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


   Acknowledgments
 
We thank Dr. A. Blank for critical reading of the article, Dr. Kristy Seidel for guidance with statistical analysis, and Bobby Stevens and Douglas Kolstoe for technical assistance.


   Footnotes
 
Grant support: American Cancer Society grant RPG-97-019 CN and NIH grants CA70790, CA82622, and CA104593.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

8 J.R. Silber and M.S. Bobola, unpublished results. Back

Received 5/13/05; revised 7/12/05; accepted 7/21/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. McNeil DE, Cote TR, Clegg L, Rorke LB. Incidence and trends in pediatric malignancies medulloblastoma/primitive neuroectodermal tumor: a SEER update. Surveillance Epidemiology and End Results. Med Pediatr Oncol 2002;39:190–4.[CrossRef][Medline]
  2. Rood BR, Macdonald TJ, Packer RJ. Current treatment of medulloblastoma: recent advances and future challenges. Semin Oncol 2004;31:666–75.[CrossRef][Medline]
  3. Koeller KK, Rushing EJ. From the archives of the AFIP: medulloblastoma:a comprehensive review with radiologic-pathologic correlation. Radiographics 2003;23:1613–37.[Abstract/Free Full Text]
  4. Giordana MT, Schiffer P, Lanotte M, Girardi P, Chio A. Epidemiology of adult medulloblastoma. Int J Cancer 1999;80:689–92.[CrossRef][Medline]
  5. Ellison D. Classifying the medulloblastoma:insights from morphology and molecular genetics. Neuropathol Appl Neurobiol 2002;28:257–82.[CrossRef][Medline]
  6. Packer RJ, Rood BR, MacDonald TJ. Medulloblastoma: present concepts of stratification into risk groups. Pediatr Neurosurg 2003;39:60–7.[CrossRef][Medline]
  7. Chintagumpala M, Berg S, Blaney SM. Treatment controversies in medulloblastoma. Curr Opin Oncol 2001;13:154–9.[CrossRef][Medline]
  8. Eberhart CG, Burger PC. Anaplasia and grading in medulloblastomas. Brain Pathol 2003;13:376–85.[Medline]
  9. Ellison DW, Clifford SC, Gajjar A, Gilbertson RJ. What's new in neuro-oncology? Recent advances in medulloblastoma. Eur J Paediatr Neurol 2003;7:53–66.[CrossRef][Medline]
  10. Cohen BH, Packer RJ. Chemotherapy for medulloblastomas and primitive neuroectodermal tumors. J Neurooncol 1996;29:55–68.[CrossRef][Medline]
  11. Mulhern RK, Merchant TE, Gajjar A, Reddick WE, Kun LE. Late neurocognitive sequelae in survivors of brain tumours in childhood. Lancet Oncol 2004;5:399–408.[CrossRef][Medline]
  12. Demple B, Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem 1994;63:915–48.[CrossRef][Medline]
  13. Krokan HE, Nilsen H, Skorpen F, Otterlei M, Slupphaug G. Base excision repair of DNA in mammalian cells. FEBS Lett 2000;476:73–7.[CrossRef][Medline]
  14. Lhomme J, Constant J-F, Demeunyck M. Abasic DNA structure, reactivity and recognition. Biopol (Nucleic Acid Sci) 1999;52:65–83.
  15. Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet 1986;20:201–30.[CrossRef][Medline]
  16. Sutton MD, Walker GC. Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination. Proc Natl Acad Sci U S A 2001;98:8342–9.[Abstract/Free Full Text]
  17. Boiteux S, Guillet M. Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae. DNA Repair 2004;3:1–12.[CrossRef][Medline]
  18. Evans AR, Limp-Foster M, Kelley MR. Going APE over ref-1. Mutat Res 2000;461:83–108.[Medline]
  19. Fritz G, Grosch S, Tomicic M, Kaina B. APE/Ref-1 and the mammalian response to genotoxic stress. Toxicology 2003;193:67–78.[CrossRef][Medline]
  20. Bobola MS, Emond MJ, Blank A, et al. Apurinic endonuclease activity in adult gliomas and time to tumor progression after alkylating agent-based chemotherapy and after radiotherapy. Clin Cancer Res 2004;10:7875–83.[Abstract/Free Full Text]
  21. Silber JR, Bobola MS, Blank A, et al. The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by oxidative stress. Clin Cancer Res 2002;8:3008–18.[Abstract/Free Full Text]
  22. Bobola MS, Blank A, Berger MS, Stevens BA, Silber JR. Apurinic/apyrimdinic endonuclease activity is elevated in human adult gliomas. Clin Cancer Res 2001;7:3510–18.[Abstract/Free Full Text]
  23. Silber JR, Bobola MS, Ghatan S, Blank A, Kolstoe DD, Berger MS. O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics. Cancer Res 1998;58:1068–73.[Abstract/Free Full Text]
  24. Chen D, Herman V, Demple B. Two distinct human diesterases that act on 3'-fragments of deoxyribose in radical damaged DNA. Nucleic Acids Res 1991;19:5907–14.[Abstract/Free Full Text]
  25. Rossi O, Carrozzino F, Cappelli E, Carli F, Frosina G. Analysis of repair of abasic sites in early onset breast cancer patients. Int J Cancer 2000;85:21–6.[CrossRef][Medline]
  26. Keles GE, Berger MS, Srinivasan J, Kolstoe DD, Bobola MS, Silber JR. Establishment and characterization of four human medulloblastoma-derived cell lines. Oncol Res 1995;7:493–503.[Medline]
  27. Wang D, Luo M, Kelley MR. Human apurinic endonuclease 1 (APE1) expression and prognostic significance in osteosarcoma:enhanced sensitivity of osteosarcoma to DNA damaging agents using silencing RNA APE1 expression inhibition. Mol Cancer Ther 2004;3:679–86.[Abstract/Free Full Text]
  28. Bobola MS, Blank A, Berger MS, Silber JR. Contribution of O6-methylguanine-DNA methyltransferase to monofunctional alkylating agent resistance in human brain tumor-derived cell lines. Mol Carcinog 1995;13:70–80.[Medline]
  29. Flaherty DM, Monick MM, Hunninghake GW. AP endonucleases and the many functions of Ref-1. Am J Respir Cell Mol Biol 2001;25:664–7.[Free Full Text]
  30. Ono Y, Furuta T, Ohmoto T, Akiyama K, Seki S. Stable expression in rat glioma cells of sense and antisense nucleic acids to a human multifunctional enzyme, APEX nuclease. Mutat Res 1994;315:55–63.[CrossRef][Medline]
  31. Walker LJ, Craig RB, Harris AL, Hickson ID. A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress. Nucleic Acids Res 1994;22:4884–9.[Abstract/Free Full Text]
  32. Chen DS, Olkowski ZL. Biological response of human apurinic endonuclease to radiation-induced DNA damage. Ann NY Acad Sci 1994;29:306–8.
  33. Gaiddon C, Moorthy NC, Prives C. Ref-1 regulates the transactivation and pro-apoptotic functions of p53 in vivo. EMBO J 1999;18:5609–21.[CrossRef][Medline]
  34. Roth TJ, Xu Y, Luo M, Kelley MR. Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection. Cancer Gene Ther 2003;10:603–10.[CrossRef][Medline]
  35. Ono Y, Watanabe M, Inoue Y, et al. Developmental expression of APEX nuclease, a multifunctional DNA repair enzyme, in mouse brains. Brain Res Dev Brain Res 1995;86:1–6.[CrossRef][Medline]
  36. Wilson TM, Rivkees SA, Deutsch WA, Kelley MR. Differential expression of the apurinic/apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis. Mutat Res 1996;362:237–48.[Medline]
  37. Asai T, Kambe F, Kikumori T, Seo H. Increase in Ref-1 mRNA and protein by thyrotropin in rat thyroid FRTL-5 cells. Biochem Biophys Res Commun 1997;236:71–4.[CrossRef][Medline]
  38. Suzuki S, Nagaya T, Suganuma N, Tomoda Y, Seo H. Inductions of immediate early genes (IEGS) and ref-1 by human chorionic gonadotropin in murine Leydig cell line (MA-10). Biochem Mol Biol Int 1998 Feb;44:217–24.[Medline]
  39. Flaherty DM, Monick MM, Carter AB, Peterson MW, Hunninghake GW. GM-CSF increases AP-1 DNA binding and Ref-1 amounts in human alveolar macrophages. Am J Respir Cell Mol Biol 2001;25:254–9.[Abstract/Free Full Text]
  40. Tanner B, Grimme S, Schiffer I, et al. Nuclear expression of apurinic/apyrimidinic endonuclease increases with progression of ovarian carcinomas. Gynecol Oncol 2004;92:568–77.[CrossRef][Medline]
  41. Bobola MS, Silber JR, Ellenbogen RG, Geyer JR, Blank A, Goff RD. O6-methylguanine-DNA methyltransferase, O6-benzylguanine, and resistance to clinical alkylators in pediatric primary brain tumor cell lines. Clin Cancer Res 2005;11:2747–55.[Abstract/Free Full Text]
  42. Mendez F, Goldman JD, Bases RE. Abasic sites in DNA of HeLa cells induced by lucanthone. Cancer Invest 2002;20:983–91.[CrossRef][Medline]
  43. Luo M, Kelley MR. Inhibition of the human apurinic/apyrimidinic endonuclease (APE1) repair activity and sensitization of breast cancer cells to DNA alkylating agents with lucanthone. Anticancer Res 2004;24:2127–34.[Abstract/Free Full Text]
  44. Del Rowe JD, Bello J, Mitnick R, et al. Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone. Int J Radiat Oncol Biol Phys 1999;43:89–93.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Mol Cancer ResHome page
D. R. McNeill, W. Lam, T. L. DeWeese, Y.-C. Cheng, and D. M. Wilson III
Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and Antimetabolites
Mol. Cancer Res., June 1, 2009; 7(6): 897 - 906.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
R. Chattopadhyay, S. Das, A. K. Maiti, I. Boldogh, J. Xie, T. K. Hazra, K. Kohno, S. Mitra, and K. K. Bhakat
Regulatory Role of Human AP-Endonuclease (APE1/Ref-1) in YB-1-Mediated Activation of the Multidrug Resistance Gene MDR1
Mol. Cell. Biol., December 1, 2008; 28(23): 7066 - 7080.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
D. R. McNeill and D. M. Wilson III
A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents
Mol. Cancer Res., January 1, 2007; 5(1): 61 - 70.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
W. Lam, S.-Y. Park, C.-H. Leung, and Y.-C. Cheng
Apurinic/Apyrimidinic Endonuclease-1 Protein Level Is Associated with the Cytotoxicity of L-Configuration Deoxycytidine Analogs (Troxacitabine and beta-L-2',3'-Dideoxy-2',3'-didehydro-5-fluorocytidine) but Not D-Configuration Deoxycytidine Analogs (Gemcitabine and beta-D-Arabinofuranosylcytosine)
Mol. Pharmacol., May 1, 2006; 69(5): 1607 - 1614.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bobola, M. S.
Right arrow Articles by Silber, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bobola, M. S.
Right arrow Articles by Silber, J. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online