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Tumor Biology |
Herman B. Wells Center for Pediatric Research, Departments of Pediatrics [K. A. R., H. A. B., Y. X., R. T., E. Z., M. R. K.], Biochemistry and Molecular Biology [M. R. K.], Pathology [T. M. U.], Medicine [L. H. E.], and Urology [R. S. F.], James Whitcomb Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1
, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2–3-fold increase in protection against bleomycin. Lesser protection was observed with 
-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preliminary investigations have been performed examining GCTs that are refractory to cisplatin and by establishing cell lines from these cells (4) . These initial studies have demonstrated that cisplatin-resistant GCT cell lines are able to repair DNA-platinum adducts and cross-links better than cisplatin-responsive cell lines. These two studies suggested a hypothesis that resistance to cisplatin may be related to the relative efficacy of DNA repair (5) . More recently, cisplatin adducts have been found to inhibit DNA glycosylases (6) , which may alter the BER pathway.
The human AP endonuclease (Ape1 or ref-1) DNA BER enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions, including oxidative signaling, transcription factor regulation, cell cycle control, and cancer (7)
. It acts on mutagenic AP (baseless) sites in DNA as a major member of the DNA BER repair pathway. Simple glycosylases, such as methylpurine DNA glycosylase, excise damaged/alkylated bases, resulting in AP sites that are subsequently incised 5' to the AP site by Ape1/ref-1, allowing repair to be completed by deoxyribose phosphatase activity, provided by DNA ß-polymerase, to remove the deoxyribose phosphate termini, followed by insertion of the correct base and ligation. Additionally, Ape1/ref-1 is able to repair the 3' phosphate and phosphoglycolate lesions generated in single-strand breaks by ionizing radiation and bleomycin (8)
. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, NFB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression (9, 10, 11, 12, 13, 14, 15)
. Ape1/ref-1 has also been shown to be closely linked to apoptosis (16)
, associated with thioredoxin (17
, 18)
, and altered levels of Ape1/ref-1 have been found in some cancers (19, 20, 21)
. More recently, in a B-lymphocyte system, Ape1/ref-1 has demonstrated a response to reactive oxygen species by rapid translocation from the cytoplasm to the nucleus (22)
.
In a pilot study, we found Ape1/ref-1 to be expressed at a relatively high level in a small group of GSTs (23)
. Because of the multifaceted role of Ape1/ref-1 in cells, we have begun to try and determine whether the elevated level of Ape1/ref-1 in GCTs is responsible for an increase in repair, redox, or both activities. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with LAPESN. Using an oligonucleotide cleavage assay and IHC to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines to be directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2–3-fold increase in protection against bleomycin. Lesser protection was observed with -irradiation. We conclude that elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. This is the first example of overexpressing Ape1/ref-1 in a mammalian model system that leads to cellular protection from chemotherapeutic agents.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IHC.
Tissue sections were stained for Ape1/ref-1 expression using an anti-Ape1 monoclonal antibody that has been characterized extensively (19
, 20)
and is available commercially (Novus Biologicals, Littleton, CO). The staining process used was identical to that described previously (19
, 20)
, with the exception that a Dako Universal Staining System was used to automate the process (Dako Corp., Carpinteria, CA). Sections were treated with a 10-min incubation in 3% H2O2 to block endogenous peroxidases, incubated with the anti-Ape1 antibody (1:1000) for 25 min, the biotinylated goat antimouse IgG secondary antibody for 10 min, streptavidin-horseradish peroxidase for 10 min, and diaminobenzidine for 5 min, per Dako recommendations and empiric determination. Preimmune IgG was used as a control for antibody specificity in place of the antihuman Ape1 antibody at a concentration of 50 µg/ml. No antigen unmasking techniques were used, nor are they necessary with this antibody.
Cell Lines.
NT2 cells and the PG13 GALV-based retrovirus packaging cell line were obtained from the American Type Culture Collection (Rockville, MD). The 833K GCT cell line was obtained from George Sledge (Indiana University). Both cell lines were maintained as adherent cells in polystyrene, six-well Costar tissue culture trays (Costar, Corning, NY) in DMEM tissue culture media (Life Technologies, Inc., Rockville, MD) plus 10% Cosmic Calf serum (Hyclone, Logan, UT) plus antibiotics. Cells were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% air.
Molecular Analysis.
Southern, Northern, and Western blotting were performed as described previously (16
, 20)
. Southern blots were probed with a 32P-labeled random primed 1-kb human Ape1/ref-1 cDNA. Northern blots were hybridized with 32P-labeled Ape1/ref-1, Neo, and human ß-actin, as described previously (16
, 20)
.
Retroviral Construction, Production, and Infection of Cell Lines.
The 1-kb human Ape1/ref-1 cDNA was cloned into the EcoRI (5') and XhoI (3') sites of the retroviral vector LXSN as described previously (16)
. Virus was generated from the LXSN plasmid harboring Ape1/ref-1 (now designated LAPESN), as performed previously (24)
. Briefly, 10 µg of agarose gel purified LAPESN were transfected into PE501 producer cells, followed by selection in G418 (Life Technologies, Inc.) at 1 mg/ml. Supernatants were harvested and used to infect PG13 GALV-based amphotrophic packaging cells (25)
and selected in G418. Total RNA was extracted from the G418-resistant cells and analyzed by Northern blot hybridization with an Ape1/ref-1 probe to demonstrate the 1.7-kb Ape1/ref-1 retroviral transcript. Supernatants from G418-resistant PG13 + LAPESN cells were used to transduce NT2 cells. After selection in G418 (1 mg/ml), individual clones were isolated and expanded. Screening by Southern blot analysis revealed two clones with distinct single inserts that were selected for further study. NT2 cells transduced with the empty LXSN vector were produced in a similar manner as controls.
Cell Culture Studies.
NT2, NT2-LXSN, and NT2-LAPESN cells were seeded (3–5 x 10 4 cells/well) in six-well trays and allowed to adhere overnight. The following day, cells were treated with bleomycin (Blenoxane; Bristol Myers Squib Company, Princeton, NJ) as indicated or radiation. Bleomycin was dissolved in sterile water (250 µg/ml) and filter-sterilized through a 0.22 µm filter prior to being added to cultures, and radiation was delivered from a Cesium 137 irradiator at a rate of 11.1 cGy/min. After 3, 5, and 7 days, control and treated cells were enumerated by trypsinizing the adherent cells, pipetting up and down to generate a single-cell suspension in trypan blue, and counting using a hemocytometer.
The determination of AP endonuclease activity was performed using an oligonucleotide cleavage assay as described previously (20 , 26 , 27) . Briefly, cell extracts were incubated with a 5'-32 P-end-labeled 26-mer oligonucleotide containing a single THF artificial AP site at position 14, which, in the presence of AP endonuclease activity, cleaves the 32P-labeled 26-mer to a 14-mer. The abasic analogue is resistant to cleavage by 3'-acting AP lyase activity, which is generally possessed by DNA glycosylases/AP lyases. Therefore, this assay is specific for Ape1/ref-1 activity in cells. Reaction mixtures (10 µl) containing cell extracts of interest, 2.5 pmol of 5' 32P end-labeled, double-stranded THF oligonucleotide, 50 mM HEPES, 50 mM KCl, 10 mM MgCl2, 1 µg/ml BSA, and 0.05% Triton X-100 (pH 7.5) were allowed to proceed for 15 min in a 37°C water bath. Reactions were halted by adding 10 µl of 96% formamide, 10 mM EDTA, xylene cytanol, and bromphenol blue. AP assay products (5 µl) were separated on a 20% polyacrylamide gel containing 7 M urea. Gels were wrapped in saran wrap and exposed to film for visualization. The amount of 14-mer to 26-mer was determined by scanning the exposed film into Sigma Scan (Jandel Scientific, San Rafael, CA) for analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Infiltrating lymphocytes had little Ape1/ref-1 expression. Tumor cells identified in each section displayed an increased level of expression of nuclear Ape1/ref-1 compared with stromal elements on the same slides (Fig. 1
and Table 1
). The distribution of Ape1/ref-1 staining in the GCTs was predominantly nuclear in 50% of the cases and mixed nuclear-cytoplasmic in the other 50% (Table 1)
. There appeared to be a trend for the more differentiated subtypes (teratomas) to have less cytoplasmic expression of Ape1/ref-1 compared with other subtypes (Table 1)
. Given the small number of samples examined in this pilot study, no statistical analysis was performed; however, a larger trial has been initiated that should permit more conclusive analysis of Ape1/ref-1 expression in GCTs and its relationship to clinical parameters, such as relapse and chemotherapy resistance.
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). This vector includes a convenient cloning site downstream from the Moloney murine leukemia virus long terminal repeat as well as the neomycin phosphotransferase gene (neo) for use as a selectable marker. NT2 clones transduced with LAPESN were selected in 10-cm dishes and isolated with cloning rings. Screening by Southern blot (Ape1/ref-1 probe) identified two clones (data not shown) with single inserts, which were used for further study. It was difficult to distinguish on Northern blot analysis the mRNA from NT2, NT2-LXSN, and NT2-LAPESN clones 1 and 2 of the endogenous Ape1/ref-1 (1.6 kb) from the retroviral-derived transcript (1.7 kb); however, probing for neo revealed the full-length, 2.9-kb LXSN transcript in the LXSN vector-only transduced cells and the truncated 1.6-kb Neo transcript from the LAPESN-expressing cells (Fig. 2B)
. The absence of a full-length LAPESN transcript likely results from an occult polyadenylation signal within the 3' sequence of the Ape1/ref-1 cDNA, similar to the transcript pattern observed with other LXSN constructs (24)
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, similar to the pattern described above in sections of primary teratomas (Table 1)
. The nuclei displayed a distinctive punctate staining pattern that we observed previously in cervical carcinoma cells (19)
. The NT2 cells overexpressing Ape1/ref-1 demonstrated an elevated level of nuclear staining for Ape1/ref-1 again with little cytoplasmic staining, similar to the wild-type NT2 cells (Fig. 3A)
. Vector-only transduced cells were identical to the wild-type NT2 cells in Ape1/ref-1 staining, and morphologically, the transduced cells (LXSN and LAPESN) were indistinguishable from the wild-type cells (Fig. 3A)
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) containing an artificial THF AP site, which when cleaved by AP endonuclease produces a labeled 14-mer (Fig. 3B)
. Sequential dilutions of cell extracts from each of the cell types were assayed for endonuclease activity and are shown in Fig. 3B
. The increasing expression of Ape1/ref-1 noted in parental NT2 cells, 833K cells, and NT2-LAPESN cells (Fig. 3A)
was reflected in increasing AP endonuclease activity, as measured with the oligonucleotide cleavage assay. Thus, Ape1/ref-1 repair activity appears to correlate with the degree of Ape1/ref-1 expression by IHC.
Ape1/ref-1 Overexpression Protects GCT Cells from Bleomycin and Radiation.
To determine whether Ape1/ref-1 expression correlated with sensitivity to therapeutic agents, we quantitated the AP-site cleavage activity of parental NT2 cells and NT2-LAPESN clones 1 and 2 (Fig. 4, A–C)
. Both clones as well as the bulk-infected NT2 cells from which the clones were selected displayed a 2-fold higher endonuclease activity compared with NT2 cells (Fig. 4D)
. We then examined the response of NT2 and NT2-LAPESN cells to bleomycin, an antitumor antibiotic known for its effectiveness in treating GCTs. Cells transduced with the empty vector, LXSN, and the parental NT2 cells responded similarly to treatment (Fig. 5)
. The NT2 cells overexpressing Ape1/ref-1 demonstrated a 2-fold increase in protection from bleomycin in a dose-dependent fashion (Fig. 5A)
, paralleling the 2-fold increase in endonuclease activity observed in the oligonucleotide cleavage assay (Fig. 4)
. A similar trend for protection from -irradiation was observed but with not quite the same degree of protection that was afforded to bleomycin (Fig. 5B)
. Thus, the AP endonuclease activity of Ape1/ref-1 as measured by the oligonucleotide cleavage assay appears to reflect the degree of protection from bleomycin and radiation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Irradiation produces a variety of damage to DNA, including oxidative damage to bases resulting in base excision and the formation of mutagenic/cytotoxic AP sites, which are substrates for Ape1/ref-1 (33)
. Thus, we conclude that elevated expression of Ape1/ref-1 in GCTs likely contributes to resistance to therapeutic agents that induce damage responsive to BER.
However, we must recognize that Ape1/ref-1 is a complex enzyme with multiple functions including a well-documented redox function (7)
. Although Ape1/ref-1 appears to protect from bleomycin and -irradiation presumably by means of repair activity, another aspect, and somewhat contrary to these results, is its potential role in augmentation of the response to certain therapeutic agents. Ape1/ref-1 involvement in activation of p53 and induction of cyclin G may be integral to the apoptotic response to cisplatin in GCTs (9
, 40)
. The high level expression of Ape1/ref-1 in GCTs in conjunction with its role in activation of apoptosis through p53/cyclin G may account in part for the sensitivity of GCTs to therapy. Additionally, elevation of Ape1/ref-1 may be important in maintaining the malignant phenotype through redox activation of transcription factors such as Fos, Jun, and HIF-1 (7)
. One way to sort out these issues is the use of site-specific Ape1/ref-1 mutants to characterize the role of the repair/redox domains in response of GCTs to specific therapeutic agents. Mechanistic studies should be complemented with the examination of Ape1/ref-1 expression in a large sample of GCT patients to determine the correlation, if any, of expression with response to therapy. We have an ongoing protocol to collect such data retrospectively and prospectively, with the objective to analyze the relationship of Ape1/ref-1 expression to histological subtypes of GCTs, risk of relapse, response to therapy, and role in tumor progression. Other questions also exist, such as: Does the cytoplasmic distribution of Ape1/ref-1 observed in some GCTs result from an alteration in the nuclear localization signal harbored in the 5'-end of Ape1/ref-1? How does this change in distribution affect repair and redox function? In one primary teratoma of the testis, we examined only the intratubular GCT cells marked with antihuman Ape1/ref-1 antibody, sparing the rest of the normal cellular elements, suggesting that in certain settings, staining with anti-Ape1 may be useful in detecting tumor cells. Similar observations have been made in staining sections of cervical carcinoma in situ for Ape1/ref-1 (19)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grants CA76643 (to K. A. R., M. R. K., and Y. X.), NS38506 (to M. R. K.), R43 CA83507 (to M. R. K.), P30-DK49218, PO1-CA74295 (to T. M. U. and L. H. E.), and P01-CA75426 (to M. R. K. and R. T.); the Lance Armstrong Foundation (to K. A. R. and M. R. K.); the Gynecologic Oncology Group (to M. R. K.); and the Riley Memorial Association (to K. A. R. and M. R. K.). 
2 To whom requests for reprints should be addressed, at The Herman B. Wells Center for Pediatric Research, Riley Hospital for Children, Room 2600, 702 Barnhill Drive, Indianapolis, IN 46202.
3 The abbreviations used are: GCT, germ cell tumor; Ape1/ref-1, apurinic/apyrimidinic endonuclease; BER, base excision repair; IHC, immunohistochemistry; AP, apurinic/apyrimidinic; THF, tetrohydrofuran; NT2, NTERA-2/D1 human embryonal carcinoma.
Received 8/28/00. Accepted 12/27/00.
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and the HIF-like factor. J. Biol. Chem., 275: 4618-4627, 2000.This article has been cited by other articles:
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