[CANCER RESEARCH 61, 2220–2225, March 1, 2001]
Altered Expression of Ape1/ref-1 in Germ Cell Tumors and
Overexpression in NT2Cells Confers Resistance to Bleomycin and
Radiation1
Kent A. Robertson, Heather A. Bullock, Yi Xu, Renee Tritt, Erika
Zimmerman, Thomas M. Ulbright,Richard S. Foster, Lawrence H.
Einhorn, and Mark R. Kelley2
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, Indiana46202
ABSTRACT
The human AP endonuclease (Ape1 or ref-1) DNA base excision
repair(BER) enzyme is a multifunctional protein that has an impact
on a widevariety of important cellular functions including
oxidative signaling, tran-scription factor regulation, and cell
cycle control. It acts on mutagenic AP(baseless) sites in DNA as a
critical member of the DNA BER repairpathway. Moreover, Ape1/ref-1
stimulates the DNA-binding activity oftranscription factors
(Fos-Jun, nuclear factor-kB, Myb, ATF/cyclic AMP-responsive element
binding protein family, HIF-1a, HLF, PAX, and p53)through a redox
mechanism and thus represents a novel component ofsignal
transduction processes that regulate eukaryotic gene
expression.Ape1/ref-1 has also been shown to be closely linked to
apoptosis associatedwith thioredoxin, and altered levels of
Ape1/ref-1 have been found in somecancers. In a pilot study, we
have examined Ape1/ref-1 expression byimmunohistochemistry in
sections of germ cell tumors (GCTs) from 10patients with testicular
cancer of various histologies including seminomas,yolk sac tumors,
and malignant teratomas. Ape1/ref-1 was expressed atrelatively high
levels in the tumor cells of nearly all sections. We hypoth-esized
that elevated expression of Ape1/ref-1 is responsible in part for
theresistance to therapeutic agents. To answer this hypothesis, we
overex-pressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1
using retroviralgene transduction with the vector LAPESN. Using an
oligonucleotidecleavage assay and immunohistochemistry to assess
Ape1/ref-1 repairactivity and expression, respectively, we found
that the repair activity andrelative Ape1/ref-1 expression in GCT
cell lines are directly related.NT2/D1 cells transduced with
Ape1/ref-1 exhibited 2-fold higher APendonuclease activity in the
oligonucleotide cleavage assay, and this wasreflected in a 2–3-fold
increase in protection against bleomycin. Lesserprotection was
observed withg-irradiation. We conclude that: (a) Ape1/ref-1 is
expressed at relatively high levels in some GCTs; (b)
elevatedexpression of Ape1/ref-1 in testicular cancer cell lines
results in resistanceto certain therapeutic agents; and (c)
Ape1/ref-1 expression in GCT celllines determined by
immunohistochemistry and repair activity assaysparallels the level
of protection from bleomycin. We further hypothesizethat elevated
Ape1/ref-1 levels observed in human testicular cancer maybe related
to their relative resistance to therapy and may serve as
adiagnostic marker for refractory disease. To our knowledge, this
is thefirst example of overexpressing Ape1/ref-1 in a mammalian
system result-ing in enhanced protection to DNA-damaging
agents.
INTRODUCTION
Therapy for disseminated testicular cancer/GCT3 has been
success-ful, with 70–80% of patients being cured with front-line
chemother-apy (1). However, for those 20–30% of patients with
extragonadalprimaries or relapsed/refractory disease, the response
to therapy ispoor, with only 3–30% surviving disease free after
second-line agents(1). Active chemotherapeutic agents against GCTs
include vinblas-tine, etoposide, bleomycin, cisplatin,
cyclophosphamide, and ifos-famide (2). Except for vinblastine and
etoposide, each of these agentsexerts their toxic effect on tumor
cells by directly reacting with DNA,inducing damage including
baseless mutagenic AP sites, and ulti-mately cell death. Although
little is known about the expression androle of DNA repair systems
in GCTs and their response to therapeuticagents, it is of interest
that biopsies of GCTs reveal that patients withtumors responding
(complete remission) to cisplatin have a highernumber of
platinum-DNA adducts/tumor cell than patients who re-spond poorly
(partial remission; Ref. 3). This observation suggeststhat
resistant cells are able to repair cisplatin-induced DNA damagemore
effectively than sensitive GCT cells.
Preliminary investigations have been performed examining
GCTsthat are refractory to cisplatin and by establishing cell lines
from thesecells (4). These initial studies have demonstrated that
cisplatin-resist-ant GCT cell lines are able to repair DNA-platinum
adducts andcross-links better than cisplatin-responsive cell lines.
These two stud-ies suggested a hypothesis that resistance to
cisplatin may be relatedto the relative efficacy of DNA repair (5).
More recently, cisplatinadducts have been found to inhibit DNA
glycosylases (6), which mayalter the BER pathway.
The human AP endonuclease (Ape1 or ref-1) DNA BER enzyme isa
multifunctional protein that has an impact on a wide variety
ofimportant cellular functions, including oxidative signaling,
transcrip-tion factor regulation, cell cycle control, and cancer
(7). It acts onmutagenic AP (baseless) sites in DNA as a major
member of the DNABER repair pathway. Simple glycosylases, such as
methylpurine DNAglycosylase, excise damaged/alkylated bases,
resulting in AP sites thatare subsequently incised 59to the AP site
by Ape1/ref-1, allowingrepair to be completed by deoxyribose
phosphatase activity, providedby DNA b-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
39phosphate and phosphoglycolatelesions generated in single-strand
breaks by ionizing radiation andbleomycin (8). Moreover, Ape1/ref-1
stimulates the DNA-bindingactivity of transcription factors
(Fos-Jun, NFkB, Myb, ATF/cyclicAMP-responsive element binding
protein family, HIF-1a, HLF, PAX,and p53) through a redox mechanism
and thus represents a novelcomponent of signal transduction
processes that regulate eukaryoticgene expression (9–15).
Ape1/ref-1 has also been shown to be closelylinked to apoptosis
(16), associated with thioredoxin (17, 18), and
Received 8/28/00; accepted 12/27/00.The costs of publication of
this article were defrayed in part by the payment of page
charges. This article must therefore be hereby
markedadvertisementin accordance with18 U.S.C. Section 1734 solely
to indicate this fact.
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 (toT. M. U. and L. H. E.), and P01-CA75426
(to M. R. K. and R. T.); the Lance ArmstrongFoundation (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 Centerfor 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/apyrimidinicendonuclease; BER, base excision repair; IHC,
immunohistochemistry; AP, apurinic/apyrimidinic; THF,
tetrohydrofuran; NT2, NTERA-2/D1 human embryonal carcinoma.
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altered levels of Ape1/ref-1 have been found in some cancers
(19–21).More recently, in a B-lymphocyte system, Ape1/ref-1 has
demon-strated a response to reactive oxygen species by rapid
translocationfrom the cytoplasm to the nucleus (22).
In a pilot study, we found Ape1/ref-1 to be expressed at a
relativelyhigh level in a small group of GSTs (23). Because of the
multifacetedrole of Ape1/ref-1 in cells, we have begun to try and
determinewhether the elevated level of Ape1/ref-1 in GCTs is
responsible for anincrease in repair, redox, or both activities. We
hypothesized thatelevated expression of Ape1/ref-1 is responsible
in part for the resist-ance to therapeutic agents. To answer this
hypothesis, we overex-pressed the Ape1/ref-1 cDNA in the GCT cell
line NT2/D1 usingretroviral gene transduction with LAPESN. Using an
oligonucleotidecleavage assay and IHC to assess Ape1/ref-1 repair
activity andexpression, respectively, we found that the repair
activity and relativeApe1/ref-1 expression in GCT cell lines to be
directly related.NT2/D1 cells transduced with Ape1/ref-1 exhibited
2-fold higher APendonuclease activity in the oligonucleotide
cleavage assay, and thiswas reflected in a 2–3-fold increase in
protection against bleomycin.Lesser protection was observed
withg-irradiation. We conclude thatelevated expression of
Ape1/ref-1 in testicular cancer cell lines resultsin resistance to
certain therapeutic agents. We further hypothesize thatelevated
Ape1/ref-1 levels observed in human testicular cancer maybe related
to their relative resistance to therapy and may serve as
adiagnostic marker for refractory disease. This is the first
example ofoverexpressing Ape1/ref-1 in a mammalian model system
that leads tocellular protection from chemotherapeutic agents.
MATERIALS AND METHODS
Patients and Tumors. Tissue sections of biopsy material from
treated anduntreated patients with disseminated GCTs were obtained
from the IndianaUniversity Medical Center, University Hospital,
under an Indiana UniversityInstitutional Review Board approved
protocol (IU Study No. 9908-47) as 4%buffered formaldehyde-fixed
tissues embedded in paraffin blocks, which weresectioned at 3mm and
fixed onto slides. Diagnosis was made from morpho-logical
examination of H&E-stained sections of biopsy material.
IHC. Tissue sections were stained for Ape1/ref-1 expression
using ananti-Ape1 monoclonal antibody that has been characterized
extensively (19,20) and is available commercially (Novus
Biologicals, Littleton, CO). Thestaining process used was identical
to that described previously (19, 20), withthe exception that a
Dako Universal Staining System was used to automate theprocess
(Dako Corp., Carpinteria, CA). Sections were treated with a
10-minincubation in 3% H2O2 to block endogenous peroxidases,
incubated with theanti-Ape1 antibody (1:1000) for 25 min, the
biotinylated goat antimouse IgGsecondary antibody for 10 min,
streptavidin-horseradish peroxidase for 10 min,and diaminobenzidine
for 5 min, per Dako recommendations and empiricdetermination.
Preimmune IgG was used as a control for antibody specificityin
place of the antihuman Ape1 antibody at a concentration of 50mg/ml.
Noantigen unmasking techniques were used, nor are they necessary
with thisantibody.
Cell Lines. NT2 cells and the PG13 GALV-based retrovirus
packaging cellline were obtained from the American Type Culture
Collection (Rockville,MD). The 833K GCT cell line was obtained from
George Sledge (IndianaUniversity). Both cell lines were maintained
as adherent cells in polystyrene,six-well Costar tissue culture
trays (Costar, Corning, NY) in DMEM tissueculture media (Life
Technologies, Inc., Rockville, MD) plus 10% Cosmic Calfserum
(Hyclone, Logan, UT) plus antibiotics. Cells were maintained in
ahumidified incubator at 37°C in an atmosphere of 5% CO2 and 95%
air.
Molecular Analysis. Southern, Northern, and Western blotting
were per-formed as described previously (16, 20). Southern blots
were probed with a32P-labeled random primed 1-kb human Ape1/ref-1
cDNA. Northern blotswere hybridized with32P-labeled Ape1/ref-1,
Neo, and humanb-actin, asdescribed previously (16, 20).
Retroviral Construction, Production, and Infection of Cell
Lines. The1-kb human Ape1/ref-1 cDNA was cloned into theEcoRI (59)
andXhoI (39)
sites of the retroviral vector LXSN as described previously
(16). Virus wasgenerated from the LXSN plasmid harboring Ape1/ref-1
(now designatedLAPESN), as performed previously (24). Briefly, 10mg
of agarose gel purifiedLAPESN were transfected into PE501 producer
cells, followed by selection inG418 (Life Technologies, Inc.) at 1
mg/ml. Supernatants were harvested andused to infect PG13
GALV-based amphotrophic packaging cells (25)and selected in G418.
Total RNA was extracted from the G418-resistant cellsand analyzed
by Northern blot hybridization with an Ape1/ref-1 probe
todemonstrate the 1.7-kb Ape1/ref-1 retroviral transcript.
Supernatants fromG418-resistant PG131 LAPESN cells were used to
transduce NT2 cells. Afterselection in G418 (1 mg/ml), individual
clones were isolated and expanded.Screening by Southern blot
analysis revealed two clones with distinct singleinserts that were
selected for further study. NT2 cells transduced with theempty LXSN
vector were produced in a similar manner as controls.
Cell Culture Studies. NT2, NT2-LXSN, and NT2-LAPESN cells
wereseeded (3–53 10 4 cells/well) in six-well trays and allowed to
adhereovernight. The following day, cells were treated with
bleomycin (Blenoxane;Bristol Myers Squib Company, Princeton, NJ) as
indicated or radiation. Bleo-mycin was dissolved in sterile water
(250mg/ml) and filter-sterilized througha 0.22mm filter prior to
being added to cultures, and radiation was deliveredfrom 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, andcounting using a hemocytometer.
The determination of AP endonuclease activity was performed
using anoligonucleotide cleavage assay as described previously (20,
26, 27). Briefly,cell extracts were incubated with a 59-32
P-end-labeled 26-mer oligonucleotidecontaining a single THF
artificial AP site at position 14, which, in the presenceof AP
endonuclease activity, cleaves the32P-labeled 26-mer to a 14-mer.
Theabasic analogue is resistant to cleavage by 39-acting AP lyase
activity, whichis generally possessed by DNA glycosylases/AP
lyases. Therefore, this assayis specific for Ape1/ref-1 activity in
cells. Reaction mixtures (10ml) containingcell extracts of
interest, 2.5 pmol of 5932P end-labeled, double-stranded
THFoligonucleotide, 50 mM HEPES, 50 mM KCl, 10 mM MgCl2, 1 mg/ml
BSA, and0.05% Triton X-100 (pH 7.5) were allowed to proceed for 15
min in a 37°Cwater bath. Reactions were halted by adding 10ml of
96% formamide, 10 mMEDTA, xylene cytanol, and bromphenol blue. AP
assay products (5ml) wereseparated on a 20% polyacrylamide gel
containing 7M urea. Gels werewrapped in saran wrap and exposed to
film for visualization. The amount of14-mer to 26-mer was
determined by scanning the exposed film into SigmaScan (Jandel
Scientific, San Rafael, CA) for analysis.
RESULTS
Expression of Human Ape1/ref-1 in GCTs.We examined theexpression
of Ape1/ref-1 in 10 GCT cases from a variety of histolog-ical
subtypes. Four tumors were yolk sac tumors, four were
metastaticteratomas, including one with primitive neuroectodermal
tumor ele-ments and one with cystic trophoblastic elements, and two
wereseminomas. Normal cells, including fibroblasts and vascular
endothe-lium, had a predominant nuclear staining pattern with
occasional cellsdemonstrating a mixed nuclear-cytoplasmic staining
pattern (Fig. 1).Infiltrating lymphocytes had little Ape1/ref-1
expression. Tumor cellsidentified in each section displayed an
increased level of expression ofnuclear Ape1/ref-1 compared with
stromal elements on the sameslides (Fig. 1 and Table 1). The
distribution of Ape1/ref-1 staining inthe GCTs was predominantly
nuclear in 50% of the cases and mixednuclear-cytoplasmic in the
other 50% (Table 1). There appeared to bea trend for the more
differentiated subtypes (teratomas) to have lesscytoplasmic
expression of Ape1/ref-1 compared with other subtypes(Table 1).
Given the small number of samples examined in this pilotstudy, no
statistical analysis was performed; however, a larger trialhas been
initiated that should permit more conclusive analysis ofApe1/ref-1
expression in GCTs and its relationship to clinical param-eters,
such as relapse and chemotherapy resistance.
Retroviral Gene Transfer of Ape1/ref-1 into NT2 Cells.
Asdetailed in “Materials and Methods,” we inserted a cDNA
fragment
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APE1 PROTECTS NT2 CELLS FROM BLEOMYCIN/RADIATION
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with multiple functions including a well-documented redox
function (7).Although Ape1/ref-1 appears to protect from bleomycin
andg-irradiationpresumably by means of repair activity, another
aspect, and somewhatcontrary to these results, is its potential
role in augmentation of theresponse to certain therapeutic agents.
Ape1/ref-1 involvement in activa-tion of p53 and induction of
cyclin G may be integral to the apoptoticresponse to cisplatin in
GCTs (9, 40). The high level expression ofApe1/ref-1 in GCTs in
conjunction with its role in activation of apoptosisthrough
p53/cyclin G may account in part for the sensitivity of GCTs
totherapy. Additionally, elevation of Ape1/ref-1 may be important
in main-taining the malignant phenotype through redox activation of
transcriptionfactors such as Fos, Jun, and HIF-1a (7). One way to
sort out these issuesis the use of site-specific Ape1/ref-1 mutants
to characterize the role ofthe repair/redox domains in response of
GCTs to specific therapeuticagents. Mechanistic studies should be
complemented with the examina-tion of Ape1/ref-1 expression in a
large sample of GCT patients todetermine the correlation, if any,
of expression with response to therapy.We have an ongoing protocol
to collect such data retrospectively andprospectively, with the
objective to analyze the relationship of Ape1/ref-1expression to
histological subtypes of GCTs, risk of relapse, response totherapy,
and role in tumor progression. Other questions also exist, suchas:
Does the cytoplasmic distribution of Ape1/ref-1 observed in
someGCTs result from an alteration in the nuclear localization
signal harboredin the 59-end of Ape1/ref-1? How does this change in
distribution affectrepair and redox function? In one primary
teratoma of the testis, weexamined only the intratubular GCT cells
marked with antihuman Ape1/ref-1 antibody, sparing the rest of the
normal cellular elements, suggest-ing that in certain settings,
staining with anti-Ape1 may be useful indetecting tumor cells.
Similar observations have been made in stainingsections of cervical
carcinomain situ for Ape1/ref-1 (19).
ACKNOWLEDGMENTS
We thank Steve Parsons for technical assistance.
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