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Molecular Oncology, Markers, Clinical Correlates |
Departments of Obstetrics and Gynecology [D. H. M.], Pathology [H. M.], and Pediatrics [R. T., S. H. P., M. R. K.], Section of Hematology/Oncology, Herman B Wells Center for Pediatric Research [R. T., S. H. P., M. R. K.], Indiana University School of Medicine, Indianapolis, Indiana 46202-5274
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DNA base excision repair involves two major classes of repair enzymes, the DNA glycosylases and APEs (3, 4, 5) . Glycosylases remove damaged bases, creating AP sites that in turn are incised by APE (APE/ref-1) 5' to the AP site, followed by removal of the abasic residue and insertion of a new base by DNA ß-polymerase and ligation. The rate-limiting step in the base excision repair pathway has not been defined in vivo (6, 7, 8) . However, in vitro evidence suggests that APE/ref-1 activity is the rate-limiting step in the repair of DNA oxidative damage (9) .
There is recent evidence that APE/ref-1 expression and base excision repair capabilities may not be equivalent in all cell types and tissues. APE/ref-1 is differentially expressed during fetal development and in the adult rat brain and testis, as well as in various human tissues (10, 11, 12) . Little is known about base excision repair functions in malignant tumors. A significant elevation in APE/ref-1 expression has been demonstrated in cervical cancer tissues and cell lines, as well as in germ cell tumors (13 , 14) . The expression of APE/ref-1 in ovarian tissues has not been characterized and is the focus of this investigation.
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MATERIALS AND METHODS |
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Antibody Preparation.
The initial antibody used in these studies was produced using
overexpression of the human APE/ref-1 protein in a
pGEX-glutathione-S-transferase Escherichia coli
system (Pharmacia, Uppsala, Sweden), as described previously (12
, 13
, 15)
, and was obtained from Novus Biologicals (Littleton,
CO). However, we have recently produced a monoclonal antibody that was
subsequently used in the studies presented here. Both polyclonal and
monoclonal antibodies yielded identical results. Antibody purity was
confirmed with Western blot analysis before each use (12
, 13)
, and each batch of slides was simultaneously processed using
the same antibody concentration. Tissues determined previously to
express APE/ref-1 were used as positive controls.
Immunohistochemistry.
Tissue sections were coated with anti-APE antibody (mouse anti-human
APE/ref-1 monoclonal) and incubated overnight at 4°C at a 1:200
dilution in 10% goat serum in PBS. The following day, sections were
washed three times for 5 min in PBS and incubated with biotinylated
goat antimouse IgG (Vector Labs, Burlingame, CA) at 15 µg/ml in 10%
goat serum for 1 h. After two PBS washes for 5 min each, sections
were incubated with avidin and biotinylated horseradish peroxidase
complex (ABC elite kit; Vector Labs) for 45 min. Slides were then
incubated with diaminobenzidine (Vector Labs). After the development of
color signal, the sections were briefly washed in distilled
H2O, counterstained with eosin, dehydrated
through a graded alcohol to xylene sequence, coverslipped, analyzed,
and photographed. As a negative control, preimmune IgG (50 µg/ml) was
used as the primary antibody in place of anti-APE.
Cell Lines.
Two ovarian cancer cell lines, Hey and Hey-C2, were supplied to us by
Dr. Gordon Mills (M. D. Anderson Cancer Center, Houston, TX). The Hey
cells were originally grown from an ovarian adenocarcinoma. By serially
passing Hey cells through athymic mice as i.p. tumors, Dr. Mills
et al. (16)
developed lines, A8 (not shown
here) and C2, that yield aggressive tumors capable of producing ascites
and killing hosts within 4–5 weeks.
Western Blot Analysis.
Western blots of Hey and Hey-C2 cells were performed using whole-cell
extracts electrophoresed on 12% SDS-PAGE and electroblotted onto 0.2
µm nitrocellulose. Protein levels in the extracts were quantitated
using the Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA), and
equal amounts (20 µg) of protein were loaded/lane. The gels were
transferred for 1 h onto nitrocellulose and stained with
Ponceau Red to confirm equal loading between lanes. The filter
was incubated in blocking buffer that contains 5% nonfat milk for
1 h. Mouse monoclonal antihuman APE/ref-1 (see
"Immunohistochemistry") was used at a dilution of 1:1000 and
incubated with the filter overnight at 4°C. The filter was washed in
TBST and incubated in TBST containing 5% milk and detected
using the Amersham chemiluminescence kit. The bands were visualized
using autoradiographic film and quantitated, after scanning, using
Jandel Scientific SigmaScan software.
AP Oligonucleotide Assay for APE/ref-1 DNA Repair Activity.
A 26-mer tetrahydrofuran-containing oligonucleotide was
32P end-labeled as described previously
(10
, 17, 18, 19)
. Reaction mixtures (10 µl) containing
protein extracts of Hey or Hey-C2 cells, 2.5 pmol of 5'
32P end-labeled double-stranded tetrahydrofuran
oligonucleotide, 50 mM HEPES, 50 mM KCl, 10
mM MgCl2, 2 mM DTT, 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 cyanol, 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 after scanning the exposed film into
SigmaScan.
Cellular Fractionation.
Fractionation of the Hey and Hey-C2 cells into nuclear and cytoplasmic
components was performed according to the method of Tell et
al. (20)
Briefly, cells (106)
were washed in PBS and resuspended in 5 ml of hypotonic lysis buffer A
(10 mM HEPES, 10 mM KCl,
0.1 mM MgCl2, 0.1
mM EDTA, 2 mg/ml leupeptin, 2 mg/ml pepstatin,
and 0.5 mM phenylmethylsulfonyl fluoride, pH
7.9). The cells were allowed to sit for 10 min and were then
homogenized in a Dounce homogenizer for 10 strokes. The nuclei were
collected by centrifugation for 5 min at 500 x g at
4°C in a microcentrifuge. The supernatant (cytoplasmic fraction) was
decanted from the nuclei, and nuclear proteins were extracted with 10
ml of buffer B (10 mM HEPES, 400
mM NaCl, 1.5 mM
MgCl2, 0.1 mM EDTA, 2 mg/ml
leupeptin, 2 mg/ml pepstatin, and 0.5 mM
phenylmethylsulfonyl fluoride, pH 7.9). After incubating for 20 min at
4°C, samples were centrifuged at 12,000 x g at 4°C
for 15 min. Nuclear and cytoplasmic extracts were quantitated for
protein levels (Bio-Rad assay) and used immediately for Western blot
analysis.
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RESULTS |
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. Normal ovarian tissues consistently
demonstrated strong nuclear staining of the surface epithelium,
epithelial inclusions, corpora lutea and albicantia, and stroma (Fig. 1)
. In one specimen obtained from a
postmenopausal woman, there were luteinized stromal cells with strong
nuclear staining. Cytoplasmic staining was absent in normal ovarian
tissues.
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. Endometriosis demonstrated moderate
to strong nuclear staining in both glands and stroma elements (Fig. 3)
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. Ovarian cancers were remarkably
different from all other tissues studied in the intensity and
subcellular localization of APE/ref-1 immunoreactivity. Nuclear
staining of the malignant epithelium ranged from strong to weak, with
considerable staining heterogeneity noted within the same tumor (Fig. 5)
. Cytoplasmic staining of the malignant
epithelium was frequently encountered and often predominant as the
subcellular location of immunoreactivity (Fig. 6)
. In general, considerable variation
was seen within and between malignant ovarian tumors with respect to
intensity and predominance of nuclear versus cytoplasmic
staining. There appeared to be no relationship between the presence,
intensity, or subcellular location of APE/ref-1 immunostaining and
either tumor grade or histological subtype.
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. We
detected a >4-fold increase in the level of APE/ref-1 protein. Western
blot analysis of protein extracts from nuclear versus
cytoplasmic components indicated that the overall increased level of
APE/ref-1 in the Hey-C2 cells, relative to the Hey cells, was mainly
attributable to an increase in cytoplasmic levels of the enzyme (Fig. 7)
. These data complemented the clinical data, showing an elevated and
altered expression of APE/ref-1 in the epithelial ovarian tumors and in
repair activity shown in the following experiments.
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. As can be seen by the activity assay,
the Hey cell line had no detectable levels <l50 ng of total cell
extract, whereas the Hey-C2 cells (same cell extract as shown in Fig. 7
, Lane 2) still had detectable activity at 40 ng of
cellular extract. Therefore, the Hey-C2 cells demonstrated a >4-fold
increase in APE activity in the AP oligonucleotide assay compared with
the Hey cells. The AP-site oligonucleotide cleavage DNA repair assay
results indicated that APE/ref-1 protein levels (Fig. 7)
correlated
well with appropriate APE/ref-1 repair activity changes (Fig. 8)
.
Assuming this in vitro relationship of increased APE/ref-1
protein signifying increased APE/ref-1 repair activity, then the
increase of APE/ref-1 observed in the immunohistochemical analysis of
human samples could correlate with increased in vivo DNA
repair activity. However, these observations do not preclude additional
alterations in redox functions of the APE/ref-1 protein, given that DNA
repair and redox functions reside in separate domains of the protein.
Thus, if the APE/ref-1 seen in ovarian cancer cells is primarily
functioning as a DNA repair enzyme, its movement out of the nucleus and
into the cytoplasm would have an impact on the ability of this protein
to carry out nuclear DNA repair activities. This could lead to
increased sensitivity of these tumor cells to DNA-damaging agents, or
the tumor cells may accumulate mutations because of decreased base
excision repair, leading to a more aggressive oncogenic phenotype.
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DISCUSSION |
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There is accumulating evidence that APE has additional cellular
functions beyond DNA repair. Xanthoudakis et al.
(25)
identified a human gene encoding a product involved
in the redox-activated DNA binding of several transcription factors
including FOS, JUN, and nuclear factor-
B. The gene product (ref-1)
proved to be identical to APE. A number of other transcriptional
activators are also regulated by redox modulation including: c-myb,
Ets, early growth response-1, the glucocorticoid receptor, members of
the activating transcription factor/cAMP-responsive element binding
family, and HIF-1
(26, 27, 28, 29, 30, 31)
. Redox regulation of
transcription factors may therefore exert an enormous influence on
cellular physiology, the end result being a net effect of positive and
negative interactions between the various transcription factors and
their downstream targets or effector genes.
Recently, it has been shown that the redox state of p53 affects its ability to bind to specific DNA sequences and subsequently regulate the transcription of adjacent genes (32) . The p53 redox state can be altered in vivo with increased oxidation, correlating with an impaired ability to effect downstream functions (33) . It is not surprising that APE/Ref-1 has been shown to be an important regulator of p53 function through both redox-independent and -dependent means (34 , 35) . These findings, along with the fact that hypoxic stresses induce the accumulation of both APE/ref-1 and p53, suggest plausible links between oxidative damage, the activation of DNA repair and redox transcriptional functions, and consequent influences on cellular proliferation and apoptotic processes.
In this study, we found marked differences in the pattern and intensity of APE/ref-1 immunostaining in ovarian cancer specimens versus all other ovarian conditions: normal ovaries, benign lesions, and LMP ovarian cancers. Strong nuclear staining was found in cells of the normal ovary surface epithelium, the epithelium of benign tumors and endometriosis, and the epithelium of LMP tumors. In contrast, nuclear staining of cells in the epithelium of ovarian malignancies ranged from strong to weak, with considerable staining heterogeneity frequently found within the same tumor. Cytoplasmic staining was frequently encountered in ovarian malignancies and often the predominant site of APE/ref-1 immunoreactivity. Two LMP tumors also had infrequent areas of cytoplasmic staining. These immunohistochemical differences parallel the clinical differences in the respective ovarian states. Although LMP tumors may metastasize, they are generally associated with a good prognosis and may be treated with conservative surgery and without need for adjuvant chemotherapy (36 , 37) . Clinical data for the patients from whom specimens were obtained are unknown. It would be interesting to know whether the two LMP tumors with (infrequent) APE/ref-1 cytoplasmic immunoreactivity came from patients with metastatic disease.
On the basis of our observations, wide variations in nuclear staining intensity and the presence of strong cytoplasmic staining in ovarian tissues are correlates of malignant behavior. In previous studies, APE/ref-1 expression during fetal development and in a variety of adult tissues was predominantly nuclear, although cytoplasmic staining was seen in certain parts of the brain and in the liver (10 , 12) . Using animal models, others have reported both nuclear and cytoplasmic APE/ref-1 expression in certain brain tissues (pyramidal neurons of the hippocampus and granular cells of the dentate gyrus) in response to ischemia (38) . APE/ref-1 expression is markedly elevated in preinvasive and invasive cervical neoplasia (13) and in germ cell tumors (14) but is exclusively nuclear. Our observations in ovarian tumors parallel those by Kakolyris et al. (39) , who reported a marked disruption in the APE/ref-1 staining pattern in colonic adenomas/carcinomas. In the normal colonic mucosa, APE/ref-1 staining was predominantly nuclear in the lower part of the crypts (less differentiated cells) and cytoplasmic in the superficial colonic epithelium (more differentiated cells). The expression and localization of APE/ref-1 in hyperplastic polyps were similar to the normal colonic epithelium. For adenomas and carcinomas, however, immunostaining was exclusively cytoplasmic in 37 and 50%, respectively, and both nuclear and cytoplasmic staining were seen in 53% of adenomas and 39% of carcinomas (39) .
The implications of cytoplasmic staining in ovarian tumors are unknown. We cannot determine by these studies the in vivo functional status of APE/ref-1 but can only speculate whether the observed differences in ovarian malignancies represent alterations in DNA repair functions, redox functions, or both. Increased expression does not signify that both DNA repair and redox functions are changed. The DNA repair and redox activities of APE/ref-1 reside in two separate domains, with the NH2 terminus primarily involved in redox regulation and the COOH terminus primarily involved in DNA repair (30) . The DNA repair activity of APE/Ref-1 may be inactivated by phosphorylation (19) . Furthermore, AP sites may occur in mitochondrial DNA as a result of oxidative damage, and base excision repair is important for the repair of AP sites in mitochondrial DNA (40 , 41) . Thus, APE/ref-1 may have both cytoplasmic and nuclear functions in base excision repair.
Given the abundance of APE/ref-1 and its pleiotropic cellular functions, further work will likely identify a number of posttranslational events for regulating specific APE/ref-1 activities. A greater understanding of alterations in base excision repair and redox functions of APE/ref-1 in human cancers has epidemiological and therapeutic significance.
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FOOTNOTES |
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1 Supported by NIH/National Cancer Institute
Program Project Grant PO1-CA75426 and NIH Grants RR09884 and ES07815
(all to M. R. K.). 
2 To whom requests for reprints should be
addressed, at Department of Obstetrics and Gynecology, Indiana
University School of Medicine, 535 Barnhill Drive, RT 433,
Indianapolis, IN 46202-5274. Phone: (317) 274-2422; Fax:
(317) 274-4878.
3 The abbreviations used are: AP,
apurinic/apyrimidinic; APE, AP endonuclease; ref-1, redox factor-1;
LMP, low malignant potential.
Received 7/ 9/99; revised 11/ 9/99; accepted 11/15/99.
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REFERENCES |
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