bcl-2, bax, bcl-xL, and bcl-x Expression in Normal and ... · Table 1 Distribution of bcl-2, bax,...
Transcript of bcl-2, bax, bcl-xL, and bcl-x Expression in Normal and ... · Table 1 Distribution of bcl-2, bax,...
Vol. 4, 517-524, February 1998 Clinical Cancer Research 517
3 The abbreviations used are: PCD, programmable cell death; RT, re-
verse transcriptase; ER, estrogen receptor; PR, progesterone receptor.
bcl-2, bax, bcl-xL, and bcl-x� Expression in Normal and Neoplastic
Ovarian Tissues’
Maria Marone, Giovanni Scambia,
Simona Mozzetti, Gabriella Ferrandina,
Sonia lacovella, Anna De Pasqua,
Pierluigi Benedetti-Panici, and
Salvatore Mancuso2
Laboratory of Antineoplastic Pharmacology, Department of Obstetrics
and Gynecology. Catholic University, 00168 Rome, Italy
ABSTRACT
The bcl-2 family of proteins includes some important
regulators of apoptosis. Among these, bcl-2 and bcl-xL pre-
vent cells from entering apoptosis, whereas bax and bcl-x�can induce cell death. Alterations in the control of this
process can lead to a decrease in cell death, thus contribut-
ing to neoplastic growth. Diminished susceptibility to chem-otherapy has also been attributed, in in vitro systems, toalterations in the levels of bcl-2, bax, or bcl-x. We analyzedthe expression of bcl-2, bax, bcl-xL, and bcl-x� in normal andneoplastic ovarian tissues by reverse transcriptase-PCR andWestern blotting. The RNA and protein levels were signifi-
cantly correlated for all genes. Interestingly, the levels ofthese genes in normal and neoplastic tissues were signifi-
cantly different: bcl-2 was higher in normal tissue (P <
0.002), whereas bax and bcl-xL were higher in carcinoma
(P < 0.018 and P < 0.030, respectively). bcl-x� was present
at low levels in 83% of neoplastic samples and was unde-
tectable in normal tissue. Reverse transcriptase-PCR anal-
ysis of 74 tumors showed no major correlation with chico-pathological parameters or with response to chemotherapy.
Only bax and bcl-xL were correlated with progesterone re-ceptor levels (n = 29, r = +0.44, P < 0.0189, and r = -0.40,
P < 0.035, respectively). No correlation was found withestrogen receptor levels or with p53 immunostaining. Ourdata indicate that the regulation of the bcl-2 family of pro-
teins differs between normal and neoplastic ovarian tissues.Moreover, the modulation of these genes in ovarian carci-
noma is different compared to other tissues; therefore, tissue
specificity is very important in regulation of the bcl-2 family
of proteins.
Received 7/25/97; revised 10/20/97; accepted 1 1/7/97.
The costs of publication of this article were defrayed in part by thepayment 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.
1 This work was partially supported by Associazione Italiana per la
Ricerca sul Cancro.
2 To whom requests for reprints should be addressed, at Department ofObstetrics and Gynecology, Catholic University, L.go A. Gemelli 8,00168 Rome, Italy. Phone: 39-6-35508736; Fax: 39-6-35508736; E-mail: [email protected].
INTRODUCTION
Cell turnover in normal tissues is regulated by the balance
between the rates of cell proliferation and cell death (1). Con-
sequently, uncontrolled neoplastic growth can be caused not
only by increased proliferation but also by a diminished rate of
cell death, which can result from the failure of cells to undergo
apoptosis or from PCD3 in response to physiological stimuli (1,
2). In addition to this role in the onset or development of cancer,
the modulation of apoptosis can also influence the outcome of
cancer treatment because drug resistance can be attributed partly
to a decreased cellular susceptibility to PCD (3). bcl-2 was the
first characterized gene that was clearly involved in the regula-
tion of PCD by inhibiting apoptosis, thus promoting cell sur-
vival (4). It was shown that transfection of bcl-2 into immature
pre-B cells allowed prolonged survival in the absence of sur-
vival factors (5) and that high levels of bcl-2 protect cells from
apoptosis induced by y irradiation and by a variety of chemo-
therapeutic agents (6).
bcl-2 belongs to a still growing family (7, 8), the members
of which are able to form homo- and/or heterochimers among
themselves; their association and the relative ratio between pro-
and antiapoptotic proteins are responsible for directing the cells
toward death or survival (9, 10). Overexpression of bax was
shown to accelerate PCD by inhibiting the death repressor
activity of bcl-2, probably by forming bcl-2-bax complexes or
by competing with other bcl-2 targets (1 1, 12). The bcl-x gene
mainly gives rise, by alternative splicing, to two mRNA species,
yielding two protein products: bcl-x�, endowed with death re-
pressor activity, and a shorter variant, bcl-x�, which functions as
a dominant inhibitor of bcl-2, thus inducing apoptosis (13).
Although bcl-2 has been described as a molecule involved in a
variety of biochemical pathways (14, 15), the mechanisms by
which the interactions between the various members of the bcl-2
family finally lead to apoptosis are still unknown.
Expression of bcl-2 has been measured in a variety of
human neoplastic tissues, including melanoma (16), non-small
cell lung (17), prostate (18), and breast (19, 21). Several studies,
mostly on breast cancer, reported a direct correlation between
bcl-2 and the steroid hormone receptor status (21, 22), and a
correlation between bcl-2 levels and survival has been reported
but is still controversial (23, 24). Moreover, low bax expression
in breast cancer was shown to be associated with poor response
to chemotherapy and shorter survival (25). Expression of bcl-2
alone has been studied in ovarian cancer as well, suggesting
that, as in breast, high bcl-2 levels may be associated with
longer survival (26, 27).
Here, we determined the protein and RNA expression
levels of bcl-2, bax, bcl-xL, and bcl-x� in a series of normal and
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Table 1 Distribution of bcl-2, bax, bcl-xL and bcl-x5 RNA levels in normal and neoplastic ovarian tissues
The values are the ratio between the relative adsorbance of the RNA of interest and aldolase-A RNA obtained by RT-PCR.
n
bcl-2 bax
Mean Median SD RangeMean Median SD Range
Normal 6 6.36 6.91 2.73 4.07-10.0” 0.90 0.88 0.23 0.59�1,01”
All carcinomas 70 2.23 2.04 1.61 0.01-8.69 2.17 1.75 2.10 0.40-13.00Primary cancers 44 2.18 2.14 1.34 0.10-7.20 2.06 1.81 2.46 0.40-13.00Metastases 7 2.23 2.36 0.91 1.01-3.72 2.37 1.87 1.51 0.83-5.41
Recurrences 19 2.61 1.79 1.59 0.38-8.69 1.73 1.61 0.70 0.45-3.02
n
665
42
6
17
bcl-xL bcl-x�ND cases
(n)
6
11
8
1
2
Range Mean
0. 13�0.20c ND”
0.01-0.76 0.13
0.01-0.71 0.13
0.24-0.47
0.11-0.76 0.16
Median
ND
0.14
0.14
0.08
0.15
SD
ND
0.08
0.08
0.07
0.06
Range
ND
0.02 -0.30
0.02 -0.30
0.07 -0.23
0.04 -0.26
Mean
0. 160.31
0.33
0.32
0.31
Normal SD
0. 15 0.070.29 0.19
0.28 0.18
0.30 0.08
0.28 0.19
518 bcl-2 Family in Normal and Neoplastic Ovary
Normal
All carcinomasPrimary cancers
Metastases
Recurrences
a p < 0.002.bp < 0.018.
C p < 0.030.d ND, not detectable.
cancer ovarian samples and analyzed their correlation with some
clinicopathological and other biological parameters.
PATWNTS AND METHODS
Patients and Tumor Specimens. The patients involved
in this study underwent surgery between 1994 and 1996 at the
Department of Obstetrics and Gynecology of the Catholic Urn-
versity (Rome, Italy). Necrotic, hemorragic and fat tissues were
trimmed out from the tumor samples, which were then cut in
half so that the same histology and grade represented in the
samples was used for histopathological analysis and RNA and
protein extraction. One half was processed for histopathological
examination to determine the percentage of tumor cells in the
samples, which was never lower than 80%. The other half was
used for RNA preparation and, if there was a sufficient amount,
for protein extraction. Tissues were frozen in liquid nitrogen
immediately after surgical resection and stored at -80#{176}Cuntil
processed. Total RNA was extracted from six normal ovarian
tissue biopsies from patients with uterine fibromatosis and from
70 neoplastic specimens (Table 1). Protein lysates were pre-
pared from 1 1 normal ovarian tissue biopsies and from a subset
of 20 neoplastic tissue samples taken from the same population
used for RNA extraction. Due to the limited size of the bioptic
specimens, in most cases, the material was sufficient for RNA
extraction only. Normal tissues came from 1 1 patients with
uterine fibromatosis, who ranged in age from 38 to 52 years old
(mean, 48 years; median, 47 years), 7 of whom were premeno-
pausal and 4 who were postmenopausal. The neoplastic tissues
included mostly primary ovarian cancers and also some meta-
static and recurrent tumors (Table 1). Histological classification
of tumors was carried out according to the WHO system (28)
and tumors were graded as well (Gl), moderately (G2) or poorly
(G3) differentiated. Stage of disease was established according
to the FIGO staging system. All patients received cisplatin-
containing chemotherapy, which was instituted 2-3 weeks after
surgery. Specimens of recurrent disease were obtained from
pelvic or peritoneal tumor masses occurring after a period of
clinical and pathological negative follow-up after intensive sur-
gery and chemotherapy. Recurrent and metastatic tissues shared
the same histological type and grade as their respective primary
tumors. The clinicopathological features of the patients bearing
primary ovarian tumors are described in Tables 2 and 3.
Semiquantitative RT-PCR Analysis. Total cellular
RNA was obtained from tissue biopsies by the method of
Chomczynsky and Sacchi (29). Five p.g of RNA per sample
were separated on 1 % formaldehyde-agarose gels to assess their
integrity. The Perkin-Elmer Gene Amp RNA PCR kit was used
for all the RT-PCRs, which were performed in the Gene Amp
PCR system 9600 (Perkin-Elmer/Cetus, San Diego, CA). After
removal of contaminating chromosomal DNA with DNase I
treatment, 1 p�g of RNA was reverse-transcribed with 25 units of
Moloney murine leukemia virus RT at 42#{176}Cfor 30 mm. Two p.1
of cDNA products were used in each PCR. The PCR were
carried out using different cycling parameters for each set of
primers. The number of cycles and the reaction conditions were
chosen so that none of the target cDNAs reached a plateau and
that the two pairs of primers did not compete with each other.
The sequences of the specific primers for bcl-2 and bax were
described previously (30), and the primers corresponding to the
bcl-x cDNA were chosen so that the two specific products,
bcl-xL and bcl-x�, migrated as distinct bands on a 2% agarose
gel (36). The target cDNAs were coamplified with aldolase-A as
an internal control using the following pair of primers: 5’-
CGCAGAAGGGGTCCTGGTGA-3’ and 5’-CAGCTCCTFCT-
TCTGCTCCGGGGT-3’, which yielded a band of 176 bp. All
oligonucleotide primers were synthesized by Pharmacia Biotech
(Uppsala, Sweden).
bcl-2 was amplified with 1 unit of AmpliTaq DNA polym-
erase in 1 mM MgC12, and a first cycle of 2 mm at 95#{176}C,45 s at
55#{176}C,and 1 mm at 72#{176}Cwas followed by 32 cycles of 45 s at
95#{176}C,45 5 at 55#{176}C,and 1 mm at 72#{176}C.bax was amplified using
1 unit of AmpliTaq DNA polymerase in 5 mr�i MgC12 as
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Clinical Cancer Research 519
Table 2 Distribution of bcl-2 and bax RNA levels in primary ovarian carcinomas according to clinico-pathological features
The values are the ratios between the relative adsorbance of the RNA of interest and aldolase-A RNA obtained by RT-PCR.
Clinicopathological
feature n
bcl-2 bax
Mean Median Range SD Mean Median Range SD
Histology
Serous 30 1.98 1.91 0.01-3.55 0.87 2.55 1.54 0.40-13.00 2.78Mucinous 1 2.04
Endometrioid 6 3.78 2.23 0.54-7.20 2.51 1.98 2.15 0.73-2.77 0.75
Undifferentiated 4 2.39 2.48 2.02-7.13 2.82 2.52 2.84 1.46-3.26 0.94
Other 3 2.87 1.73 1.60-3.85 1.26 5.17 5.74 3.99-5.80 1.02
Stage
I-Il 6 2.11 2.20 0.54-3.33 1.78 4.29 3.38” 1.55-9.44 3.02
HI 30 2.28 2.04 0.01-7.13 1.22 2.45 1.60 0.40-13.00 2.47
IV 8 2.45 2.21 0.54-7.20 2.01 1.54 1.50 0.64-2.35 0.65Grading
1-2 9 2.36 2.48 0.54-3.33 0.52 2.65 2.15 1.55-9.44 1.02
3 29 2.21 2.04 0.01-7.20 1.43 2.48 1.64 0.50-13.00 2.78
Ascites
No 24 2.01 2.03 0.54-3.33 0.81 2.69 1.95 0.64-9.44 2.13
Yes 20 2.69 2.19 0.01-7.20 1.78 2.48 1.61 0.50-13.00 2.84
Residual tumor”�2 cm 23 2.35 2.11 0.54-7.13 1.27 1.99 1.60 0.40-6.15 1.33>2 cm 15 2.26 2.02 0.01-7.20 1.67 2.63 1.61 0.50-13.00 3.14
Response to
chemotherapyc
CR 14 2.55 2.19 1.30-7.13 1.52 1.97 1.54 0.40-6.15 1.47PR 12 1.79 1.58 0.54-3.13 0.83 2.07 2.04 0.85-3.96 0.98NC-P 12 2.60 2.24 0.01-7.20 1.79 2.45 1.22 0.50-13.00 3.50
Age (yr)
�60 29 2.26 2.11 0.54-7.13 1.31 2.39 2.04 0.64-6.26 1.52
>60 15 1.84 1.87 0.01-2.97 0.77 3.69 1.60 0.50-13.00 4.07
P < 0.009.a Stage I-Il vs. stage III-IV,
b Only stage III-IV patients.C CR, complete response; PR, partial response; NC-P. no change-progression.
follows: 2 ruin at 95#{176}C,45 s at 65#{176}C,and 1 mm at 72#{176}Cfor the
first cycle and then 45 s at 95#{176}C,45s at 65#{176}C,and 1 mm at 72#{176}C
for 26 cycles. One unit of AmpliTaq Gold in 5 mivi MgC12 was
used for bcl-x using the same cycling parameters applied for
bax, except that the first denaturation step was extended to 10
mm, which was then followed by 28 cycles, as described above.
The PCR products were loaded onto 2% agarose gels and
stained with ethidium bromide. For bcl-2 and bcl-x, images of
the gels were acquired with a Cohu charged coupled device
camera, and quantification was performed with Phoretix ID
(Phoretix International Ltd., Newcastle upon Tyne, United
Kingdom). bax was undetectable by ethidium bromide staining
in most neoplastic samples; hence, the PCR products were
separated on 1 .2% agarose gels, blotted onto nylon membranes
(Duralon; Stratagene, La Jolla, CA), UV-cross-linked and hy-
bndized at 42#{176}Cfor 18 h in 50% formamide, 5 X SSC, 1 X
Denhardt’s solution, 0.2% SDS, and 200 p.g/ml denatured
salmon sperm DNA. About 50,000 cpm/ml of 32P PCR-labelcd
bax and aldolase-A probes were added to the reaction. After
washing, the blots were exposed to an Instant Imager Electronic
Autoradiography Instrument, and band intensity was measured
using the Imager software provided with the instrument (Pack-
ard Instrument Company, Meridian, CT). Each set of reactions
and each blot included the ovarian cancer cell line A2780 as a
positive control and a no-sample negative control. The ratio
between the sample RNA to be determined and aldolase-A was
calculated to normalize for initial variations in sample concen-
tration and as a control for reaction efficiency. All samples were
normalized to their control. Reproducibility of the method was
established by analyzing the level of variability in the control
sample A2780 in different experiments and different gels, which
was never greater than 20%.
Preparation of the Tissue Lysates and Western BlottingAnalysis. Frozen tissues were homogenized in S volumes of
lysis buffer [20 mivi Tris-HC1 (pH 7.4), 0.1 M NaC1, Smsi MgC12,
1% Nonidet P-40, 0.5% sodium deoxycholate, and 2 kallikrein
inhibitor units/mi aprotinin]. The protein concentration was
determined using the Bio-Rad Protein Assay (Bio-Rad Labora-
tories, Hercules, CA). SDS-PAGE and Coomassie blue staining
was performed for all samples as a control for degradation prior
to Western blotting.
For bax and hcl-x detection, 50 p.g of each protein sample
were separated on a 12% SDS-polyacrylamide gel and electro-
blotted onto nitrocellulose membranes (Bio-Rad). After electro-
blotting, the membranes were incubated with 6% nonfat dry
milk in 1 X TBST [0. 1 M Trizma base, 0. 15 M NaC1, and 0.05%
Tween 20 (pH 7.4)] for blocking and then with the primary
antibody in 3% nonfat dry milk in 1 X TBST. Following incu-
bation with an alkaline phosphatase-conjugated goat antirabbit
antibody, visualization of the bound antibody was performed
with the BCIPINBT Phosphatase Substrate System (Kirkegaard
& Perry Laboratories, Gaithersburg, MD). For bcl-2 detection,
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Table 3 Distribution of bcl-XL and bcl-x5 RNA levels in primary ovarian carcinomas according to clinicopathological features
The values are the ratios between the relative adsorbance of the RNA of interest and aldolase-A RNA obtained by RT-PCR.
Clinicopathological
feature
bcl-xL bcl-x5
n Mean Median Range SD n Mean Median Range SD
Histology
Serous 29 0.33 0.27 0.10-0.71 0.18 23 0.14 0.13 0.02-0.30 0.09
Mucinous I 0.19 1 0.27
Endometrioid 6 0.29 0.29 0.21-0.50 0.12 5 0.10 0.10 0.02-0.19 0.07
Undifferentiated 4 0.34 0.39 0.18-0.46 0.14 3 0.09 0.12 0.02-0.14 0.06
Other 2 0.34 0.34 0.09-0.60 0.36 2 0.14 0.14 0.06-0.22 0.11
Stage
I-I! 5 0.28 0.32 0.21-0.34 0.15 4 0.04 0.04 0.02-0.07 0.08
III 29 0.32 0.27 0.09-0.71 0.17 23 0.14 0.14 0.02-0.30 0.09IV 8 0.33 0.33 0.14-0.71 0.18 7 0.13 0.14 0.02-0.21 0.07
Grading
1-2 9 0.24 0.27 0.14-0.34 0.06 8 0.12 0.12 0.02-0.27 0.09
3 29 0.31 0.28 0.09-0.71 0.18 24 0.11 0.08 0.02-0.30 0.09
Ascites
No 22 0.33 0.25 0.09-0.71 0.20 19 0.1 1 0.10 0.02-0.27 0.09
Yes 20 0.33 0.32 0.10-0.60 0.14 15 0.16 0.16 0.04-0.30 0.08
Residual tumor”�2 cm 22 0.36 0.35 0.09-0.71 0.18 18 0.16 0.19 0.02-0.30 0.09>2 cm 15 0.28 0.19 0.10-0.60 0.15 12 0.12 0.14 0.02-0.22 0.06
Response to
chemotherapy”
CR 13 0.32 0.35 0.09-0.71 0.19 10 0.11 0.12 0.02-0.25 0.06PR 12 0.39 0.43 0.14-0.71 0.18 9 0.18 0.17 0.02-0.27 0.08
NC-P 12 0.28 0.26 0.13-0.60 0.14 10 0.13 0.13 0.02-0.30 0.09Age (yr)
�60 29 0.31 0.28 0.01-0.70 0.17 23 0.13 0.12 0.02-0.27 0.06
>60 13 0.29 0.21 0.09-0.7 1 0. 18 1 1 0.09 0.05 0.02-0.27 0.08
a Only stage III-IV patients.S CR, complete response; PR, partial response; NC-P. no change-progression.
520 bcl-2 Family in Normal and Neoplastic Ovary
100 p.g of each protein sample were separated on a 15% SDS-
polyacrylamide gel and, after clcctroblotting onto polyvinyli-
dene fluoride (Millipore Co., Bedford, MA), the membranes
were incubated with the primary antibody, as described above.
The membranes were then transferred first to a biotinylated
secondary antibody and then to an avidin-biotin-horseradish
peroxidase complex (ABC Vectastain Elite; Vector Laborato-
rics, Burlingame, CA), and detection was performed with the
3,3-diaminobenzidinc peroxidase substrate kit (Vector Labora-
tories) in 1 x TBST. Images of the blots were acquired with a
Cohu charged coupled device camera, and quantification of the
bands was performed by Phoretix 1 D (Phoretix International).
Band intensity was expressed as relative adsorbance. The fol-
lowing antibodies were used: mouse antihuman bcl-2 (clone
124; DAKO, A/S, Glostrup, Denmark), rabbit antihuman bax,
and rabbit antihuman bcl-x (Santa Cruz Biotechnology, Santa
Cruz, CA). The latter antibody reacts with both bcl-xL and
bcl-x5, which can be distinguished by their different molecular
weights on a SDS-polyacrylamidc gel (Mr �30,000 and 18,000,
respectively).
p53 Analysis. p53 positivity was evaluated by immuno-
histochemistry on formalin-fixed, paraffin-embedded sections
using the antimouse antibody (clone DO-7; Oncogene, Manhas-
set NY; Ref. 31).
ER and PR Analysis. The cytosolic fractions of tissue
samples, prepared as described earlier (32), were used for ER
and PR assays, which were carried out by the dextran-coated
charcoal method according to the EORTC protocol (32).
Statistical Analysis. The Pearson’s correlation test was
used to analyze the relationship between RNA and protein levels
and the correlations among the bcl-2 family members. The
Mann-Whitney and Kruskal-Wallis nonparametric tests were
used to analyze the distribution of bcl-2, bax, bcl-xL, and bcl-x�
levels according to the clinicopathological characteristics of the
cases.
RESULTS
bcl-2, bax, bcl-xL, and bcl-x� RNA Expression Levelsare Different in Normal and Neoplastic Ovarian Tissues.bcl-2 and box RNA expression levels were measured by semi-
quantitative RT-PCR on 76 ovarian samples, which included 6
normal tissues, 44 primary carcinomas, 7 omental metastases,
and 19 recurrent carcinomas. bcl-x RNA levels were assessed on
a total of 71 samples, comprising six normal tissues, 42 primary
tumors, 6 omental metastases, and 17 recurrent tumors. The
reliability of the technique was established by comparing the
values obtained in six different experiments for the ovarian
cancer cell line A2780, which was used as a control. The values
were: bcl-2, mean, 2.11, SD, 0.21; bax, mean, 1.22, SD, 0.24;
bcl-xL, mean, 0.46, SD, 0.03, bc1-x�, mean, 0.09, SD, 0.02. All
the tissue samples showed a detectable band of the expected
molecular size of 385 bp for bcl-2 (Fig. lA) and of 538 bp for
bax (Fig. 1B). bcl-xL and bcl-x5 were amplified by the same pair
of primers and were visible as two distinct bands of 780 and 591
bp, respectively, in the same gel (Fig. lC). Although bcl-xL was
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. bcl-2
- . aldolaseA A w-w �
I 23456789
�::=� � � bcl-2
B bax
� �LbcI�x
Fig. 2 Western blot of bcl-2 (A), bax (B). and bcl-x�� (C) in normal(Lanes 1-4) and neoplastic (Lanes 5-8) ovarian tissues. Lane 9, positivecontrols, which are the leukemic T cell line Jurkat (A) and the ovariancancer cell line A2780 (B and C).
Clinical Cancer Research 521
I 2 3 4 5 6 7 9 9101112131415 1817
A � � --
B ��:::�±�:: . -bax
#{149}.i:� � #{149}- �- - -� 6 - aldolase A
I- ‘ - �.bcI-x1I -- � � . .-‘-� . “bcl-x9
� . . aldolase A
Fig. 1 Semiquantitative RT-PCR analysis of bcl-2 (A), box (B), andbcl-x,� (C) in primary ovarian cancers (lanes 1-8), mctastases (lanes
9-11) and recurrent diseases (lanes 12-15). The histological types ofthe tumors were: Lanes 3, 5, and 10, endometrioid; Lane 4, undifferen-tiated. The remaining lanes were all serous. Lane 16, ovarian cancer cellline A2780, used as positive control; Lane 17, negative (no sample)control. A and C, photographs of ethidium bromide-stained gels. B,
autoradiography of the RT-PCR analysis, which was transferred to filter
and hybridized with a bax cDNA probe.
present in all samples, bcl-x5 was detectable in only 83% of the
tumors (54 of 65) and was undetectable in normal samples
(Table 1). The RNA levels of these genes, when analyzed in the
whole population, were not normally distributed and were
slightly skewed to the left (data not shown). Median and range
values of bcl-2, bax, bcl-xL and bcl-s5 did not show any signif-
icant variation according to type of disease (Table 1). bcl-2
levels were significantly higher in normal than in neoplastic
ovarian tissues (P < 0.002); conversely, both bax and bcl-xL
RNA levels were lower in normal than in neoplastic samples
(P < 0.018 and P < 0.030, respectively; Table 1). The RNA
levels of bcl-2, box, bcl-xL, and bcl-x5 were not significantly
correlated with each other (data not shown). The distributions of
bcl-2, box, bcl-xL, and bcl-x� values according to the clinico-
pathological features of the cases examined are shown in Tables
2 and 3. box levels were higher in stage I and II than they were
in advanced-stage tumors (P < 0.009). bcl-2, bcl-xL, and bcl-x5
were not associated with any of the clinicopathological features
considered.
Protein Levels of bcl-2, bax, and bcl-xL Correlate with
RNA Levels. The protein expression levels of bcl-2, bax,
bcl-x�, and bcl-x5 were determined by Western blotting. bcl-2
and bax were measured on 20 ovarian carcinomas and 11
normal ovarian samples, whereas bcl-x expression was meas-
ured on 16 carcinomas and 8 normal ovarian samples. A band of
the expected M1 of 26,000 for bcl-2 (Fig. 2A) and of 21,000 for
bax (Fig. 2B) was detectable in all samples. Both isoforms of
bcl-x, bCl-XL as a Mr 30,000 band and bc1-x� as a Mr 18,000
band, were present in the A2780 control cell line, together with
an additional band of higher molecular weight (Fig. 2C, Lane 9).
In normal samples, bcl-xL was present together with this higher
molecular weight form (Lanes 1-4). Only the Mr 30,000 band
was visible in the neoplastic samples (Fig. 2C, Lanes 5-8),
some of which also contained additional bands that could cor-
respond to different isoforms (as in Lanes 5 and 8). We analyzed
only the Mr 30,000 band. bcl-x� was not present in any of the
tissue samples analyzed. The difference between normal and
neoplastic tissues is shown in the box and whisker plots in Fig.
3. bcl-2 levels were significantly lower in neoplastic (median,
112.0; range, 23.0-328.0) than in normal samples (median,
175.3; range, 97.0-265.9; P < 0.038; Fig. 3A). bax protein
levels followed the opposite trend (Fig. 3B), i.e., they were
higher in neoplastic (median, 1040.0; range, 398.0-1523.0) than
in normal samples (median, 236.0; range, 103.3-1040.0; P <
0.006). bcl-xL (Fig. 3C) was expressed at lower levels in normal
(median, 4,164.9; range, 1,036-8,967) than in neoplastic sam-
ples (median, 14,365; range, 7,544-17,485; P < 0.0033). Anal-
ysis of these proteins in normal and cancer tissues confirmed the
trend observed at the RNA level. The protein levels of the bcl-2
family members analyzed were directly correlated with their
corresponding RNA levels (Fig. 4), as follows: bcl-2, r =
+0.50, P < 0.047; bax, r = +0.44, P < 0.038; bcl-xL, r 0.53,
P < 0.023. bcl-x5, which was present at very low levels, as
measured by RT-PCR, was undetectable by Western blot in all
tissue samples.
Biological Correlation. p53 positivity was determinedby immunohistochemistry on 27 cases, 8 of which were positive
(30%). The RNA levels of bcl-2, bax, bcl-xL, and bcl-x� did not
show any significant correlation with p53 positivity. ER levels,
measured on 29 cases, ranged from 0.01 to 73.6 fmol/mg of
protein (mean, 16.6; median, 10.3; SD, 17.7) and were not
correlated with the RNA level of the bcl-2 family members
analyzed. The PR levels, which were measured on 29 samples
(range, 0.01-159.9 fmol/mg of protein; mean, 19.4; median,
10.6; SD, 10.6), although not correlated with bcl-2, were di-
rectly correlated with box (r +0.44, P < 0.019) and inversely
correlated with bcl-xL (r = -0.40, P < 0.035). In an attempt to
take advantage of the biological information gathered from each
of the parameters examined, we tried to combine bcl-2, box,
bcl-xL, and bcl-x� to define the susceptibility of each sample to
apoptosis. Positivity for the four parameters was defined on the
basis of the median levels for each of them. Cases that were
Research. on September 20, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Normal Neopiastic
A
B
C
.
300
250
c� 200
U
� 150
100
50
0
2000
1600
)( 1200
� 800
400
0
20000
16000
12000
�8000
4000
0
#{149}_.,_-s_-
500
400
I:100
C 20000
16000
.c 12000
4000
Normal Neopiastic
Fig. 3 Box and whisker plots showing the difference in protein levels
between normal and neoplastic ovarian tissues of bcl-2 (A), bax (B), andbcl-x1 (C). -. Median values.
1 2 3 4 5 6
RNA
0 10 20 30 40 50 60 70 80
ANA
522 bcl-2 Family in Normal and Neoplastic Ovary
Normal Neoplastic
negative for bcl-2 and bcl-xL and positive for box and bcl-xs
were considered to represent a situation such that apoptosis
would be maximally favored. Cases that were positive for bcl-2L
and bcl-xL and negative for box and bcl-x� were considered to
represent a situation of maximal resistance to apoptosis. Sub-
groups representing intermediate situation were also taken into
consideration. No case fell into the category of maximal sus-
ceptibility to apoptosis. Nineteen of 70 cases (27%) fell into the
category of maximal resistance to apoptosis, and 5 1 of 70 cases
(73%) belonged to an intermediate situation, in which any
combination of the four genes is possible, so that apoptosis
would be determined by a mixture of contrasting inputs. These
subgroups were not differently distributed according to response
to chemotherapy.
DISCUSSION
The aim of this study was to elucidate the role of bcl-2 and
some bcl-2-rclated proteins (bax, bcl-xL, and bcl-x5) in ovarian
tissues. bcl-2, bax, and bcl-xL were present to a variable degree
in all samples tested, and their RNA and protein levels were
A 400
350
300C
:B 2500
�200
150
100
50
0 1 2 3 4 5
RNA
Fig. 4 Correlations between the RNA and protein levels of bcl-2 (A),
bax (B), and bd-XL (C).
directly correlated. bcl-x5, the RNA of which was detectable at
low levels in most tumor samples, was undetectable by Western
blotting, which could be indicative of its marginal role in ovar-
ian biology. The significant correlation between RNA and pro-
tein levels suggests that, although further posttranslational pro-
cesses could interfere with the regulation and the activity of
these proteins, their overall levels arc predominantly determined
by their respective RNA expression levels. This allowed us to
perform all the subsequent analyses on the data obtained by
RT-PCR, for which a larger number of samples was available.
In agreement with previous works on breast (22) and ovary
(26), we found that normal ovarian tissues have significantly
higher levels of bcl-2 than do neoplastic ovarian tissues. More-
over, normal tissues expressed lower bax, bcl-XL, and bcl-x�
levels than did neoplastic tissues. These data suggest the possi-
bility that the regulation and, possibly. the role of these genes in
apoptosis may differ in carcinoma, as compared with normal
tissue (33). The increase in bcl-xL levels concomitant with the
decrease in bcl-2 in ovarian tumor supports the hypothesis that
bcl-xL could take over the role ofbcl-2 in carcinoma (33). bcl-xL
Research. on September 20, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 523
has been shown to be a stronger protector against apoptosis than
bcl-2 under certain circumstances (34, 35), and its presence in
tumor is suggestive of an important role in cell turnover regu-
lation. The complex picture, in which the proapoptotic genes
box and bcl-x5 follow the same trend, whereas the antiapoptotic
genes bcl-2 and bcl-xL follow opposite trends in normal and
neoplastic ovary, is very similar to the one recently described in
gastric tissues (33). Conversely, other studies on breast (36) and
prostate (37) described a different trend, which is indicative of
the existence of a tissue-specific mechanism of regulation of the
expression of the bcl-2 family. The distributions of bcl-2, box,
bcl-xL, and bcl-x� levels did not show any variations among
primary, metastatic, or recurrent tumors, suggesting that modi-
fications of these apoptosis related genes are likely to influence
the acquisition of a malignant phenotype but seem to have no
further impact on tumor progression.
bcl-2 has been shown to be inversely associated with tumor
grade or stage in some but not all reports on breast cancer (21,
22), whereas bcl-2 and bax were shown to increase with tumorgrade in prostate cancer (37). In our series, high box levels were
associated with advanced tumor stage, whereas none of the otherparameters showed any major correlation with the clinicopath-
ological features we analyzed.
In vitro studies on ovarian cancer cell lines reported that
overcxprcssion of bcl-2 could prevent cells from cisplatin-in-
duced apoptosis, whereas bax was ineffective (38, 39); however,
no in vivo data was available. Our data showed that the degree
of response to chemotherapy did not vary according to the levels
of the bcl-2 family members analyzed, which emphasizes that
caution is required in transposing in vitro-generated results to an
in vivo setting.
p53, classically known as a tumor suppressor gene, is also
clearly involved in some major apoptotic pathways and has been
shown to regulate both bcl-2 and bax expression in vitro (40).
An inverse correlation between bcl-2 and p53 has been de-
scribed in some carcinomas but has not been unequivocally
reported in ovarian carcinoma (26, 41), and no correlation
between bcl-2 RNA levels and p53 immunostaining was present
in our series of samples.
Several studies on solid tumors (22, 24, 37, 42, 44) mdi-
cated that the regulation of bcl-2 could be hormone dependent,
but no information had yet been reported on ovarian cancer.
bcl-2 levels did not correlate with steroid receptors levels in our
series of samples. Conversely, we observed a direct correlation
between box and PR, which is paralleled by an inverse correla-
tion between bcl-xL and PR. Interestingly, hormonal regulation
seems to affect the two genes box and bcl-xL with high expres-
sion in carcinomas.
Our attempt to combine the data obtained on the four genes
to evaluate the susceptibility to apoptosis did not lead to any
solid conclusions, although, interestingly, we did not find in our
samples, the scenario that maximally favored apoptosis, which
is highly unlikely to be compatible with tissue viability. We are
still at an early stage because more bcl-2 family members are
still being discovered and it has yet to be defined how they are
regulated and to what extent their interaction is modulated in a
tissue-specific manner (45). It had already been hypothesized by
White (45) that it is the global balance between proapoptotic and
antiapoptotic proteins that is relevant for cell survival, and
different bcl-2 family members and different complexes or
different pathways may become predominant in different tis-
sues. Furthermore, we have to consider that various functions
arc being described for each of these genes (8, 46, 47); thus,
thinking of their role exclusively in term of regulation of apop-
tosis may be restrictive. Their expression in tumors could be
directed to pathways that are partially distinct from apoptosis.
The differences we reported in the pattern of expression of
bcl-2, bax, bcl-xL and bcl-x5 in carcinoma compared to normal
ovary point to a role of the bcl-2 family in the acquisition or
maintenance of the transformed features of the tissue, that could
not be drawn from in vitro studies, which arc not representative
of tissue complexity and heterogeneity. More work will, there-
fore, be required in vivo, to fully characterize the expression of
these and other apoptosis-related genes to evaluate their com-
plex interactions, which may influence tumor evolution, and
long-term studies will be required to determine their prognostic
role.
ACKNOWLEDGMENTS
We thank Dr. A. Pontecorvi for providing the aldolase-A primers.
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1998;4:517-524. Clin Cancer Res M Marone, G Scambia, S Mozzetti, et al. neoplastic ovarian tissues.bcl-2, bax, bcl-XL, and bcl-XS expression in normal and
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