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From the Department of Laboratory Medicine and
Pathology,*
Mayo Clinic and Mayo Foundation, Rochester,
Minnesota, and the Department of Pathology,
St. Michael's Hospital, University of Toronto, Toronto,
Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Bcl-2 family of proteins regulate various steps in apoptosis.10,11 Some of the members of this gene family, including Bcl-2, Bcl-XL, and Mcl-1, block cell death whereas others, such as Bax, Bad, and Bcl-Xs, promote programmed cell death.10,11 These proteins often interact with each other as homo- and heterodimers in which the relative proportions of the anti-apoptotic and pro-apoptotic members determine the ultimate susceptibility of cells to apoptosis. A recent study has shown overexpression of Bcl-2 reported in nine (30%) of the pituitary adenomas examined.12 However, expression of other pro-apoptotic and anti-apoptotic proteins have not been previously examined in human pituitary adenomas. Overexpression of Bcl-2 provides protection against various apoptotic stimuli, including growth factor deprivation, oncogenes such as c-myc, tumor suppressor genes such as p53, radiation, and chemotherapeutic drugs.1-3 In this report, we examined a large series of pituitaries, including normal pituitaries, pituitaries from pregnant and postpartum patients, pituitary adenomas with and without drug therapy, and pituitary carcinomas for evidence of apoptosis and for expression of the Bcl-2-related pro-apoptotic and anti-apoptotic proteins to determine the role of physiological alterations and neoplastic development and progression on apoptosis in pituitary cells. An immortalized human pituitary cell line, developed in our laboratory, was also studied to analyze regulation of expression of the Bcl-2 family of proteins in pituitary cells during induction of apoptosis.
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Materials and Methods |
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Formalin-fixed and paraffin-embedded human pituitary tissues (n = 95) retrieved from the files of the Mayo Clinic and the St. Michael's Hospital were used for these studies. These included postmortem pituitaries from women who died during pregnancy (n = 5) and women who died during the postpartum period (n = 20). Ten postmortem pituitary tissues from young women were used as a control for pituitaries from pregnant and postpartum women. In addition, resected pituitary adenomas (n = 35) from untreated patients, growth hormone (GH)-producing adenomas from patients treated with octreotide before surgery (n = 9), prolactin (PRL)-producing tumors from patients treated with dopamine agonist before surgery (n = 8), and pituitary carcinomas with proven metastases (n = 8) were also studied. All pituitary adenomas and carcinomas were classified by immunohistochemistry (IHC). Electron microscopy was performed on some adenomas to analyze for apoptotic bodies and for other ultrastructural changes associated with apoptosis.
TUNEL Reaction
Apoptotic activity on paraffin sections of the pituitary tissue was analyzed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) with a commercial kit (Boehringer Mannheim, Indianapolis, IN), following the manufacturer's instructions. Briefly, pituitary sections were treated with proteinase K (25 µg/ml in phosphate-buffered saline (PBS), Boehringer Mannheim) for 15 minutes at 37°C before the TUNEL reaction. After incubation with anti-digoxigenin alkaline phosphatase, the sections were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate for 5 to 10 minutes. Apoptotic cells had blue nuclear staining. Omission of the TdT enzyme in the TUNEL reaction was used as a negative control and resulted in no staining. Tonsil tissue was used as a positive control. Apoptosis was evaluated by randomly counting 3000 cells from each section with a microscope containing a 1 x 1 cm2 grid in the ocular, and the results were expressed as an apoptotic index (AI = number of apoptotic cells per 100 cells). Apoptotic bodies were also detected on H&E-stained sections.
Immunohistochemistry
The expression of various apoptotic regulatory proteins was analyzed on paraffin sections of pituitaries from all groups after IHC. The anti-human antibodies used in this study included Bax (used at a 1/1000 dilution; from Pharmingen, San Diego, CA), Bcl-X (1/250) and Bad (1/100; both from Transduction Laboratories, Lexington, KY), and Bcl-2 (1/25; from Dako, Carpinteria, CA). Sections were treated with antigen retrieval by incubating for 15 minutes in a 700-W microwave oven in 10 mmol/L citric acid, pH 6.0, to increase the sensitivity of antigen detection. All immunostains were performed with the avidin biotin peroxidase method as previously reported.13 Substitution of normal serum for primary antibodies, as a negative control, resulted in no staining of the cells. Spleen tissue was used as a positive control. The specificity of each antibody was also checked by Western blotting. Immunostaining results were graded as follows: 0, negative; 1+, weakly positive; 2+, moderately positive; 3+, strongly positive.
IHC for pituitary hormones was done as previously described.13 Immunostaining for Ki67 with the MIB-1 antibody (AMAC, Westbrook, ME) at a 1:50 dilution was done as previously described.14
Combined immunostaining for S100 protein (Sigma Chemical Co., St. Louis, MO) and Bcl-X was done to co-localize Bcl-X in folliculo-stellate cells. The antibody co-localizations were visualized with diaminobenzidine and then with alkaline phosphatase after sequential staining.
Apoptotic Activity in Vitro
To analyze the apoptotic activity of pituitary tumors in vitro, a cell culture study was performed using six pituitary adenomas (three gonadotroph (GTH) and three null cell adenomas) and the HP75 cell line. The immortalized HP75 cell line, originally derived from a human nonfunctional pituitary adenoma, was developed in our laboratory using a replication-defective recombinant human adenovirus that contains an SV40 early large T antigen.15 The HP75 cells were grown in serum-free Dulbecco's modified essential medium (DMEM; Life Technologies, Grand Island, NY) for 1 to 3 days, at 37°C in an atmosphere of 5% CO2 and 95% air. At the start of the experiment the HP75 cells were treated with the protein kinase C (PKC) inhibitor hypericin16,17 (2 to 20 µmol/L) or chelerythrine chloride (1 to 5 µmol/L)18 and transforming growth factor (TGF)-ß1 (10-9 mol/L; R&D Systems, Minneapolis, MN). After culture, the tumor cells were harvested and used for protein extraction. Cytocentrifuge preparations were also made for IHC and TUNEL staining.
Immunoblot Analysis
Cultured HP75 pituitary cells and spleen tissue were used for protein extraction. One-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed with a 12% gel using the discontinuous buffer system of Laemmli (Bio-Rad Laboratories, Richmond, CA), as previously reported.14 The electrophoresed proteins were transferred to a polyvinylidene difluoride membrane and subjected to immunoblot analysis with anti Bcl-X (used at a 1/500 dilution), Bax (1/4000), Bcl-2 (1/200), and Bad (1/400). The reaction was detected with enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). The membranes were reblotted with a ß-actin antibody (1/2500; Sigma) after washing to check for equal loading of the gel. Spleen tissue was used as a positive control.
Statistical Analysis
Results were expressed as the mean ± SEM. Student's t-test was used to evaluate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cases studied are summarized in Table 1
. The 10 control pituitaries were
obtained from premenopausal women obtained at autopsy from patients
without endocrine diseases. The five pituitaries from pregnant women
who died during the last trimester of pregnancy showed prolactin cell
hyperplasia. The 20 pituitaries from postpartum women included glands
from patients who died between 28 days and 399 days after delivery. It
was not known how many of these patients were nursing at the time of
death. The 35 adenomas from patients who had surgery without previous
treatment included 6 GH, 6 PRL, 6 ACTH, 3 TSH, 6 GTH, and 8 null cell
adenomas. Nine cases of GH adenomas treated with octreotide and eight
cases of prolactinomas treated with bromocriptine or parlodel before
surgery were studied. The eight carcinomas with proven metastatic
disease included five PRL and three ACTH tumors.
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Ultrastructural studies of two pituitary adenomas (ACTH and GTH)
that had apoptotic cells detected with the TUNEL assay showed
condensation of the nuclear chromatin and occasional apoptotic bodies
(Figure 1)
.
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. Apoptotic cells were
identified in most carcinomas (7/8 cases), but only in 40% or less of
the cases in all other groups (Table 1)
. Relatively few apoptotic cells
were present in the normal pituitaries or in adenomas. Quantitation of
the AI showed that pituitaries from pregnant and postpartum women had
four- to fivefold higher AI compared with control nonpregnant women
(Figure 3)
. There were no significant
differences in the AI in functioning (PRL, GH, ACTH, and TSH) adenomas
compared with the nonfunctioning (GTH and null cell) adenomas. There
was no difference in the AI of GH-secreting adenomas or the
octreotide-treated GH adenomas. Prolactin adenomas treated with
bromocriptine did not contain any detectable apoptotic cells.
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Immunostaining of the pituitary tissues in paraffin sections
showed moderate (2+) Bcl-2 staining in most groups, except for the
carcinomas in which weak staining (1+) was present in only 2/8 (25%)
cases (Table 1)
. Two of four null cell adenomas and six of nine GTH
adenomas had weak staining for Bcl-2, and the remaining null cell and
GTH tumors were Bcl-2 negative. Other adenomas had strong to moderate
staining for Bcl-2. Immunostaining for Bcl-X, Bax, and Bad were not
very different among the various groups except that Bax and Bcl-X
stained more weakly in carcinomas (Figure 4)
. Bcl-X staining was very strong in
~10% of the S100-positive folliculo-stellate cells in normal
pituitaries and in pituitaries from pregnant and postpartum women
(Figure 4)
. Co-localization studies confirmed that some of these
Bcl-X-positive cells were angular folliculo-stellate cells with
positive nuclear stains for S100 protein and brown cytoplasmic staining
for Bcl-X (Figure 4D
, inset).
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Analysis of the HP75 cell line cultured in serum-free medium
showed an AI of 3% to 5% by TUNEL analysis, whereas pituitary adenoma
cells in primary cultures consisting of three null cells and three GTH
adenomas had AI values of 0.17 ± 0.09% and 0.10 ± 0.06%,
respectively. Apoptotic bodies were readily identified by light and
electron microscopic analysis (Figure 5)
.
Treatment of the cells with 10-9 mol/L TGF-ß1 for 1 to 3
days produced an increase in the number of apoptotic cells, with an AI
of ~10% on day 1 to 15% on day 3 (Figure 6)
. The protein kinase inhibitor
hypericin stimulated apoptosis in the HP75 cells (Figure 7)
. Because hypericin is not a specific
inhibitor of PKC, the experiments were repeated with chelerythrine
chloride, which also stimulated apoptosis in the HP75 cells but at a
much lower concentration of the drug. Cells treated with chelerythrine
chloride had a lower AI than with hypericin but significantly higher
than controls (Figure 7)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The apoptotic indices in this study and others8,22 were significantly lower than those observed in a recent report of 85 human pituitary adenomas.7 This discrepancy probably reflects technical differences in detecting and quantifying apoptotic cells with the TUNEL reaction. We also used the presence of apoptotic bodies and ultrastructural evidence of apoptosis to support the light microscopic observations.23 Comparison of functioning and nonfunctioning adenomas in this study showed no significant difference in apoptotic indices between the two groups of tumors in contrast to a previous report.7 Functioning somatotropinomas in the study of Saitoh et al22 and Green et al 8 did not have increased apoptotic indices, which agrees with our observations. GH-producing adenomas treated with octreotide had a similar rate of apoptosis as untreated adenomas, whereas apoptotic cells were not detected in prolactin cell adenomas after treatment with dopamine agonist. This observation is in agreement with the study of Kontogeorgos et al.7 Similarly, Saitoh et al 22 found no significant change in apoptosis in octreotide-treated somatotropinomas when compared with untreated tumors. However, they reported an increased apoptotic index in bromocriptine-treated prolactinomas. Divergent findings about the effects of bromocriptine on apoptosis in prolactin cells have also been reported in animal model studies4 in which hyperplasia was induced by high doses of estrogen. Withdrawal of estrogen induced cell death, which was further increased by application of bromocriptine. In the mouse AtT20 corticotroph cell line, bromocriptine stimulated apoptosis in up to 40% of the cells after 3 days of treatment.5 In vitro studies using SMS-201-995, a drug similar to octreotide, showed induction of apoptosis in AtT20 cells from 8% to 41% after 24 hours.6 The effect of SMS-201-995 on MCF-7 breast cancer cells was similar and caused an increase in the percentage of apoptotic cells from 8% to 34% after 24 hours.24 The contradictory reports about the effects of dopamine and somatostatin analogues on pituitary cell apoptosis may result from different doses and duration of treatment in particular groups of patients and in animal and/or in vitro experiments.
In this report we studied several members of the Bcl-2 family of
apoptosis-regulating genes in normal and neoplastic human pituitary
tissues. Bcl-2 expression has been analyzed in pituitary
adenomas12
but not in physiological stages such as
pregnancy and lactation and in pituitary carcinomas. In our study,
Bcl-2 was moderately expressed in all groups of tumors and normal
pituitary and was much weaker in pituitary carcinomas (Table 1)
, which
correlates with the increased rate of apoptosis. Bax, Bcl-X, and Bad
were expressed in most pituitary tissues with less intensive staining
in carcinomas, indicating that both pro-apoptotic and anti-apoptotic
proteins are expressed concurrently in the same cells. The Bcl-X
antibody recognizes the anti-apoptotic Bcl-XL and the
pro-apoptotic Bcl-Xs forms of the protein. However, the predominant
form of Bcl-X seen in most tissues to date is Bcl-XL, and
Bcl-Xs is usually absent or present in very low amounts,25
so Bcl-X may be acting as an anti-apoptotic protein in these tissues.
The expression of the Bcl-2 family of proteins has been studied in several neoplastic tissues, indicating its importance in tumor development.26 A recent model of a transgenic mouse in which p53-mediated apoptosis modified brain tumor growth showed that p53-dependent expression of Bax was induced in slow-growing tumors.27 In Bax-deficient mice, tumor growth was accelerated and apoptosis decreased by 50%, suggesting that Bax acts as a tumor suppressor gene.27 In primary colorectal adenocarcinomas,28 Bcl-2 immunostaining was weaker than in adjacent normal epithelium, which is similar to our observations in pituitary carcinomas. In the study of colonic adenocarcinomas, fewer cells were Bcl-2 positive in carcinomas than in adenomas. Expression of Bax was not significantly altered in colorectal carcinomas whereas the intensity of Bcl-X immunostaining was increased. Bcl-2 was expressed in ~70% of pituitary adenomas in our study, which is significantly more than in previous report.12 Two of four null cell adenomas in our study of resected pituitary adenomas stained weakly (1+) for Bcl-2 protein. This is in contrast to a previously published report in which all null cell adenomas were Bcl-2 negative.12 These differences may be related to the sensitivity of the assay techniques and the Bcl-2 antibodies used. We used microwave antigen retrieval in our studies, which increases the sensitivity of the assay. Recent studies have described markers of the Bcl-2 family of proteins in other endocrine tumors, including thyroid tumors.29 Both Bcl-2 and Bax were weakly expressed in undifferentiated thyroid tumors, which is similar to our observations in pituitary carcinomas.
The immortalized human pituitary cell line HP75 was derived from a nonfunctional adenoma15 by overexpression of SV40 T antigen. Analysis of the Bcl-2 family of proteins in this cell line revealed stronger expression of Bcl-X and Bax and decreased levels of Bcl-2 and Bad. Interestingly, induction of apoptosis in the HP75 cells by TGF-ß or PKC inhibitors correlated with significantly decreased Bcl-X expression whereas Bcl-2 levels remained unchanged. It suggests that Bcl-X may function as a protective protein in this cell line.
Relatively few TUNEL-positive cells were observed in our study and in
other in vivo studies of normal pituitaries and in pituitary
adenomas.8,22
Hyperplastic pituitaries in pregnant women
showed intermediate values of proliferation rates and cell death
compared with pituitary adenomas (Figure 9)
. There may be several reasons for
these low apoptotic indices in pituitary and other tissues; low values
of apoptotic indices in vivo may result from very rapid
kinetics of induction of apoptosis and elimination of dead cells by
phagocytosis.30
Active phagocytosis of apoptotic cells in
rat pituitary by folliculo-stellate cells has been
reported.4
In the rat liver, histologically detectable
apoptosis lasts only ~3 hours.31
Thus, the rapid
clearance of apoptotic cells in this study may result in low numbers of
measured apoptotic indices in vivo, despite the dramatic
changes in the size of the pituitary.4,19
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Pituitary carcinomas, in contrast to adenomas, have been shown to have
a higher proliferation rate and to overexpress p53
protein.33-35
Carcinonomas also had an increased rate of
apoptosis compared with adenomas in this study. The levels of Bcl-2
protein are significantly decreased in carcinomas compared with
adenomas. Comparison of normal and hyperplastic pituitaries, pituitary
adenomas, and carcinomas in vivo indicates that an increased
proliferative index and lower Bcl-2 expression correlate with a higher
rate of apoptosis in pituitary tissues (Figure 9)
. In a recent review
on the evaluation of apoptosis in various tumor tissues,36
the authors reported a positive correlation between the grade of tumor,
proliferation rates, and apoptotic indices in hormone-dependent
epithelial tumors, which agrees with our findings.
Immortalized HP75 pituitary cell lines have a higher rate of apoptosis
as well as proliferation than that observed in primary cultures of
pituitary adenomas. However, positive correlation between the rate of
proliferation and cell death was not observed by us in the mouse
pituitary gonadotroph cell lines 
T3-1 and
LßT2 (unpublished data). These observations suggest that
established cell lines cannot be directly compared with primary tumors,
although they may serve as a very useful tool for studies of the
molecular mechanisms of apoptosis.
TGF-ß is known to stimulate apoptosis in various tissues,37 and this growth factor also stimulated apoptosis in the HP75 cell line, which has been shown to have the three principal TGF-ß receptors.14 Recent studies indicate that TGF-ß activates multiple signaling pathways, including the caspase family of protease leading to apoptosis in various cell types.38 The observation that hypericin and the more specific PKC inhibitor chelerythrine chloride stimulated apoptosis in pituitary tumor cells in vitro agrees with a recent study17,18 and suggests that these molecules may have potential utility in the therapy of pituitary adenomas.
In summary, apoptosis is relatively infrequent in normal and neoplastic human pituitaries, but the AI is higher in pituitary carcinomas than in adenomas. Apoptosis is stimulated by TGF-ß1 and PKC inhibitors in the HP75 cell lines, implicating these molecules in the regulation of programmed cell death in the pituitary.
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Acknowledgements |
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Footnotes |
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Supported in part by NIH grant CA42951.
Accepted for publication November 20, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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