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Endocrinological Oncology |
Division of Metabolism and Endocrinology, School of Clinical Medicine (D.-G.W., C.F.J., K.D.B.), Molecular & Cell Biology Unit, School of Dentistry (J.J.M., K.V.P.), The Queen’s University of Belfast, and Sir George E. Clark Metabolic Unit (A.B.A.), Department of Endocrine Surgery (C.F.J.R.), Royal Victoria Hospital, Belfast, United Kingdom
Address all correspondence and requests for reprints to: Dr Da-Gong Wang, c/o Colin F. Johnston, Wellcome Research Laboratories, Department of Medicine, Institute of Clinical Science. Grosvenor Road, Belfast BT12 6BJ, United Kingdom.
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Abstract |
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Introduction |
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Recent evidence suggests that the genetic regulation of apoptosis is of critical importance during tumorigenesis and that oncogenes and tumor suppressor genes can regulate the rate or susceptibility of cells to undergo apoptosis (3, 4). The protein product of the Bcl-2 oncogene blocks programmed cell death without affecting cell proliferation (5). Previously, a high frequency of Bcl-2 expression has been described in neuroblastoma (6, 7) and carotid body tumors (8), which are believed to arise from cells derived from the neural crest, and in other human tumor cell lines of neural origin (7). It has also been reported that Bcl-2 inhibits the death of central nervous system cells induced by multiple agents (9). Despite the apparent importance of c-Myc in the induction of cell proliferation, expression of c-Myc has recently been linked to the induction of cell death in different cells (10, 11), suggesting that this may be a general function of c-Myc. Moreover, documented evidence demonstrates a marked synergy between Bcl-2 and c-myc in myc/Bcl-2 double-transgenic mice during tumorigenesis (12, 13).
The present investigation was undertaken to determine whether a disturbance of the apoptotic pathway might occur in the generation of human pheochromocytomas. We assessed a bank of archival, wax-embedded, and frozen, pheochromocytoma tissue samples for the presence of Bcl-2 and c-Myc protein and for the expression of Bcl-2 gene transcripts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cases of pheochromocytoma were reviewed retrospectively from
the Northern Ireland Neuroendocrine Tumor
Register1 established in 1978 in the
Department of Medicine, the Queen’s University of Belfast (14).
Detailed clinicopathological data on each patient were available, and
the patient’s clinical progress had been followed since initial
diagnosis (Table 1
). Patients were diagnosed by standard
clinical, biochemical, and imaging criteria. All familial tumors were
validated by family history, and malignant tumors were identified by
confirming the presence of tumor metastases. The tumor identity was
made in each case by the diagnostic pathology service of the Royal
Group of Hospitals, Belfast, by histological methods, electron
microscopy, and immunohistochemistry for general neuroendocrine markers
and specific regulatory peptides.
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Samples of primary pheochromocytoma (n = 22) were
collected, either fresh at surgical biopsy or within 6 h of death
at necropsy, sequentially over the period 1980–1994. All tumors
originally came from the adrenals, except patient 17, who underwent
surgical resection of a pheochromocytoma from the para-aortic region
and patient 18 who had undergone excision of a bladder tumor. One
patient, patient 8, had a recurrent tumor 6 yr after removal of a
pheochromocytoma. Tumor tissues were fixed in modified Susa and
embedded in paraffin wax according to a constant protocol (15). In
addition, 8 frozen tumor samples, derived from the original 22 samples
of primary pheochromocytoma (patient 1–8 Table 1
), were available for
protein and RNA isolation.
Immunohistochemistry
Immunohistochemical studies were performed by the avidin-biotin complex (ABC) method as previously described (8). Briefly, 4 µm consecutive paraffin sections were dewaxed and subjected to microwave antigen retrieval by immersion in citrate buffer pH 6.0. Oncoprotein immunoreactivity was detected by incubation with primary antibodies to Bcl-2 (1:50) (clone 124, Dakopatts, Glostrup, Denmark), or c-Myc (1:100) (clone 9E11, Novocastra, Newcastle upon Tyne, UK) for 1 h at room temperature according to the manufacturers’ instructions. Sections were incubated with a biotinylated secondary antibody (Dakopatts). After 35 min incubation in ABC (Dakopatts), the reaction product was visualized using 3,3-diaminobenzidine tetrahydrochloride (Sigma, Dorset, UK).
Controls utilized throughout the staining procedure included staining
of normal human tonsil cells and breast cancer cells that were
previously determined as positive for Bcl-2 and c-Myc, respectively,
plus incubation and development of sections using normal blocking serum
in place of primary antibody. The immunocytochemical results were
evaluated semiquantitatively. The sections were scored according to the
number of positively immunostaining cells (Table 1).
Protein isolation and Western blotting
Protein was extracted from eight frozen tumor samples using RNA ISOLATOR (Genosys, Cambridge, UK). Protein was run on 10% SDS PAGE and transferred onto nitrocellulose along with prestained molecular weight markers (Sigma, Dorset, UK). The membranes were saturated with 3% BSA (Sigma, Dorset, UK) and incubated with monoclonal anti-Bcl-2 antibody (124, Dakopatts) or monoclonal anti-c-Myc antibody (9E11, Novocastra). Immune complex was visualized by Nitro blue tetrazolium (NBT) and 5-bromo-4-choro-3-indolyl phosphate (BCIP) (Sigma).
RNA isolation and Northern blotting
RNA isolation and Northern blotting were performed as
described (16). Briefly, total RNA was isolated from eight frozen tumor
samples using RNA ISOLATOR (Genosys). 20 µg total RNA per lane from
each sample was fractionated by 1% agarose-formaldehyde gel
electrophoresis. Human Bcl-2 genomic comlementary DNA probe (Oncogene
Science, Cambridge, UK) was labeled with [
-32P]dCTP
using Rediprime labeling system (Amersham, Little Chalfont,
UK). The membrane was prehybridized for 15 min in Quichhyb solution
(Stratagene, La Jolla, CA), and hybridized for 1 h at 68 C in
Quichhyb solution. The membrane was then washed twice at room
temperature for 15 min in 2 x SSC (1 x SSC is 150 mmol/L
NaCl and 15 mmol/L sodium citrate, pH 7) containing 0.1% SDS, followed
by 45 min stringency washes in a solution of 0.1 x SSC containing
0.1% SDS at 60 C, and then exposed for 4 days to Hyperfilm (Amersham)
at -70 C with an intensifying screen. To control for variability in
the loaded quantity of RNA, the membrane was reprobed with human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (Clontech, Palo
Alto, CA). The RNA ladder was used to mark size (kb) (Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twenty-two primary (including 5 malignant) pheochromocytoma specimens were examined for Bcl-2 and c-Myc by immunoperoxidase staining. Eighteen tumors (82%) were sporadic (including 2 extra-adrenal tumors), 4 tumors (17%) were from MEN 2 patients.
Immunostaining for Bcl-2 was positive in 18 (82%), including 5
malignant, of the 22 pheochromocytoma specimens examined (Table 1).
This included 14 of 18 sporadic (including both extra-adrenal) and all
4 familial tumors. All of the tumor specimens that expressed Bcl-2
showed a perinuclear cytoplasmic staining pattern (Fig. 1) that was heterogeneous in intensity.
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Western blots
To confirm the immunohistochemical results on paraffin tissue
sections, the expression of the Bcl-2 and c-Myc proteins in eight
frozen pheochromacytoma samples was examined by immunoblotting. As
shown in Fig. 2 and Fig. 3, five (63%)
of eight tumors co-expressed the 26 kDa Bcl-2 and 64 kDa c-Myc
proteins. One tumor sample was negative for both, and two tumor samples
apparently expressed 64 kDa c-Myc protein in the absence of obvious 26
kDa Bcl-2 protein.
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To examine whether there was an association between the expression of Bcl-2 and c-Myc, consecutive sections of all pheochromocytoma samples were immunostained for both proteins. Of 18 Bcl-2 positive tumors all demonstrated c-Myc immunoreactivity. On Western blotting five tumors that expressed Bcl-2 protein also expressed c-Myc. There was, therefore, an association of the expression of Bcl-2 and c-Myc in these tumors.
Analysis of RNA expression
The Bcl-2 transcript levels in eight pheochromocytoma samples that
were used for Western blotting were examined to determine whether Bcl-2
protein expression correlated with the expression of Bcl-2 transcripts.
There were three Bcl-2 transcripts, 8.5 kb, 5.5 kb, and 3.5 kb, as
described by Tsujimoto and Croce (17). Northern blot analysis revealed
that the 8.5 kb and 5.5 kb Bcl-2 transcripts were present in six out of
eight tumors (Fig. 4).
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Discussion |
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Recently Mah et al. (18) and Lindenboim et al. (19) reported that Bcl-2 inhibits apoptosis in rat pheochromocytoma (PC12) cells induced by deprivation of growth factors and cytotoxic drugs. It is hypothesised that Bcl-2 expression in PC 12 cells abrogates the requirement for stimulation by nerve growth factor (NGF) for survival (18). We know that Bcl-2 can prolong cell life by suppressing programed cell death in various tissues. This protein also protects neoplastic cells from undergoing apoptosis, which is induced by various therapeutic modalities such as irradiation and chemotherapy, although the suppression is incomplete (20, 21, 22).
Moreover, our observation that Bcl-2 is frequently expressed in pheochromocytomas, which are usually slow-growing and relatively unaggressive, suggests that in these tumors, Bcl-2 expression might lead to indolent tumor growth. This has been borne out by recent studies in transgenic mice, generated from Bcl-2 minigene constructs, which were used to assess the contribution of Bcl-2 gene deregulation to lymphomogenesis (5, 23). The mice initially exhibited a marked hyperplasia that was not a consequence of increased cellular proliferation; rather, Bcl-2 overexpression resulted in enhanced longevity secondary to an inhibition of apoptosis. The disease process was initially indolent, as is also seen in follicular lymphoma, but after an extended latency period the mice developed clonal, high-grade B-cell malignancies (24). Data obtained from this model, therefor also suggest that Bcl-2 overexpression may contribute to indolent tumor growth.
Furthermore, because this condition was generated by the use of Bcl-2 constructs, it may be that Bcl-2 overexpression might be an early event in the wild-type condition. Korsmeyer (25) has recently suggested that alterations of a gene, such as Bcl-2, which controls cell death, could be a frequent primary aberration not limited to follicular lymphoma but occurring in other types of neoplasms. A growth advantage due to cell survival with a low mitotic rate, together with slower acquisition of additional genetic defects, could explain the indolent progress of pheochromocytoma as is the case with follicular lymphomas (13, 16), in which Bcl-2 expression is a frequent primary aberration.
Although Bcl-2 alone is insufficient to cause transformation (16), the surviving cells could provide the substrate for further genetic changes to conventional oncogenes, such as c-myc. Eventually a malignant clone could be produced. That this can, indeed, occur has been confirmed in transgenic mice that constitutively express Bcl-2 in their lymphocytes (12, 13, 24). Although it has been demonstrated recently that constitutive c-myc expression can result in the induction of apoptotic cell death in cell lines under growth limiting conditions (10), it has also been shown that c-Myc-induced apoptotic cell death is inhibited by Bcl-2, while cell proliferation enhanced by deregulated and overexpressed c-Myc is not reduced (27, 28). In the present investigation, a marked association was observed between the presence of Bcl-2 and c-Myc immunoreactivities. This coexpression of Bcl-2 and c-Myc could indicate that Bcl-2, by mitigating the apoptotic effects of deregulated c-Myc expression without affecting its ability to promote continuous cell growth, so provides a mechanistic basis for the oncogenic synergy between these two proto-oncogenes in pheochromocytomas.
Although the initial genetic changes in the development of the major MEN2 syndromes have now been characterized, it is clear that additional changes must also occur during the progression of endocrine hyperplasia through phases of benign and malignant growth. That the expression of Bcl-2 and c-Myc was also found in MEN2, malignant and recurrent pheochromocytomas indicates that additional complementary genetic events are apparently required for the development of pheochromocytoma in MEN2 and progression to high-grade disease, just as tumor formation in transgenic mice harboring both deregulated c-myc and Bcl-2 requires additional somatic mutations (13).
The present study has demonstrated that deregulation of programed cell death may be a critical component in multistep tumorigenesis of pheochromocytoma and that the frequent expression of the oncoprotein Bcl-2 in pheochromocytomas may contribute to their pathogenesis. The genetic complementation of simultaneously deregulated Bcl-2 and c-myc is therefore implicated in the multistep tumorigenesis of human pheochromocytomas.
Received November 27, 1996.
Revised February 27, 1997.
Accepted March 5, 1997.
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