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Endocrinological Oncology |
Department of Endocrinology (P.L.M.D., A.M., R.A.J., S.L.C., G.M.B., A.B.G.), St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom; Neurochirurgische Klinik der Universität Erlangen-Nürnberg (J.H., R.F.), 91054 Erlangen, Germany; and Klinikum der Bayerischen Julius-Maximilians-Universität Würzburg (M.R), D-97080 Würzburg, Germany
Address all correspondence and requests for reprints to: Prof. Ashley Grossman, Department of Endocrinology, St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom. E-mail: a.b.grossman{at}mds.qmw.ac.uk
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Abstract |
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. Alternatively, a change in overall GR expression, or
mutations of some functional domains of the GR gene, might be involved
in the pathogenesis of corticotroph tumors.
We studied 22 tumors (17 pituitary ACTH-secreting tumors, 2 ectopic
ACTH-producing tumors, 2 prolactinomas, and 1 nonfunctioning adenoma)
and three normal pituitaries. RT-PCR was performed with primers
specific to GR and GRß complementary DNA, followed by Southern
blotting using an internal probe, and the ratio of the two bands
quantitated by densitometry. We also assessed the overall expression of
GR relative to the message of both the POMC gene and a housekeeping
gene. Single-strand conformation polymorphism analysis of the
DNA-binding domain and splice junction region of the gene was also
performed.
GR messenger RNA was expressed at 37.3-fold ± 5.7 (range, 32
to 46) excess, as compared with the GRß subform. This pattern was
observed both in the tumor samples and in the normal pituitaries used
as controls. A majority of the ACTH-secreting tumors (16/19), including
the ectopic secretors, showed variable but increased overall GR
expression, whereas 3 tumors showed an expression approximately
equivalent to the normal controls; however, no correlation was found
between these two groups and the response to the high-dose
dexamethasone test, nor was there any correlation with tumor histology.
No mutations were found in any of the tumors by PCR-single-strand
conformation polymorphism analysis.
In conclusion, although both pituitary and ectopic ACTH-secreting tumors are at least partially glucocorticoid-resistant, no significant abnormalities in the relative expression of the two main GR subforms were observed in a series of such tumors. Additionally, mutations of regions critical to normal function of the receptor do not seem to be a frequent event in these tumors.
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Introduction |
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One of the most characteristic biochemical features of corticotroph tumors is their resistance to corticosteroid feedback, as demonstrated by a failure to suppress circulating cortisol or its urinary metabolites during a dexamethasone suppression test (5, 6, 7). Nevertheless, this resistance is only partial, and most patients will show considerable suppression when high doses are used, suggesting that the cardinal feature of their deranged biochemistry is a resetting of their steroidal feedback (6). Unlike pituitary tumors, ectopic ACTH secretors show, in general, a more pronounced degree of dexamethasone resistance, and this may indicate distinct mechanisms of glucocorticoid resistance in these two groups of tumors. Because dexamethasone, similar to cortisol, binds to the type II glucocorticoid receptor (GR), it is possible that a somatic mutation involving the GR gene may be a fundamental event in tumor pathogenesis. Indeed, mutations of the GR gene have been reported in cell lines derived from ectopic ACTH-secreting tumors (8, 9), and recently, one tumor from a patient with Nelson’s syndrome was shown to carry an insertion of the GR gene that would result in a truncated GR protein (10).
The human GR is known to have two transcripts as a result of
alternative splicing of the gene, giving rise to two highly homologous
isoforms, GR and GRß, which differ only at the carboxy-terminus
(11, 12). In contrast to the well-characterized GR isoform,
transfected GRß does not bind glucocorticoids or antiglucocorticoids
and seems to reside primarily in the nucleus (11, 13), although some
studies also have demonstrated its presence in the cytoplasm (14).
GRß heterodimerizes with ligand-bound GR, either in the cytoplasm,
with subsequent translocation into the nucleus, or in the nucleus
itself, to act as a dominant negative inhibitor of the classic
receptor. In addition, GRß can repress the activity of GR on
glucocorticoid-responsive promoters (13, 15). It recently has been
demonstrated that both GRß messenger RNA (mRNA) and protein are
expressed at low levels in virtually all human tissues and may be found
complexed with heat shock protein (hsp90) and other proteins (14, 15, 16).
The GRß isoform therefore seems to participate in determining the
sensitivity of target tissues to glucocorticoids.
We speculated that an increase in the relative production of the GRß splice variant might play a role in the development of Cushing’s disease, specifically in determining relative corticosteroid resistance, and have investigated this in a series of such tumors. Our technique also has allowed us to determine whether the reported mutation in Nelson’s syndrome is a common causative factor in Cushing’s disease, and finally, whether changes in overall GR expression are characteristic of these tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied tumors from 22 patients, of which 17 were pituitary
ACTH-secreting tumors, 2 secreted ACTH ectopically (both were bronchial
carcinoids), and 3 were non-ACTH-secreting pituitary tumors (2
prolactinomas and 1 nonfunctioning adenoma). Clinical details,
including responses to low- and high-dose dexamethasone where
applicable, are shown in Table 1
. Detailed histological
data were available for a number of tumors and revealed them to be a
heterogeneous population, with regard to the presence of intermediary
filaments, degree of pleomorphism, and mitotic activity. One of the
tumors (no. 21 in Table 1) showed a high degree of mitotic activity and
invasion of adjacent structures. Both ectopic neoplasms were classified
as intermediate types of carcinoid tumor (between classical central and
peripheral forms). All corticotroph and ectopic tumors stained positive
for ACTH on immunohistochemistry. Tumors were obtained at surgery and
kept at -70 C until assay.
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Total RNA was obtained and reverse-transcribed into
complementary DNA (cDNA), as previously published (17, 18). Two regions
of the GR gene were targeted: one common area including the DNA-binding
domain (DBD), spanning exons 2 to 4 (primers GRCs and GRCa, see
sequences in Table 2); and the splice junction region,
using the sense primer at exon 7 (GR7s), common to both primers, and
either exon 9 (GRa) or exon 9ß (GRßa) as antisense sequences
to assess the two different subforms of GR (Fig. 1).
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The specific GR and GRß products were amplified in a duplex PCR,
in which the specific antisense primers GRa and GRßa shared the
same sense primer, GRSs, each of them at 0.5 µmol/L concentration.
All other components of the reaction were as described above. Because
the expression of the GRß subform is at a much lower level than the
GR subform, the two products could not be visualized on a gel at the
same phase of the PCR. Therefore, we performed a Southern blot analysis
of the amplified products after a 28-cycle PCR.
Southern blotting of GR and GRß
After 28 cycles of reaction, both GR and GRß products were
still on the exponential phase of the PCR, with only the GR products
visible by ethidium bromide staining. The gels were then transferred to
nylon membranes (N+, Amersham, UK) with the Mini-Electron Transfer Blot
(Bio-Rad), and hybridized to an end-labeled internal oligoprobe
directed against exon 8, as previously described (19). Two bands were
expected to be generated from such a procedure. The relative intensity
of the two bands resulting from the respective abundance of the two
subforms was assessed by densitometric analysis, as described above.
The GR/GRß ratio of ACTH-secreting pituitary and ectopic tumors
was compared with the ratio obtained in pituitaries from three
autopsies in individuals without endocrine disease or glucocorticoid
therapy, and also to three non-ACTH-secreting tumors.
SSCP (single-strand conformation polymorphism) analysis
To search for mutations of the DBD and the splice junction area
of the GR gene, PCR-SSCP analysis was performed. A single PCR was
performed as described above, and the products were labeled with
[32P]-dCTP (6000 Ci/mmol S.A., ICN Biochemicals,
Thame, UK), as previously described (17). The radioactive PCR products
were diluted in a denaturing dye and run on polyacrylamide gels under
two different conditions: with or without 10% glycerol. Products
greater than 400 bp in length were digested with two different
restriction enzymes, AvaII and ClaIII (New
England Biolabs, Taunus, Germany), according to the manufacturer’s
guidelines, giving rise to bands of smaller size before running on the
gels.
Three different pituitaries obtained from autopsy in individuals without endocrine disease were used as normal controls. The small cell lung cancer-derived cell line COR L24 (kindly provided by Dr. Adrian Clark), known to carry abnormalities of the GR sequence (8), was used as a positive control for the SSCP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). No clinical, histological, or
biochemical feature of the patients [in particular, the degree of
suppressibility to high-dose dexamethasone (Fisher’s exact test =
0.65; P = 0.75)], correlated with increased expression
of GR. The non-ACTH-secreting tumors showed intermediate expression of
GR between the corticotroph tumors and normal pituitaries, in relation
to both GAPDH and POMC transcripts.
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subform was expressed at 37.3- ± 5.7- (range, 32- to 46-)
fold optical density units (nonlinear values), as compared with the
GRß subform; this pattern was observed in the tumor samples and the
normal pituitaries used as controls (Fig. 3). The
expression of GR varied among the tumors, with 70% showing a higher
expression, and 30% a similar expression, compared with the
controls.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GR is a more plausible candidate gene potentially involved in
Cushing’s disease, as dexamethasone resistance is one of the most
robust biochemical indicators of the syndrome (6, 7, 22). Other
clinical models of glucocorticoid resistance, such as rare cases of
glucocorticoid-resistant asthma, recently have begun to be explored at
the molecular level (23). There is evidence that a shift in the
proportions of the alternate splice variants occurs in such cases, such
that the GRß transcript is overexpressed (24). Similarly, it has been
shown that New World primates overexpress the GRß isoform, compared
with humans, which may account for the glucocorticoid resistance seen
in these species (25, 26). It has been suggested that the GRß excess
may disrupt the receptor dimerization process; alternatively, a
predominance of GR-GRß heterodimers may contribute to a decrease
in the transactivating process or may even actively transrepress
GR-responsive genes, as compared with the GR-GR homodimers. We
therefore investigated whether the nonligand-binding form, or GRß,
might be involved in determining the tissue-specific glucocorticoid
resistance seen in ACTH-secreting tumors. However, we were unable to
demonstrate any abnormality in the level of expression of the two
subforms of GR, GR and GRß, in a panel of tumors of both pituitary
and ectopic origin. The active or ligand-binding form, GR, was
expressed in greater excess than the GRß subform in our tumor
samples, as well as in three normal pituitaries used as controls.
Nevertheless, as we have not attempted to quantify the expression of
the two isoforms at the protein level, possible posttranslational
modifications affecting the relative amount of GR and GRß cannot
be entirely excluded. In fact, a discrepancy between the mRNA and
protein expression levels of the two GR isoforms recently has been
suggested (14).
We found that total GR mRNA expression was variable, with the majority (>80%) of tumors showing increased expression, whereas the few remaining cases had expression equivalent to the pituitary controls. Chronic exposure to high cortisol levels, such as that occurring in Cushing’s disease, might be expected to down-regulate GR if feedback mechanisms were intact. In fact, previous studies in vitro have described a decrease in GR expression after glucocorticoid exposure (27, 28, 29, 30). In addition, a study performed in cultured cells from corticotroph adenomas revealed maintenance of the feedback mechanism by showing a decrease in the GR expression after exposure to dexamethasone (31). The reasons for these apparently contradictory findings amongst our tumors are unknown. It is possible that, owing to the small size of the pituitary tumors, some degree of contamination with normal tissue could have occurred, and thus, the variable extent of normal cells in the tumor preparation could account for differential expression levels of the GR gene amongst the different samples. In the normal pituitary, the corticotroph population accounts for approximately 12–20% of the whole pituitary population and may be diluted by other cell types. As the other cell types may also express the GR (it has been shown that most GH-secreting, at least 50% of LH-, FSH-, and TSH-secreting, and some PRL-secreting cells also express GR (32, 33)), it is difficult to estimate the normal corticotroph GR density. We have attempted to normalize our data by relating the GR expression to another corticotroph product, that of POMC gene, although POMC expression in corticotroph tumors has not been defined clearly in comparison with normal corticotrophs. Nevertheless, our data clearly suggest that in corticotroph tumors, the GR message is not decreased and may even be overexpressed in some tumors. We also were unable to correlate the degree of dexamethasone suppression to the relative expression of GR message. It is conceivable that the regulation of GR in response to high cortisol levels does not occur at the transcriptional level, but instead, posttranscriptional or posttranslational regulatory mechanisms might account for a putative down-regulation of GR.
We also were unable to detect any mutation of the region of the GR analyzed. This included the DBD and part of the ligand-binding domain, two important functional regions of the GR protein. The technique employed, SSCP, has been widely used for mutation screening. Two different gel conditions were used, and the fragments were digested with restriction enzymes to enhance sensitivity of the SSCP. This technique allowed us to detect a mutation in the GR previously reported in a small-cell lung carcinoma cell line (8). Although we cannot rule out the occurrence of mutations in other regions of the gene, recent reports have suggested that mutations of the GR regions examined in our study, in particular the DBD, seem to correlate with clinically relevant conditions (10). It has been proposed that mutations of this region may play a part in the pathogenesis of certain cases of Nelson’s syndrome, but we were unable to find mutations of this area in either of our two cases of Nelson’s syndrome. More recently, another mutation of the GR-coding region has been described in a very uncommon clinical setting: Cushing’s disease developed in a subject previously shown to be glucocorticoid resistant (34), indicating that structural abnormalities of the GR may contribute to, but are not essential for, the corticotropin tumor phenotype.
In conclusion, although ACTH-secreting tumors commonly show glucocorticoid resistance on clinical testing, no significant abnormalities in the relative expression of the two main GR subforms were observed in a series of such tumors. Additionally, mutations of regions critical to normal function of the receptor do not seem to be a frequent event of these tumors. However, other mechanisms of regulation of the GR gene cannot be ruled out as mediators of the development of ACTH-secreting tumors.
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Acknowledgments |
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Footnotes |
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Received October 23, 1996.
Revised December 6, 1996.
Accepted December 16, 1996.
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