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Endocrinological Oncology |
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH
Address all correspondence and requests for reprints to: Dr. N. J. L. Gittoes, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH. E-mail n.j.gittoes{at}bham.ac.uk
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Abstract |
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-subunit, which, under
normal physiological conditions, is under negative feedback control by
thyroid and gonadal steroid hormones. We postulate that inappropriately
elevated levels of expression of -subunit in the face of normal
levels of these target organ hormones may reflect an abnormality of
thyroid hormone receptors (TRs) and/or gonadal steroid receptors in
NFTs. Using immunocytochemistry and Western blotting we have examined
TR and estrogen receptor (ER) protein expression in normal human
anterior pituitary glands and NFTs. Pretranslational expression of
these receptors was examined using semiquantitative reverse
transcriptase-PCR. Expression of all TR variant and ER proteins was
reduced in pituitary tumors compared with that in normal pituitaries.
The expression of messenger ribonucleic acids encoding the
TRß1 and TRß2 isoforms and ER was also
significantly reduced in tumors compared with normal tissues, although
there was no difference between tumors and normals in the level of
expression of TR1 and 2 messenger
ribonucleic acids. We suggest that reduced expression of TRs and ER may
account for inappropriate expression of the glycoprotein hormone
-subunit gene in some NFTs and may contribute to uncontrolled tumor
growth. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit expression is reported in between
40–70% (8, 9).
Expression of the glycoprotein hormone subunits is normally under
negative feedback regulation by the target organ hormones, gonadal
steroids and thyroid hormone. In NFTs, however, elevated circulating
concentrations of free -subunit have been reported in approximately
25% of cases (4, 9, 10), whereas elevated levels of -subunit
protein (8, 9) and messenger ribonucleic acid (mRNA) (6) have likewise
been described in tumor tissue. These observations are made in the face
of normal circulating concentrations of the target organ hormones in
the majority of cases, suggesting an abnormality of negative feedback
regulation of the -subunit gene by these hormones. Furthermore,
administration of pharmacological doses of thyroid hormone or gonadal
steroids fails to suppress the inappropriately elevated -subunit
expression (11), lending support to this view.
The etiology of NFTs remains unclear, although the majority of pituitary tumors are thought to be derived from monoclonal expansion of cells originating from a somatic genome alteration in the parent cell (12, 13). To date, there is good evidence for such a causative genetic mutation in only a subset of GH-secreting adenomas (14, 15). Studies of loss of heterozygosity have demonstrated allelic deletions on chromosome 11 in 18% of pituitary tumors, raising the possibility of a recessively acting tumor suppressor gene in this region (16, 17).
Although mutations leading to altered expression of oncogene or tumor
suppressor gene products may play a pathogenic role in NFTs, a change
in hormonal regulation of pituitary gene expression may contribute to
uncontrolled tumor cell proliferation. The regulation of glycoprotein
hormone -subunit expression in NFTs is abnormal and may reflect
defective negative feedback by T3 and/or gonadal steroids.
T3 and estrogen mediate their effects on gene transcription
via nuclear receptors that bind to specific regulatory regions of
target genes termed hormone response elements. The two classes of
thyroid hormone receptor (TR), designated and ß, are further
diversified in man due to alternate splicing of the gene products to
generate four isoforms, TR1, TRß1,
TRß2, and the nonligand binding variant,
c-erbA2. All are ubiquitous, apart from the
ß2 variant, which is expressed predominantly in the
anterior pituitary and hypothalamic regions (18, 19). Until recently,
only one physiologically relevant isoform of estrogen receptor (ER) was
recognized, although a number of recent reports have documented
functional alternative splice variants of ER, which may be of
importance both physiologically and pathogenically (20, 21, 22, 23).
In the studies described below we have explored the hypothesis that
defective negative regulation of the -subunit gene in NFTs may be
mediated by abnormal expression and/or function of TRs and/or ER. This
hypothesis has been tested by comparing receptor expression in normal
human anterior pituitary glands and NFTs using the techniques of
immunocytochemistry and Western blotting. Pretranslational expression
of receptors has also been compared using semiquantitative reverse
transcriptase-PCR (RT-PCR).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transsphenoidal selective adenomectomies were performed on 23 patients \[11 men and 12 women; age, 57 ± 2.2 yr (mean ± SE)\] with clinically nonfunctioning pituitary macroadenomas. No patient had evidence of GH or ACTH hypersecretion, and serum PRL levels were normal in all. After surgical excision, the tumors were immediately snap-frozen in liquid nitrogen and stored at -70 C until required. Studies of receptor protein expression in normal pituitaries used both fresh fixed tissue from postmortem proceedings (13 pituitaries) and archival sections obtained after hypophysectomy in patients with breast cancer (16 pituitaries). For RT-PCR studies, 6 normal human pituitary glands were obtained from postmortem proceedings (carried out as close to death as possible and always within 24 h) and stored at -70 C.
Immunocytochemistry
Formalin-fixed sections of normal pituitary and NFTs were
dewaxed and microwaved in citrate buffer (0.01 mol/L; pH 6) at 750
watts for 10 min and allowed to cool. Sections were treated with
methanol-H2O2 (1:1000) to block endogenous
peroxidase activity. After washing in phosphate-buffered saline (PBS),
slides were incubated with specific rabbit polyclonal antibodies to
human TR1, TR2, and TRß1
(Affinity Bioreagents, Golden, CO; 1:100 in 10% normal goat serum)
(24), specific mouse monoclonal antibody to human ER (Dako, High
Wycombe, Bucks, UK; 1:100 in 10% normal goat serum), or specific
polyclonal rabbit antibody to human glucocorticoid receptor (GR;
Affinity Bioreagents; 1:100 in 10% normal goat serum; used as a
control for findings for TR and ER) overnight at 4 C. Primary antibody
was omitted during incubation of control sections and was replaced by
nonimmune serum. Biotinylated secondary antibody was added to sections
for 30 min, followed by addition of the avidin-biotin complex (Vector
Laboratories, Burlingame, CA). Slides were developed using
3,3'-diaminobenzidine for 5–10 min and counterstained with methyl
green. Photographs were taken at x400 magnification. Immunostaining
was quantified by a single experienced observer under blind conditions.
Tissue sections were scored according to the number of positive cells
and the intensity of staining within cells, and an overall assessment
of immunostaining was recorded as absent, weak, intermediate, or strong
(24).
Nuclear protein preparations of pituitary tissue and Western blot analysis
Nuclear proteins were extracted from fresh-frozen pituitary tissue using the methods of Samuels and Tsai (25). Briefly, pituitary tissue was homogenized in STM buffer (0.25 mol/L sucrose, 20 mmol/L Tris, and 1.1 mmol/L magnesium chloride, pH 7.85) at 4 C and resuspended in STM and Triton X-100 (0.5%) on two occasions to obtain a clean nuclear preparation. Purified nuclei were incubated in lysis buffer (STM, 0.5 mmol/L phenylmethylsulfonylfluoride, 5 mmol/L dithiothreitol, 0.4 mol/L potassium chloride, and 20% glycerol) and agitated vigorously for 15 min on ice. Nuclear chromatin was removed by centrifugation, and the supernatant containing nuclear proteins was quantified by optical densitometry at 595 nm (OD595) and stored at -70 C.
Western blot analysis of pituitary nuclear proteins was performed by
SDS-PAGE on discontinuous acrylamide gels according to the method of
Laemmli (26). Samples were prepared for loading by denaturing at 95 C
in 62.5 mmol/L Tris (pH 6.8), 10% glycerol, 0.1% dithiothreitol, and
2% SDS and were then electrophorized at 200 V through 4.5% stacking
and 10% resolving gels using Mini-Protean II Western apparatus
(Bio-Rad, Richmond, CA). Fifteen micrograms of nuclear proteins
(determined by OD595 readings) were loaded per lane, and
prestained mol wt markers (Amersham International, Aylesbury, UK) were
run in parallel. After electrophoretic separation, proteins were
transferred to an Immobilon-P membrane (0.4 µm; Millipore, Bedford,
MA) using a semidry blotting system. Nonspecific binding sites were
blocked with a solution of 20% nonfat milk in PBS with 0.1% Tween-20.
After washing with PBS and 0.1% Tween-20 (PBST), membranes were
incubated overnight with polyclonal antibodies against human
TR1, TR2, and TRß1 (1:500
dilution in PBST) (24) or monoclonal antibody to human ER (1:500
dilution in PBST; as used for immunocytochemistry). After additional
washes with PBST (three times, 10 min each time), membranes were
incubated for 2 h with a horseradish peroxidase-conjugated
secondary antibody (Amersham International) at a dilution of 1:50,000
in all instances. Protein bands were visualized by incubating membranes
with an enhanced chemiluminescence detection system (Amersham) and
immediately exposed to DuPont Cronex x-ray film (DuPont, Wilmington,
DE) for 30 s to 15 min.
RNA extraction
Total RNA was isolated from fresh-frozen pituitary tissue using a single step acid guanidinium phenol-chloroform extraction technique (27). Briefly, pituitary tissue was homogenized in an Ultraturax homogenizer in the presence of RNAzol B (Biotecx Laboratories, Houston, TX). Total RNA was extracted by adding 0.1 vol chloroform and vortexing before centrifugation at 8000 rpm for 20 min at 4 C. The aqueous phase was transferred into an equal volume of isopropanol, and RNA was allowed to precipitate at -20 C overnight. After centrifugation, the RNA pellet was washed with 75% ethanol and allowed to dry before being resuspended in diethyl pyrocarbonate-treated water. Total RNA was quantified by optical density measurement at 260 nm, and the integrity of the RNA was verified by agarose gel electrophoresis with ethidium bromide staining.
Reverse transcription
Reverse transcription was performed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI) in a total reaction volume of 50 µL. Two micrograms of pituitary total RNA were added to 60 pmol oligo(deoxythymidine)15 (Promega, Madison, WI) primer, and the volume was adjusted to 32 µL with diethyl pyrocarbonate-treated water. The solution was heated to 77 C for 10 min to allow for primer annealing. The mixture was cooled to room temperature, and 10 µL 5-fold concentrated AMV reverse transcriptase buffer (Promega) were added along with 5 µL deoxy-NTP mix (20 mmol of each; Boehringer Mannheim, Mannheim, Germany). Fifty units of placental ribonuclease inhibitor (RNasin, Promega) and 15 U AMV reverse trancriptase (Promega) were added to the reaction mixture. Incubation for 70 min at 42 C was carried out in a thermal cycler, and the RT reaction was terminated by heating to 77 C for 10 min. Reverse transcription was performed on each RNA sample with no AMV RT added. PCR carried out on this resultant RT product generated no PCR product, confirming absent genomic DNA contamination.
PCR
PCR was carried out in a total volume of 50 µL, and a hot
start technique was employed in all instances. For the purposes of
semiquantitation, PCR reaction components were premixed (to generate
master mixes) before addition to individual PCR tubes to minimize
pipetting errors, and all samples underwent PCR at the same time in the
same experiment. Each PCR tube contained 60 pmol each of the forward
and reverse primers (see Table 1
), 1 µL deoxy-NTP mix (20
mmol of each; Boehringer Mannheim), and between 2–10% of the RT
reaction product. Volume was adjusted to 44.5 µL with water.
The reaction constituents were pulse centrifuged, mineral oil was
overlaid, and the PCR mixture was heated to 95 C for 6 min and cooled
to 72 C before adding PCR buffer and Taq. A dilution of 2.5
U Taq DNA polymerase (Boehringer Mannheim) in 5 µL 10-fold
concentrated PCR reaction buffer (Boehringer Mannheim) was prepared and
added through the mineral oil layer to each of the PCR reaction
mixtures. PCR cycling was commenced using a melt temperature of 94 C in
all instances, and extension was carried out at 72 C. Oligonucleotide
sequence design was carried out on GCG-8 software using GenBank
published complementary DNA sequences. Table 1 lists oligonucleotide
sequences, expected PCR product sizes, annealing temperatures, and
number of PCR cycles used for each primer pair. PCR products were run
on 2% agarose gels stained with ethidium bromide. Appropriate negative
control PCR reactions were run in parallel.
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All RT reactions used oligo(deoxythymidine)15-primed RNA to minimize the variations in RT efficiency seen when using specific RT primers. Once PCR conditions for each primer pair had been optimized, comparative kinetic analyses (28, 29) were performed to determine the phase during which there was exponential generation of PCR product before reaching a plateau. It was at this point that the PCR was terminated, allowing semiquantitative data to be obtained.
Ethidium bromide-stained gels were visualized under UV light, and the image was digitized and stored on computer disk. Gelbase/Gelblot software (Ultra Violet Products, Cambridge, UK) was used to measure the luminescence of PCR-generated bands, which, in turn, is a measure of the quantity of PCR product (30). Values for each PCR product were expressed as a fraction of the quantity of ß-actin, which was used as an internal standard to correct for sample to sample variation in RNA degradation. PCR products were run in triplicate on 2% agarose gels, and the mean luminescence value was recorded in each case.
To detect any significant contamination of pituitary tumors with normal pituitary tissue, we determined mRNA expression for Pit-1 (31, 32) using RT-PCR. Three of the 23 NFTs showed evidence of the presence of Pit-1, and these 3 tumors were excluded from further analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The immunostaining characteristics of normal
pituitaries and NFTs were determined using specific antibodies to PRL,
GH, ACTH, -subunit, LHß, FSHß, and TSHß. Figure 1
shows that nearly all normal pituitaries demonstrated expression of all
anterior pituitary hormones, whereas 47% of NFTs stained for
-subunit, 41% for FSHß, 41% for TSHß, 29% for LHß, and few
samples for ACTH, GH, and PRL. The majority of normal pituitary glands
revealed staining for each TR variant (Figs. 1 and 2a; 76%
for TR1, 76% for TR2, and 62% for
TRß1) and ER (62%; Figs. 1 and 2b). In contrast,
immunostaining for TR variants was apparent in only a small proportion
of NFTs (18% for TR1, 12% for TR2, and
6% for TRß1; P < 0.0001, by
2 test, for all TR variants), and those with evidence of
receptor expression demonstrated only a weak staining pattern (Fig. 1).
No NFTs expressed demonstrable ER by immunocytochemistry
(P < 0.0001, by 2 test). In contrast to
the reduction in TR and ER protein expression, GR was demonstrated by
immunocytochemistry in 72% of normal pituitaries and 88% of NFTs
(P = NS; Figs. 1 and 2b).
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however, in NFTs, protein bands were either absent or present at a
lower level of intensity for all TR variants and ER, confirming the
findings for immunocytochemistry.
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mRNA encoding all TR variants and ER was detected in all examples
of normal pituitary tissues and NFTs examined by RT-PCR (Fig. 4). To provide a semiquantitative assessment of mRNA
expression, rigid RT-PCR protocols were established for each primer
pair as described in Materials and Methods. Figure 5 indicates the results of ratios of expression of specific
receptor mRNAs to ß-actin mRNA for normal pituitaries and NFTs. The
level of expression of both TR1 and TR2
variant mRNAs corrected for ß-actin mRNA expression was similar in
normal pituitaries and NFTs. In contrast, TRß1 and
TRß2 mRNA levels were significantly lower in NFTs than
those in normal pituitaries (P < 0.005 for
TRß1; P = 0.01 for TRß2; by
Student’s t test). ER mRNA levels in NFTs were also
significantly lower (P < 0.0001, by Student’s
t test) than those in normal pituitaries.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, TR2, and
TRß1 variants as well as ER by immunocytochemistry and
Western blot analysis. Immunocytochemistry also showed that only a
minority (11%) of NFTs expressed demonstrable TR of any isoform, and
those few that were positive exhibited only weak staining
characteristics; no ER protein could be detected in any of the NFTs
examined. These differences in receptor expression evident using
immunocytochemistry were confirmed by Western blot analysis. We also
demonstrated similar expression of GR in normal pituitaries and NFTs,
indicating that reduced expression of TRs and ER was not merely a
function of abnormal expression of all nuclear thyroid/steroid
receptors in NFTs. We, like others (31, 32), used Pit-1 mRNA expression
to detect possible contamination of NFTs with normal pituitary tissue.
There are reports of endogenous Pit-1 expression in some NFTs (33),
although by excluding those three Pit-1-positive NFTs we have excluded
the possibility of normal pituitary contamination. Several investigators have examined normal and tumorous pituitary glands for the presence of ER using various techniques. Immunocytochemistry for ER in the hands of others has revealed absent immunostaining in NFTs (31, 34), although another group demonstrated ER in a minority of tumors (35). TR expression in the pituitary has not previously been reported. The generalized reduction in TR and ER expression observed at the protein level in NFTs was not associated with a reduction in all mRNAs encoding these receptors. ER mRNA levels were significantly lower in NFTs compared to those in normal pituitaries. Expression of ER mRNA has previously been determined by others using ribonuclease protection assay and has revealed low levels in NFTs (35). Zafar et al. (31), despite showing absent ER on immunocytochemistry in NFTs, detected ER mRNA by RT-PCR and in situ hybridization.
Reduced expression of ER transcripts in NFTs may be explained by altered regulatory sequences, premature stop sequences, or gross allelic deletions involving the ER gene. The production of ER variant mRNAs in NFTs provides an alternative potential explanation for this finding. We used a single primer pair directed at a nucleotide sequence spanning exons 3 and 4 of the ER gene to examine ER mRNA expression; hence, we are unable to exclude the possibility of significant generation of ER deletion variants in our series of NFTs. ER isoforms generated by alternative exon splicing have, however, been postulated to have potentially important pathogenic implications in a number of tumor types (36, 37, 38), including pituitary adenomas (39).
We demonstrated similar levels of expression of mRNAs encoding the
TR variants in NFTs and normal pituitaries, whereas
TRß1 and TRß2 mRNA levels were
significantly reduced in NFTs compared to those in normal pituitaries.
The observation that TR1 and TR2 mRNA
levels were similar in both NFT and normal pituitaries despite a marked
reduction in TR1 and TR2 proteins
suggests that TR mRNAs may be nonfunctional, or, due to a
posttranscriptional defect, there may be inappropriate processing of
the transcribed product. Apparent discrepancies between TR protein and
mRNA expression have been noted previously by others (40, 41).
The presence of mRNAs encoding TR1, TR2,
and TRß1 variants has previously been reported in a small
number of NFTs (42), detected using nonquantitative RT-PCR. Other
workers, using quantitative techniques, have shown abnormalities in
relative levels of TR isoforms in specific cell lines and various
intact tissues. The cell line designated TSH (43) secretes large
amounts of -subunit in the presence of high levels of T3
and in this way provides a close analogy to the situation in human
NFTs. Sarapura et al. (44) have shown these cells to express
lower levels of mRNA encoding TRß1 and
TRß2, but not TR1, compared to
T3-responsive thyrotropic tumor cells (TtT97). After
transfection studies, they have also shown that it is possible for
T3 regulation of the -subunit to be reestablished by
overexpression of any one of the TRs. We have reported that
TRß1 is more potent than TR1 in mediating
negative regulation of the -subunit gene by T3 (45), and
it is, therefore, likely that defects in TRß would have
quantitatively greater effects than those in TR on -subunit
production in NFTs. Furthermore, recent work by Langlois et
al. (46) has demonstrated that TRß2 is highly
significant in mediating thyroid hormone regulation of the -subunit
gene.
Abnormalities of TR expression have been identified in other tumors and have also been implicated in tumor growth. Sarapura et al. (47) found a significant rise in TRß1 mRNA levels and a smaller reduction in the expression of other TRs in sc grown mouse thyrotropic tumors (TtT97) in response to treatment with T4. In association with this altered TR expression was a significant reduction in tumor size, raising the possibility of a causal relationship between TR isoform mRNA expression and tumor growth. Using human thyroid tissue, Bronnegard et al. (48) showed significantly reduced expression of TRß variant mRNAs in neoplastic thyroid tissue compared to that in nonneoplastic tissue. These findings imply that a change in expression of TR isoforms, as demonstrated in the present study, may be important in modulating tumor growth.
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Acknowledgments |
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Footnotes |
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2 Supported by a Smith and Nephew Research Fellowship and currently a
Sheldon Research Fellow awarded by the West Midlands Regional Research
Committee.
3 Supported by award of a Medical Research Council Project Grant to
Prof. Jayne Franklyn.
Received December 5, 1996.
Revised February 5, 1997.
Accepted February 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit gene by 1, 2 and ß1
thyroid receptor variants. J Endocrinol. 148(Suppl):OC29.
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), intermediate (
), and strong
(
) staining characteristics.
