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Endocrinological Oncology |
Department of Laboratory Medicine and Pathology (L.J., X.Q., E.K., B.W.S., R.C.-R., R.V.L.), Division of Endocrinology/Metabolism and Internal Medicine (C.A.), and the Department of Neurologic Surgery (D.H.D.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and the Department of Pathology, St. Michaels Hospital (K.K.), Toronto, Canada
Address all correspondence and requests for reprints to: R. V. Lloyd, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905.
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Abstract |
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In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded tissue sections of normal pituitaries and pituitary adenomas retrieved from the files of the Mayo Clinic were used for these studies. Three nonneoplastic pituitaries obtained within 8 h of death were studied by combined PRL-R in situ hybridization and immunostaining for pituitary hormones to localize the specific cell types with PRL-R gene expression. Twenty-eight pituitary adenomas were used for the PRL-R in situ hybridization study in the first set of experiments; these included PRL (n = 6), GH (n = 6), ACTH (n = 3), FSH/LH (n = 6), null cell adenomas (n = 6), and a TSH adenoma. Pituitary adenomas were characterized by immunostaining in all cases and by ultrastructural studies in some cases.
In another set of experiments, only PRL adenomas from 34 patients were used. The PRL adenoma cases included men (n = 10), reproductive age women ranging in age from 24–40 yr (n = 9), postmenopausal women ranging in age from 43–68 yr (n = 10), and a final group of patients who had been treated with bromocriptine before transsphenoidal surgery (n = 5).
Frozen tissues from portions of 12 pituitary adenomas and 2 normal pituitary tissues were used for RNA extraction and reverse transcription-PCR (RT-PCR) studies.
In situ hybridization (ISH)
The oligonucleotide probes for human PRL-R were synthesized with
an automated DNA synthesizer at the Mayo Foundation from the published
sequences (9). PRL-R mRNA expression was analyzed by ISH with
35S-labeled probes to human PRL-R gene (Table 1
). The probes to human PRL-R was used for all tissues
in this study, and a sense probe was used as a control. The specificity
of the probes was verified by a GenBank search. The oligonucleotide
probes were labeled at the 3'-end with 35S as previously
described (16, 17). Sections were hybridized with 3 x
106 cpm/slide at 42 C for 18 h, followed by washings
with 0.5–2 x SSC (standard saline citrate) and autoradiography
for 2–3 weeks. ISH analysis for all cases from one set of experiments
was performed together. Negative control for ISH consisted of
pretreating tissues with 200 µg/mL ribonuclease A (Sigma) before
hybridization and using a sense control probe for PRL-R.
Formalin-fixed, paraffin-embedded sections of liver tissues were used
as positive controls.
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RT-PCR
Total RNA was extracted from 10 immunohistochemically classified
pituitary adenomas and 2 normal pituitaries as previously reported
(18). First strand complementary DNA was prepared from total RNA by
using a first strand synthesis kit (Stratagene, La Jolla, CA). The RT
reaction was performed in a final volume of 50 µL with 5 µg total
RNA, 300 ng antisense primer for PRL-R (Table 1
), 1 x RT buffer,
1.0 mmol/L of each deoxyribonucleotide [deoxy (d)-ATP, dCTP, dTTP, and
dGTP], 40 U RNase inhibitor, and 50 U Moloney murine leukemia virus
reverse transcriptase at 37 C for 60 min, heated at 95 C for 5 min, and
then immediately placed on ice.
The PCR amplification was performed in 100-µL final reaction volumes
containing 10 µL RT reaction product as template DNA corresponding to
complementary DNA synthesized from 1 µg total RNA, 1 x PCR
buffer (from Promega, Madison, WI), 1.5 mmol/L MgCl2, 0.2
mmol/L of each deoxynucleotide, 300 ng of each sense and antisense
primer for PRL-R (Table 1), and 2.5 U Taq DNA polymerase
(Promega). Programmable temperature cycling (no. 480,
Perkin-Elmer/Cetus, Norwalk, CT) was performed with the following cycle
profile: 95 C for 5 min for denaturing, followed by 30 cycles of 94 C
for 1 min, 60 C for 1 min, and 72 C for 2 min. After the last cycle,
the elongation step was extended by 10 min. The housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase was used to check the
integrity of the RNA (18).
A 20-µL aliquot of PCR product was analyzed by 2% agarose gel
electrophoresis and stained with ethidium bromide. PhiX174
DNA/HaeIII digest (Life Technologies, Grand Island, NY) was
used as the mol wt standard. The PCR amplification products for PRL-R
were transferred to nylon membrane filters, and Southern hybridization
with internal probes (Table 1) that hybridized to a region within the
amplified sequences was performed. Hybridization was performed with
1 x 106 cpm/mL 33P-labeled probe at 42 C
for 18 h and washed with 6 x SSC-0.1% SDS at 23 C for 30
min and at 42 C for 15 min. Autoradiography was performed at -70 C
with Kodak Omat-AR film (Eastman Kodak, Rochester, NY) for 18 h.
Omission of Moloney murine leukemia virus reverse transcriptase in the
RT reaction was used as a negative control for the RT-PCR
procedure.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Most normal anterior pituitary cells expressed PRL-R (Table 2 and Fig. 1), as indicated by labeling
with black silver grains. The use of the sense control probe (Fig. 1)
and ribonuclease A pretreatment reduced the hybridization signal to
background levels. The liver tissue used as a control, which is known
to express PRL-R, had a positive hybridization signal (not shown). To
determine the level of PRL mRNA expressed in different pituitary cell
types, the MGC for each cell type was performed after combined ISH and
immunostaining for different pituitary hormones (Table 2). In addition,
the percentage of PRL-R mRNA-positive cells for each cell type was, in
decreasing order; LH, 73 ± 2.9; PRL, 72 ± 2.7; ACTH,
59 ± 4.7; GH, 52 ± 5.1; and TSH, 41 ± 3.0. PRL- and
LH-producing cells had significantly more PRL-R mRNA expression than
the other cell types with both methods of analysis (Table 2 and Fig. 2).
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and 4), which was significantly greater than those of the
other tumor groups. When a series of PRL adenomas was analyzed in a
separate experiment, there was no significant difference in the
labeling density among men, reproductive age women, and postmenopausal
women. In contrast, the five patients treated with bromocriptine before
surgery had significantly fewer silver grains per cell than the other
three clinical groups (Table 3 and Fig. 4).
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RT-PCR analysis of 2 normal pituitaries and 10 adenomas resulted
in amplification of a predicted 276-bp band corresponding to the PRL-R.
PRL-R was detected in all tumor groups and in the normal pituitaries
(Fig. 5), as verified by Southern hybridization with an
internal probe. RT-PCR with the glyceraldehyde-3-phosphate
dehydrogenase primers also showed a single band of amplified product of
495 bp, confirming the integrity of the starting total RNA used in the
analyses (not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The close physical relationship of PRL and gonadotroph cells has been well described in both rats and humans (19, 20), and a paracrine interaction between these cell types has been proposed (19). LH and FSH are also known to increase PRL-R expression, and PRL stimulates gonadotropin receptor expression in the ovary (21, 22). The observation that PRL tumors have significantly more PRL-R than other tumors suggests that the regulatory function of PRL-R in pituitary tumors would have a greater influence on PRL cells than any other cell type.
Recent studies indicate that PRL has an autocrine regulatory effect on PRL-secreting cell lines, such as GH3 cells, which have been shown to have PRL-R (23, 24). Krown et al. (23, 24) reported that PRL stimulated a proliferative response in GH3 cells. Preliminary studies with human pituitary adenomas in vitro have also suggested a cell proliferative effect of PRL on pituitary adenomas expressing the PRL-R (25).
This is the first reported observation that preoperative bromocriptine treatment can reduce PRL-R mRNA levels. Bromocriptine causes shrinkage of prolactinomas (26, 27, 28) and is usually associated with decreased PRL protein and mRNA production as well as decreased PRL secretion in most prolactinomas, except those that are resistant to dopaminergic therapy. However, the mechanism(s) of bromocriptine actions on pituitary tumor shrinkage and decreased PRL-R mRNA levels are probably very complex. The D2 dopamine receptors in normal and neoplastic PRL cells are negatively coupled with adenylate cyclase, and a reduction in intracellular cAMP levels is one mechanism by which dopamine and bromocriptine inhibit hormone release (28). The bromocriptine-lowered cAMP levels may prevent PRL release, and this may contribute to reduced gene transcription of PRL synthesis (28). Recent studies of human prolactinomas have shown that bromocriptine decreased DNA synthesis by prolactinomas in vitro, but not that by GH tumors, suggesting that inhibition of DNA synthesis may be related to the decreased gene transcription that is observed after bromocriptine treatment (29). Studies with rat hypothalamus have shown that PRL can induce its own receptors (30), which may also occur in human prolactinomas. However, as bromocriptine lowers PRL blood levels in most patients, this lower level of serum PRL may contribute to the down-regulation of PRL-R mRNA. Because of the existence of isoforms of the D2 receptor that are regulated by guanyl nucleotides, these isoforms might interact with different G proteins to initiate intracellular signals (31), increasing the complexity of the regulatory role of bromocriptine. Recent studies have also shown that specific trophic factors, such as nerve growth factor, can control proliferation in prolactinomas by autocrine mechanisms and that this control may be lost in dopamine-resistant prolactinomas (32). Because of the complexity of the regulatory role of bromocriptine on PRL cell function, many more studies are needed to elucidate the mechanisms by which bromocriptine decreases PRL-R mRNA levels.
Liver tissue was used as a positive control, because it has been used to purify PRL-R (5) and because various studies have demonstrated PRL-R in hepatocytes (33, 34, 35). Furthermore, PRL-R transcripts have been shown to be abundant in the liver (36, 37). The widespread distribution of PRL-R not only in all pituitary cell types and tumors, but in many other tissues as well is in keeping with the myriad functions of PRL.
In summary, we have shown that the highest levels of PRL-R are found in PRL- and LH-producing cells of the normal pituitary, whereas PRL adenomas have significantly higher levels of PRL-R than any other adenoma type. PRL adenomas from bromocriptine-treated patients have significantly decreased levels of PRL-R. These observations indicate that PRL adenomas can serve as an excellent model for in vitro studies of the interaction of PRL and its receptors and to further elucidate the mechanisms involved in the activation of the PRL-R complex. These studies are currently in progress in our laboratory.
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Acknowledgments |
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Footnotes |
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Received September 10, 1996.
Revised November 18, 1996.
Accepted November 22, 1996.
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References |
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