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DIFFERENTIAL EXPRESSION OF PROLACTIN RECEPTOR (PRLR) IN NORMAL AND TUMOROUS ADRENAL TISSUES: SEPARATION OF CELLULAR ENDOCRINE COMPARTMENTS BY LASER CAPTURE MICRODISSECTION (LCM)
A.Glasow1, A.Haidan1, J.Gillespie3, P.A.Kelly2, G.P.Chrousos4, S.R.Bornstein4
1Department of Internal Medicine III, University of Leipzig, Germany, 2Inserm Endocrinologie Moleculaire, Paris, France, 3Department of Pathology, National Cancer Institute, 4Developmental Endocrinology Branch, National Institutes of Health, NICHD, Bethesda, MD, USA.
ABSTRACT
PRL stimulates adrenal steroidogenesis. In this study, we compared the PRLR expression in normal and tumorous adrenal tissues and investigated a potential proliferative effect of PRL in adrenal cells. mRNA expression of long and intermediate forms of PRLR was detected in both normal adrenal cortex as well as benign and malignant adrenal tumors and in the human adrenocortical carcinoma cell line NCI-H295. Molecular analysis of cells procured by LCM clearly demonstrated that PRLR mRNA is expressed in the adrenal cortex but not in the medulla. Immunostaining revealed PRLR protein in all three zones of the normal adrenal cortex. Furthermore, adrenal carcinomas and adenomas stained positive for the PRLR, while in phaeochromocytomas as in the normal adrenal medulla, no specific staining was observed. By WST-1 test, we could show that PRL (10-7 M) decreased proliferation and viability of adrenal cells in primary cell culture suggesting that PRL is not a mitogenic factor of adrenocortical cells.
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INTRODUCTION
As previously described, PRL has a stimulatory effect on normal human adrenals aldosterone-producing adenomas and an adrenocortical carcinoma [1-4]. PRLR mRNA expression respectively binding sites were detected in the adrenal glands PRLR of different species [5,6]. Furthermore it has been shown that PRL is involved in the cell proliferation of normal and neoplastic tissues and in primary cultures i.e. leiomyoma cells as well as mammary breast cancer and Nb-2 cell lines by activation of the mitogen-activated protein kinase (MAPK) cascade [2]. Interestingly, PRL also activates the mitotic activity of the adrenal cortex in Snell Dwarf mice [7]. Therefore, we compared the distribution of PRLR in the normal adrenal and adrenal tumors and NCI-H295 cells. To detect effects of PRL on the proliferation rate of adrenal cells, a WST-1 assay was performed.
METHODS
Thirteen normal human adrenals, 21 adrenal tumors (4 functioning and 2 non-functioning adrenocortical adenomas, 8 adrenocortical carcinomas, and 7 phaechromocytomas), and the adrenocortical cell line NCI-H295 were analyzed. Immunohistochemistry and LCM: Immunostaining was performed by the avidin- biotin-technique as described previously [1]. For LCM frozen tissue sections were fixed in 70% alcohol and stained in hematoxylin and eosin. After dehydrating optically transparent thin film caps were placed on top of the air-dried tissue sections, and the tissue-film sandwich was viewed in an inverted microscope (Olympus Model CK2, Tokyo). Using a photodiode laser beam (Arcturus, Mountainview, CA), target cells were selected and fused with the transfer film. Total RNA was extracted from adherent cells (Stratagene, USA).
Polymerase-Chain-Reaction (PCR): DNAse A (Boehringer, Mannheim, Germany) digested total RNA was used as template for RT-PCR. Contamination by leukocyte RNA was checked by RT-PCR on CD 45. PCR amplification conditions for the human PRLR (accession No: M 31661) were as follows: 1) long form (primers 5’→3’ F1: ATGTCGTTCTCCCAATAAGG, R2: CTGGATGTAGGCTGA- GAATC) 34 cycles, denaturation (Td) 94°C/15 s, annealing (Ta) at 64℃/20 s, elongation temperature (Te) 72ºC/20 s; 2) extracellular domain (F3: CATCTTTCCGCCAGTTCCTG, R4: TCTTTACCTGCTTCATTCAAC) 35 cycles,
C
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m
b
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2
6
e
FIGURE 1 Immunostaining of the PRLR (mouse anti human PRLR antibody B6.2., courtesy of B.K. Vonderhaar), bar = 37 um as not otherwise indicated A) normal human adrenal cortex with zona glomerulosa, fasciculata and reticularis, bar = 17.5 um, B) normal adrenal medulla (m) and red stained cortical cells (c), C) adrenal cortical carcinoma, bar = 17.5 um, D) adrenocortical incidentaloma, E) phaeochromocytoma
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- 491 bp
NA
HAC
NCI
AD
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- 573 bp
B
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HAC
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RT/PCR analysis of PRLR expression. A) extracellular domain B) long form C) intermediate form. Normal adrenal tissue (NA); isolated adrenal medullary cells (M); NCI-H295 cell line (NCI); adrenal incidentaloma (AD); adrenal carcinoma (CA); negative control without cDNA template in the sample (C); 100-bp ladder (ST).
Td 94°C/15 s, Ta 63ºC/30 s, Te 72°C/40 s; 3), intermediate form [8] (F5: GACTATGAGGACTTGCTGGTGGAGTATTTA, R6: CACTTGCTTGATGTTG- CAGTGAAGTTG) cycles 35, Td 94°C/15 s, Ta 47°C/ 30 s, Te 72°C/40 s) WST-1 (Boehringer) and 3H-thymidine test were described previously [9].
RESULTS and DISCUSSION
In the human adrenal gland, chromaffin and cortical cells are highly intermingled [10]. Therefore we applied the LCM method [11] to separate adrenal medullary cells from surrounding cortical tissue, which allowed us to investigate the PRLR mRNA expression in the different cellular compartments of the adrenal gland. By RT-PCR we could detect the extracellular domain of the PRLR (included in the long, intermediate and postulated short forms) and the cytoplasmatic domain of the long form PRLR in benign and malignant adrenal tumors, NCI-H295 cells as well as in normal adrenal tissue (Fig. 1a,b). However, the long form PRLR was absent in chromaffin cells isolated by LCM (1 b). In contrast to breast tissue [8] the intermediate PRLR form was shown to be expressed in adrenal tumors and the NCI- H295 cells, as well as in normal adrenal tissue (1 c).
0.60
adsorbance [450 nm]
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0.40
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*
0.30
0.20
0.10
0.00
Basal
PRL
PRL
ACTH
10-7M 10-9 M 10-8M
By WST-1 test we could show, that PRL (10-7 M) decreased proliferation and viability of adrenal cells in primary culture to 77 ±4,9% of basal rate (mean ± SEM, n =3, p<0.01, ANOVA) while at lower concentration (10-9M) no effect was found.
By immunostaining we could show, that PRLR protein occures in all zones of the normal adrenal cortex as well as in tumors (Fig. 2). There were no differences in the intensity of staining within the cortical zones (a). Medullary cells did not stain for the PRLR (b). In accordance with our RT-PCR and staining results we found no change of catecholamine secretion by human medullary cells in vitro by PRL incubation (unpublished observations), however previous studies in the rat reported that PRL exerted a weak influence on the catecholaminergic activity [2]. Immunostaining of adrenal carcinoma (c), adrenal adenoma (d) and phaeochromocytoma (e) did not reveal different staining pattern and intensity compared to normal adrenal tissue.
By WST-1 assay we demonstrated that PRL (10”7 M) decreased the proliferation and viability of adrenal cells to 77 ± 4,9% of basal rate (mean + SEM, n =3). Inhibition of cell proliferation by corticotropin served as a quality control (Fig. 3). 3H-Thymidine test using NCI-H295 cells gave comparable results (data not shown).
In the present study we could show that (1) PRLR is expressed in the human adrenal cortex but not in the medulla; (2) PRLR is expressed in adrenocortical tumors but not in phaeochromocytomas; (3) PRL inhibits adrenal cell proliferation.
ACKNOWLEDGMENTS
We thank S. Brauer and S. Laue for her excellent technical assistance. This work was supported by the BMBF/ IZKF (01KS9504, Project B1).
REFERENCES
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