9021-972X/92/7406-1474$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

GROWTH

HORMONE-RELEASING

Vol. 74, No. 6 Printed in U.S.A.

HORMONE

TRANSCRIPTS

IN HUMAN

PITUITARY

ADENOMAS.

A. LEVY AND STAFFORD L. LIGHTMAN Char@ Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, UK ABSTRACT. We have investigated the possibility that local production of GHRH within the adenohypophysis could be an aetiological factor in the development of human pituitary somatotroph and other turnours. We examined 51 human pituitary adenomas for GHRH transcripts using in siru hybridization histochemistry. GHRH transcripts were identified in 13 of 17 somatotroph adenomas, 4 of 10 corticotrophs, 1 of 6 lactotrophs and 1 of 18 endcctinologically inactive adenomas. In 11 GHRH-expressing somatouoph adenomas, SRIH uanscripts were also identified. In all cases except one (a cotticotroph adenoma) the average number of GHRH transcripts exceeded that found in the arcuate nucleus of simultaneously hybridized rat brain sections. In some pituitary adenomas, GHRH transcripts were clearly localized to a discrete subpopulation of cells and in these, the amount of GHRH mRNA per cell was comparable to that found in the GHRH-expressing cells of the rat arcuate nucleus. Although we have not directly demonstrated an association between the two, the identification of localized, high level GHRH gene expression in somatotroph adenomas suggests that GHRH may have a role in pituitary adenoma formation 80 mM Tris-HCl (pH7.5). 4mM EDTA, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulphate, O.Zmg/mL sodium heparin, 10% dextran sulphate, 1OOmM dithiothreitol and 106 dpm of probe per slide, and washed for 1 hour in 4 changes of 1 x SSC at 55°C followed by two 30 minute washes in 1 x SSC at room temperature. Hybridization with 48mer probes complementary to SRIH, GH, PRL, alpha subunit and FSHp was carried out exactly as above (see (7.9) for further details of tissue transport and storage, probes and protocol validation).

Hypothalamic growth hormone-releasing hormone (GHRH) and somatostatin (SRIH) control the production and secretion of growth hormone (GH) from the anterior pituitary. GHRH also induces somatouoph hyperplasia (1, 2) and when cyclic AMPmediated GHRH signal transduction is defective, as in the pituitary glands of dwldw dwarf mice (3) few somatotrophs containing little releasable growth hormone are found. Conversely, constitutive activation of the cyclic AMP second messenger pathway in somatotrophs is responsible for the formation of a subgroup of human somatotroph adenomas (4) and has been shown to produce gigantism in transgenic mice (5). Inappropriate secretion of hypothalamic releasing hormones has been adduced as an unusual cause of pituitary adenoma formation. In the past, the source of these hormones has been assumed to be hypothalamic, or at least extra-pituitary. The identification of hypothalamic releasing hormone synthesis (including GHRH) in both normal and adenomatous pituitary (6). however, raises the possibility of a paracrine or autocrine mechanism of tumour formation. We have previously identified an association between SRIH gene activity and somatotroph adenomas (7) and now report the presence of GHRH transcripts in a discrete subgroup of cells in 13 of 17 somatotroph adenomas (and a smaller proportion of other pituitary adenomas) in many cases in amounts comparable to those found in GHRH neurons of the rat arcuate nucleus. Materials Hybridization

Image analysis Slides bearing hybridized sections were apposed to autoradiography film (HyperFilm. Amersham) for 7 days and the resulting images analysed densitometrically using a Macintosh II computer equipped with an image capture board (Scion, Walkersville, MD21793) running the programme ‘Image’ by Wayne Rasband, NIMH, Bethesda, MD. The mean optical density of autoradiographs was measured in dpm/mg by comparison with simultaneously exposed 35S brain paste standards. Slides hybridized to the GHRH probe were dipped in nuclear emulsion and exposed for a further 21 days before analysis. Results The autoradiographs of pituitary tumours hybridized to the GHRH probe (Figure la) can be compared to the simultaneously hybridized and exposed autoradiograph of a rat brain section (Figure lb). The localized pattern of GHRH probe binding in the rat arcuate nucleus and in a somatouoph adenoma (corresponding to somatouoph adenoma No 1 in Figure la) is shown in Figure 2 (a & b, and c & d respectively). Of the 51 tumours examined, 23 expressed the GH gene and of these 14 contained GHRH transcripts and 11, SRIH transcripts. In 28 turnours, GH mRNA probe binding was negative and in these only 5 contained GHRH uanscripts and 5, SRIH transcripts. Of the biopsy samples from the 17 Acromegalics, 8 contained GH, SRIH and GHRH mRNA. The mean optical density of the autoradiographs of GHRH transcript positive adenomas was 12 1 l&477, compared to 125fl6 dpm/mg for rat arcuate nucleus (n = 4). We were unable to detect GHRH transcripts in sections of normal human pituitary gland.

and Methods

histochemimy

Frozen 12pm thick sections of human pituitary adenomas, a normal human pituitary gland and adult male Sprague-Dawley arcuate nucleus were thaw mounted onto gelatin/chrome alum coated slides and stored at -70°C until use. Prehybridization treatment was exactly as described (8). A synthetic 27mer oligonucleotide complementary to a sequence from the 5’ end of the second exon of human and rat GHRH (5’ AAG AAC ACC CAG AGT GGC ATC ClT CAC 3’) was labelled using 35S-dATP (NEN. Boston MA) and terminal deoxynucleotidyltransferase (Boehringer Mannheim). Sections were incubated overnight at 37°C in 5OpL hybridization buffer containing 50% formamide, 600mM NaCl, 1474

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RAPID ENDGCRINOLGGICALLY INACTIVE ADENOMAS CIRCULATING HORMONES 1 HYBRIDIZATION

COMMUNICATIONS

1 RESULTS

HYBRIDIZATION

3 1.3 6.8 212 2 279 0.2 18.1 546 2.4 7.5

1567/MO.5 16 48/F 1742/M 18 28/F

1.3 3.9 1 25t 30f

I I

774 681

7

68 50

r

SOMATOTROPH CIRCULATING

RESULTS

1 52/F 1.2 560 1.2 2 26/F

Growth hormone-releasing hormone transcripts in human pituitary adenomas.

We have investigated the possibility that local production of GHRH within the adenohypophysis could be an aetiological factor in the development of hu...
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