J Neurosurg 72:879-882, 1990
Prolactin gene expression in human growth hormonesecreting pituitary adenomas TAKASHI NAGAYA, M.D., HISAO SEO, M.D., AKIO KUWAYAMA, M.D., TSUYOSHI SAKURAI, M.D., NOBUHIRO TSUKAMOTO, M.D., KENICHIRO SUGITA, M.D., AND NOBUO MATSUI~ M.D.
Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, and Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan ~" To elucidate the mechanism of hyperprolactinemia often observed in patients with growth hormone (GH)secreting pituitary adenomas, the presence of immunoreactive prolactin (ir-PRL) and prolactin (PRL) messenger ribonucleic acid (mRNA) in the tumor tissue was examined by immunohistochemistry and cytoplasmic dot hybridization. Hyperprolactinemia was observed in three of 18 patients with GH-secreting adenoma. The tumor tissue was demonstrated to contain ir-PRL in nine patients and PRL mRNA in 13. The presence of irPRL in the tumor tissue was always associated with positive PRL mRNA, indicating production of PRL in GH-secreting tumors. Among the three patients with hyperprolactinemia, both ir-PRL and PRL mRNA was revealed in the tumor tissue of one, PRL mRNA but not ir-PRL was detected in the adenoma tissue of another, and neither PRL mRNA nor ir-PRL was found in the tumor tissue of the third. The association of hyperprolactinemia with the presence of both ir-PRL and PRL mRNA or PRL mRNA alone is indicative of PRL production and secretion. However, the absence of ir-PRL and PRL mRNA in the tumor tissue may indicate that hyperprolactinemia is caused by the suppression of PRL inhibitory factor due to hypothalamic dysfunction by the tumor mass. Thus, the study of PRL gene expression and immunohistochemistry in GHsecreting adenomas is valuable to understanding the pathophysiology of pituitary tumors. KEY WORDS prolactin 9 immunohistochemistry 9 gene e x p r e s s i o n hyperprolactinemia 9 growth hormone - pituitary a d e n o m a 9
p
ATIENTSwith growth h o r m o n e (GH)-secreting pituitary a d e n o m a often exhibit hyperprolactinemia; the frequency o f this occurrence is reported to be as high as 30% to 40%. 6,9 Immunohistochemical studies have shown that immunoreactive prolactin (irPRL) can be detected in t u m o r cells in 32% to 70% of these cases.l'4'5'~3 T o further study the pathophysiology of hyperprolactinemia in patients with GH-secreting adenomas, the presence o f ir-PRL and prolactin (PRL) messenger ribonucleic acid ( m R N A ) in the t u m o r tissues was investigated by immunohistochemistry and cytoplasmic dot hybridization, respectively. Clinical M a t e r i a l and M e t h o d s
Clinical Material Eighteen patients with acromegaly, aged 21 to 61 years, were operated on by transsphenoidal adenomect o m y at the D e p a r t m e n t of Neurosurgery, Nagoya University School of Medicine. The endocrinological data and t u m o r sizes before surgery are summarized in Table 1. Serum G H and P R L levels were determined by J. Neurosurg. / Volume 72~June, 1990
9
radioimmunoassay using commercial kits, and t u m o r sizes were graded according to the classification o f H a r d y and Vezina. 3 A d e n o m a tissues were collected f r o m the patients during surgery. A portion o f the tissue was fixed with 10% formaldehyde and e m b e d d e d in paraffin for histological studies. The remainder o f the tissue was kept at - 7 0 " C for cytoplasmic dot hybridization.
Immunohistochemistry Indirect i m m u n o p e r o x i d a s e staining for G H and P R L was performed as described by Nakane and Pierce? ~ The primary antibodies against h u m a n G H a n d h u m a n P R L were provided by the National Institute of Diabetes, Digestive and Kidney Diseases. The secondary antibody conjugated with peroxidase was obtained from the Medical Biological Laboratory, Tokyo, Japan. In brief, sections of a d e n o m a tissues 5 u m thick were treated with 0.3% H2OE before incubation with primary antibody to block endogenous peroxidase activity. After 879
T. Nagaya, et al.
FIG. 1. I m m u n o h i s t o c h e m i c a l studies for prolactin (PRL) in the tissue o f growth h o r m o n e (GH)-secreting pituitary a d e n o m a s . Prolactin was d e t e c t e d in nine of the 18 patients. Left: Case 9. A single PRL-positive cell (arrow) is seen s u r r o u n d e d by G H cells. Center." Case 14. A cluster o f PRL-positive cells is surrounded by G H cells. Right." Case 8. A mosaic pattern of PRL- a n d GH-positive cells is d e m o n s t r a t e d .
incubation with the primary and secondary antibodies, v i s u a l i z a t i o n w a s a c c o m p l i s h e d b y s t a i n i n g w i t h diaminobenzidine solution and counterstaining with hematoxylin. Normal rabbit serum in place of antiserum was used as negative control.
TABLE 1
Summary of clinical data, immunohistochemical findings, and mRNA analysis* Case No.
Age, (yrs), Sex
Tumor Size1"
1
30, M
2 3 4 5 6 7 8 9 10
39, M 35, F 47, F 41, F 21, F 34, M 49, F 39, F 39, M
11
45, F
IIa IIId IIIa II II lid II I II I III IIb II II II IIa IIb II
12 28, F 13 57, M 14 51, F 15 48, F 16 49, F 17 46, F 18 61, M normal range
Serum Level (ng/ml)
Immunostaining
GH
GH
PRL
GH
PRL
+
-
+
+
+ + + + + + + + +
+ + + +
+ + + + + + + + +
+ + + + + +
PRL
14.0 14.4 35.3 2.6 75.2 6.6 31.2 10.1 42.1 20.0 323.0 23.3 80 4.8 29.8 126.9 80.0 6.1 8.2 2.0 18.5 9.7 80 2.3 55.0 7.7 53.6 6.7 39.6 5.9 64.4 15.4 80 3.8 23,3 10.2 < 5.0 < 20.0
+
+ + + + + + +
-
+ + + + +
mRNA
+
+ + + + + + +
+
+ + + + +
* Abbreviations: mRNA = messenger ribonucleic acid; GH = growth hormone; PRL = prolactin; + = present; - = absent. t Tumor size was graded according to the classification by Hardy and Vezina. 3 880
Preparation o f cDNA Probe Complementary deoxyribonucleic acid (cDNA) for h u m a n P R L 2 c l o n e d i n t o t h e P s t I site o f t h e p l a s m i d pBR 322 and human GH cDNA 8 cloned into the Hind III site o f t h e s a m e p l a s m i d w e r e u s e d . A f t e r t r e a t m e n t with respective restriction enzymes, the cDNA inserts w e r e p u r i f i e d b y a g a r o s e gel e l e c t r o p h o r e s i s a n d electroelusion. These DNA's were labeled with phosphorus32-deoxycytidine triphosphate (32p-dCTP) by nick t r a n s l a t i o n , '~ a n d t h e l a b e l e d p r o b e s (1 x 106 t o 1 • 107 disintegrations per minute (dpm)/reaction) were used for cytoplasmic dot hybridization.
Cytoplasmic Dot Hybridization C y t o p l a s m i c d o t h y b r i d i z a t i o n , as d e s c r i b e d b y W h i t e a n d B a n c r o f t , TM w a s e m p l o y e d t o d e t e c t t h e G H a n d P R L m R N A ' s i n t h e t u m o r tissues. H u m a n l i v e r as n e g a t i v e c o n t r o l a n d f e t a l p i t u i t a r y as p o s i t i v e c o n t r o l were used. In brief, tumor tissue was homogenized in 10 #1 i c e - c o l d b u f f e r ( c o n t a i n i n g 10 m M T r i s - H C l ( p H 7.0), 1 m M e t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d ( E D T A ) , a n d 0 . 5 % N o n i d e t P 4 0 / m g t u m o r tissue. T h e n u c l e i were pelleted and a 0.6 volume of 20 x standard saline citrate (SSC: 0.15 M NaC1, 0.015 M trisodium citrate) and a 0.4 volume of 37% formaldehyde were added to the cytoplasmic fraction. The mixture was then incub a t e d a t 6 0 ~ f o r 15 m i n u t e s . T h e c y t o p l a s m i c f r a c t i o n w a s h e a t e d , t h e n s e r i a l l y d i l u t e d w i t h 15 x S S C a n d applied to a nitrocellulose sheet employing a 96-hole Minifold apparatus.* Then RNA was immobilized onto * Nitrocellulose sheet BA85 a n d Minifold apparatus m a n ufactured by Schleicher & Schuell, Keene, New H a m p s h i r e .
J. Neurosurg. / Volume 72~June, 1990
Prolactin gene expression in pituitary adenomas
FIG. 2. Cytoplasmic dot hybridization demonstration of growth hormone (GH) messenger ribonucleic acid (mRNA) m GH-secreting adenomas. Growth hormone mRNA's were detected in all cases. L = liver RNA; F = fetal pituitary tissue.
the nitrocellulose sheet by baking at 80~ for 90 minutes. The sheets were prehybridized at 42~ for 2 89hours in a solution consisting of 50% deionized formamide, 744 m M NaC1, 5 m M EDTA, 0.1% sodium dodecyl sulfate (SDS), 50 m M NaH2PO4, 16.25 m M N a O H , 0.1% Ficoll, 0.1% povidone, 0.1% bovine serum albumin, and 0.01% denatured herring sperm DNA. They were then hybridized overnight with 32p-labeled G H or P R L cDNA probes. After they were washed three times for 10 minutes in 2 x SSC containing 0.1% SDS at r o o m temperature and twice for 1 hour in 0.1 x SSC with 0.1% SDS at 68"C, the washed sheets were dried on W h a t m a n n 3M paper and exposed for a few days to K o d a k X - O m a t A R film in a cassette at -70"C. We have shown in a previous paper ~2 that G H cDNA does not cross-hybridize with P R L m R N A , and PRL c D N A does not cross-hybridize with G H m R N A . Results
Serum G H and P R L Levels As shown in Table 1, all patients exhibited high levels of serum GH. High serum P R L levels were observed in three (17%) of the 18 patients (Cases 5, 6, and 8). There was no correlation between serum G H and P R L levels.
lmmunohistochemistry Immunoreactive G H (ir-GH) was demonstrated in the a d e n o m a tissues from all patients, but P R L i m m u noreactivity was detected in nine (50%) of the 18 patients. In one patient (Case 8), the association of high serum P R L level (126.9 ng/ml) and ir-PRL in the t u m o r tissue was demonstrated.
J. Neurosurg. / Volume 72/June, 1990
FIG. 3. Cytoplasmic dot hybridization demonstration of prolactin messenger ribonucleic acid (mRNA) in growth hormone-secreting adenomas. Prolactin mRNA was detected in Cases 1, 3, 5, 7, 8, 9, 10, 11, 13, 14, 15, 17, and 18. Although in some cases the signals are faint in the photograph, they are clearly present in the original autoradiogram. L = liver RNA; F = fetal pituitary tissue.
When the distribution o f the cells with positive P R L i m m u n o r e a c t i v i t y in the a d e n o m a tissue was examined, five samples (from Cases 9, 10, 13, 15, and 18) showed a single PRL-positive cell s u r r o u n d e d by i r - G H cells (Fig. 1 left). In three t u m o r samples (from Cases 7, 14, and 17), a cluster o f P R L cells was surrounded by irG H cells (Fig. 1 center). In the t u m o r tissue from Case 8, a mosaic pattern o f P R L - a n d GH-positive cells was seen (Fig. 1 right). Serum P R L levels in all patients with the two types illustrated in Fig. 1 left and center were within n o r m a l limits. A high P R L level was observed only in Case 8.
Cytoplasmic Dot Hybridization Growth h o r m o n e m R N A was detected in the adenohaa tissues f r o m all patients (Fig. 2). Prolactin m R N A was present in 13 (72%) o f the 18 cases (Fig. 3). It should be noted that P R L m R N A was demonstrated in all patients with positive P R L i m m u n o r e a c t i v i t y in the t u m o r tissues. In four instances (Cases 1, 3, 5, and 11), positive P R L m R N A was d e m o n s t r a t e d in the t u m o r tissue without P R L i m m u n o r e a c t i v i t y . In Case 6, a high serum P R L level was not associated with either i m m u noreactive P R L or P R L m R N A in the a d e n o m a tissue. Discussion A m o n g 18 cases o f GH-secreting a d e n o m a s studied, hyperprolactinemia was observed in three (17%), P R L i m m u n o r e a c t i v i t y in the t u m o r tissue was d e m o n strated in nine (50%), a n d P R L m R N A in 13 cases 881
T. Nagaya, et al. (72%). The frequency of detection of P R L m R N A in GH-secreting adenomas was high, as mentioned by Lloyd, et al. 7 Prolactin m R N A was demonstrated in all cases with positive P R L cells in the t u m o r tissues, indicating that the presence o f P R L immunoreactivity in the a d e n o m a tissue was due to P R L synthesis in the tissues. Hyperprolactinemia was not observed in patients whose t u m o r tissue showed a single or a few PRLpositive cells surrounded by GH-positive ceils. However, P R L m R N A was demonstrated in all o f these cases. Thus, some GH-secreting adenomas without hyperprolactinemia can also synthesize PRL in the tissue. The absence of hyperprolactinemia in these cases could be due to the small n u m b e r o f PRL-synthesizing cells in the adenoma. In only one patient (Case 8) was hyperprolactinemia associated with positive P R L cells and P R L m R N A in the t u m o r tissue. In Case 6, despite a high serum P R L level, both irP R L and P R L m R N A investigations were negative in the a d e n o m a tissue. It was suggested that the high serum P R L level in this patient was not caused by PRL production in the a d e n o m a tissue but by hypothalamic dysfunction due to the t u m o r mass (the disturbance of PRL-inhibitory factor). Thus, the detection o f PRL immunoreactivity and P R L m R N A in the adenoma tissue makes it possible to differentiate the causes of hyperprolactinemia in patients with GH-secreting adenoma. In this study, the presence o f P R L m R N A without P R L immunoreactivity in the t u m o r tissue was demonstrated in four cases. There are three possibilities for the absence o f immunoreactive P R L in those tissues: 1) cytoplasmic dot hybridization is more sensitive than immunohistochemistry; 2) sparse distribution of PRL cells in the a d e n o m a makes it difficult for these cells to be included in sections obtained for immunohistochemical study; and 3) P R L synthesized by the t u m o r cells has no immunoreactivity. Acknowledgments
We are indebted to Dr. J. A. Martial (University of Liege) for providing human GH and human PRL cDNA. We are also grateful to the National Institute of Diabetes, Digestive and Kidney Diseases for supplying the antisera of human PRL and human GH used in the immunohistochemistry. References
1. Bassetti M, Spada A, Arosio M, et al: Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Cliu Endocrinol Metab 62:1093-1100, 1986 2. Cooke NE, Coit D, Shine J, et al: Human prolactin. cDNA structural analysis and evolutionary comparisons.
882
J Biol Chem 256:4007-4016, 1981 3. Hardy J, Vezina JL: Transsphenoidal neurosurgery of intracranial neoplasm, in Thompson RA, Green JR (eds): Neoplasia in the Central Nervous System. Advances in Neurology, Vol 15. New York: Raven Press, 1976, pp 261-274 4. Kanie N, Kageyama N, Kuwayama A, et al: Pituitary adenomas in acromegalic patients: an immunohistochemical and endocrinological study with special reference to prolactin secreting adenoma. J Clin Endocrinol Metab 57:1093-1100, 1983 5. Kovacs K, Horvath E: Adenomas with GH and PRL production, in Kovacs K, Horvath E (eds): Tumors of the Pituitary Gland. Atlas of Tumor Pathology, Series 2, Fascicle 21. Washington, DC: Armed Forces Institute of Pathology, 1986, pp 116-133 6. Lamberts SWJ, Liuzzi A, Chiodini PG, et al: The value of plasma prolactin levels in the prediction of the responsiveness of growth hormone secretion to bromocriptine and TRH in acromegaly. Eur J Clin Invest 12:151-155, 1982 7. Lloyd RV, Cano M, Chandler WF, et al: Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization. Am J Pathol 134: 605-613, 1989 8. Martial JA, Hallewell RA, Baxter JD, et al: Human growth hormone. Complementary DNA cloning and expression in bacteria. Science 205:602-607, 1979 9. Moriondo P, Travaglini P, Rondena M, et al: Prolactin secretion in acromegaly, in Faglia G, Giovanelli MA, MacLeod RM (eds): Pituitary Mieroadenomas. London: Academic Press, 1980, pp 247-256 10. Nakane PK, Pierce GB Jr: Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histoehem Cytochem 14:929-931, 1966 (Letter) 11. Rigby PWJ, Dieckmann M, Rhodes C, et al: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237-251, 1977 12. Sakurai T, Seo H, Yamamoto N, et al: Detection of mRNA of prolactin and ACTH in clinically nonfunctioning pituitary adenomas. J Neurosurg 69:653-659, 1988 13. Trouillas J, Girod C, Sassolas G, et al: Immunocytochemistry, What does it add to clinical management?, in Landolt AM, Heitz PU, Zapf J, et al (eds): Advances in Pituitary Adenoma Research. Oxford: Pergamon Press, 1988, pp 11-20 14. White BA, Bancroft FC: Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem 257:8569-8572, 1982 Manuscript received August 3, 1989. Accepted in final form November 22, 1989. This work was supported in part by Grant-in-Aid for Scientific Research 61570695 from the Japanese Ministry of Education, and by a Research Grant for Intractable Disease from the Japanese Ministry of Health and Welfare. Address reprint requests to: Hisao Seo, M.D., Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan.
J. Neurosurg. / Volume 7 2 / J u n e , 1990
Prolactin gene expression in human growth hormonesecreting pituitary adenomas TAKASHI NAGAYA, M.D., HISAO SEO, M.D., AKIO KUWAYAMA, M.D., TSUYOSHI SAKURAI, M.D., NOBUHIRO TSUKAMOTO, M.D., KENICHIRO SUGITA, M.D., AND NOBUO MATSUI~ M.D.
Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, and Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan ~" To elucidate the mechanism of hyperprolactinemia often observed in patients with growth hormone (GH)secreting pituitary adenomas, the presence of immunoreactive prolactin (ir-PRL) and prolactin (PRL) messenger ribonucleic acid (mRNA) in the tumor tissue was examined by immunohistochemistry and cytoplasmic dot hybridization. Hyperprolactinemia was observed in three of 18 patients with GH-secreting adenoma. The tumor tissue was demonstrated to contain ir-PRL in nine patients and PRL mRNA in 13. The presence of irPRL in the tumor tissue was always associated with positive PRL mRNA, indicating production of PRL in GH-secreting tumors. Among the three patients with hyperprolactinemia, both ir-PRL and PRL mRNA was revealed in the tumor tissue of one, PRL mRNA but not ir-PRL was detected in the adenoma tissue of another, and neither PRL mRNA nor ir-PRL was found in the tumor tissue of the third. The association of hyperprolactinemia with the presence of both ir-PRL and PRL mRNA or PRL mRNA alone is indicative of PRL production and secretion. However, the absence of ir-PRL and PRL mRNA in the tumor tissue may indicate that hyperprolactinemia is caused by the suppression of PRL inhibitory factor due to hypothalamic dysfunction by the tumor mass. Thus, the study of PRL gene expression and immunohistochemistry in GHsecreting adenomas is valuable to understanding the pathophysiology of pituitary tumors. KEY WORDS prolactin 9 immunohistochemistry 9 gene e x p r e s s i o n hyperprolactinemia 9 growth hormone - pituitary a d e n o m a 9
p
ATIENTSwith growth h o r m o n e (GH)-secreting pituitary a d e n o m a often exhibit hyperprolactinemia; the frequency o f this occurrence is reported to be as high as 30% to 40%. 6,9 Immunohistochemical studies have shown that immunoreactive prolactin (irPRL) can be detected in t u m o r cells in 32% to 70% of these cases.l'4'5'~3 T o further study the pathophysiology of hyperprolactinemia in patients with GH-secreting adenomas, the presence o f ir-PRL and prolactin (PRL) messenger ribonucleic acid ( m R N A ) in the t u m o r tissues was investigated by immunohistochemistry and cytoplasmic dot hybridization, respectively. Clinical M a t e r i a l and M e t h o d s
Clinical Material Eighteen patients with acromegaly, aged 21 to 61 years, were operated on by transsphenoidal adenomect o m y at the D e p a r t m e n t of Neurosurgery, Nagoya University School of Medicine. The endocrinological data and t u m o r sizes before surgery are summarized in Table 1. Serum G H and P R L levels were determined by J. Neurosurg. / Volume 72~June, 1990
9
radioimmunoassay using commercial kits, and t u m o r sizes were graded according to the classification o f H a r d y and Vezina. 3 A d e n o m a tissues were collected f r o m the patients during surgery. A portion o f the tissue was fixed with 10% formaldehyde and e m b e d d e d in paraffin for histological studies. The remainder o f the tissue was kept at - 7 0 " C for cytoplasmic dot hybridization.
Immunohistochemistry Indirect i m m u n o p e r o x i d a s e staining for G H and P R L was performed as described by Nakane and Pierce? ~ The primary antibodies against h u m a n G H a n d h u m a n P R L were provided by the National Institute of Diabetes, Digestive and Kidney Diseases. The secondary antibody conjugated with peroxidase was obtained from the Medical Biological Laboratory, Tokyo, Japan. In brief, sections of a d e n o m a tissues 5 u m thick were treated with 0.3% H2OE before incubation with primary antibody to block endogenous peroxidase activity. After 879
T. Nagaya, et al.
FIG. 1. I m m u n o h i s t o c h e m i c a l studies for prolactin (PRL) in the tissue o f growth h o r m o n e (GH)-secreting pituitary a d e n o m a s . Prolactin was d e t e c t e d in nine of the 18 patients. Left: Case 9. A single PRL-positive cell (arrow) is seen s u r r o u n d e d by G H cells. Center." Case 14. A cluster o f PRL-positive cells is surrounded by G H cells. Right." Case 8. A mosaic pattern of PRL- a n d GH-positive cells is d e m o n s t r a t e d .
incubation with the primary and secondary antibodies, v i s u a l i z a t i o n w a s a c c o m p l i s h e d b y s t a i n i n g w i t h diaminobenzidine solution and counterstaining with hematoxylin. Normal rabbit serum in place of antiserum was used as negative control.
TABLE 1
Summary of clinical data, immunohistochemical findings, and mRNA analysis* Case No.
Age, (yrs), Sex
Tumor Size1"
1
30, M
2 3 4 5 6 7 8 9 10
39, M 35, F 47, F 41, F 21, F 34, M 49, F 39, F 39, M
11
45, F
IIa IIId IIIa II II lid II I II I III IIb II II II IIa IIb II
12 28, F 13 57, M 14 51, F 15 48, F 16 49, F 17 46, F 18 61, M normal range
Serum Level (ng/ml)
Immunostaining
GH
GH
PRL
GH
PRL
+
-
+
+
+ + + + + + + + +
+ + + +
+ + + + + + + + +
+ + + + + +
PRL
14.0 14.4 35.3 2.6 75.2 6.6 31.2 10.1 42.1 20.0 323.0 23.3 80 4.8 29.8 126.9 80.0 6.1 8.2 2.0 18.5 9.7 80 2.3 55.0 7.7 53.6 6.7 39.6 5.9 64.4 15.4 80 3.8 23,3 10.2 < 5.0 < 20.0
+
+ + + + + + +
-
+ + + + +
mRNA
+
+ + + + + + +
+
+ + + + +
* Abbreviations: mRNA = messenger ribonucleic acid; GH = growth hormone; PRL = prolactin; + = present; - = absent. t Tumor size was graded according to the classification by Hardy and Vezina. 3 880
Preparation o f cDNA Probe Complementary deoxyribonucleic acid (cDNA) for h u m a n P R L 2 c l o n e d i n t o t h e P s t I site o f t h e p l a s m i d pBR 322 and human GH cDNA 8 cloned into the Hind III site o f t h e s a m e p l a s m i d w e r e u s e d . A f t e r t r e a t m e n t with respective restriction enzymes, the cDNA inserts w e r e p u r i f i e d b y a g a r o s e gel e l e c t r o p h o r e s i s a n d electroelusion. These DNA's were labeled with phosphorus32-deoxycytidine triphosphate (32p-dCTP) by nick t r a n s l a t i o n , '~ a n d t h e l a b e l e d p r o b e s (1 x 106 t o 1 • 107 disintegrations per minute (dpm)/reaction) were used for cytoplasmic dot hybridization.
Cytoplasmic Dot Hybridization C y t o p l a s m i c d o t h y b r i d i z a t i o n , as d e s c r i b e d b y W h i t e a n d B a n c r o f t , TM w a s e m p l o y e d t o d e t e c t t h e G H a n d P R L m R N A ' s i n t h e t u m o r tissues. H u m a n l i v e r as n e g a t i v e c o n t r o l a n d f e t a l p i t u i t a r y as p o s i t i v e c o n t r o l were used. In brief, tumor tissue was homogenized in 10 #1 i c e - c o l d b u f f e r ( c o n t a i n i n g 10 m M T r i s - H C l ( p H 7.0), 1 m M e t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d ( E D T A ) , a n d 0 . 5 % N o n i d e t P 4 0 / m g t u m o r tissue. T h e n u c l e i were pelleted and a 0.6 volume of 20 x standard saline citrate (SSC: 0.15 M NaC1, 0.015 M trisodium citrate) and a 0.4 volume of 37% formaldehyde were added to the cytoplasmic fraction. The mixture was then incub a t e d a t 6 0 ~ f o r 15 m i n u t e s . T h e c y t o p l a s m i c f r a c t i o n w a s h e a t e d , t h e n s e r i a l l y d i l u t e d w i t h 15 x S S C a n d applied to a nitrocellulose sheet employing a 96-hole Minifold apparatus.* Then RNA was immobilized onto * Nitrocellulose sheet BA85 a n d Minifold apparatus m a n ufactured by Schleicher & Schuell, Keene, New H a m p s h i r e .
J. Neurosurg. / Volume 72~June, 1990
Prolactin gene expression in pituitary adenomas
FIG. 2. Cytoplasmic dot hybridization demonstration of growth hormone (GH) messenger ribonucleic acid (mRNA) m GH-secreting adenomas. Growth hormone mRNA's were detected in all cases. L = liver RNA; F = fetal pituitary tissue.
the nitrocellulose sheet by baking at 80~ for 90 minutes. The sheets were prehybridized at 42~ for 2 89hours in a solution consisting of 50% deionized formamide, 744 m M NaC1, 5 m M EDTA, 0.1% sodium dodecyl sulfate (SDS), 50 m M NaH2PO4, 16.25 m M N a O H , 0.1% Ficoll, 0.1% povidone, 0.1% bovine serum albumin, and 0.01% denatured herring sperm DNA. They were then hybridized overnight with 32p-labeled G H or P R L cDNA probes. After they were washed three times for 10 minutes in 2 x SSC containing 0.1% SDS at r o o m temperature and twice for 1 hour in 0.1 x SSC with 0.1% SDS at 68"C, the washed sheets were dried on W h a t m a n n 3M paper and exposed for a few days to K o d a k X - O m a t A R film in a cassette at -70"C. We have shown in a previous paper ~2 that G H cDNA does not cross-hybridize with P R L m R N A , and PRL c D N A does not cross-hybridize with G H m R N A . Results
Serum G H and P R L Levels As shown in Table 1, all patients exhibited high levels of serum GH. High serum P R L levels were observed in three (17%) of the 18 patients (Cases 5, 6, and 8). There was no correlation between serum G H and P R L levels.
lmmunohistochemistry Immunoreactive G H (ir-GH) was demonstrated in the a d e n o m a tissues from all patients, but P R L i m m u noreactivity was detected in nine (50%) of the 18 patients. In one patient (Case 8), the association of high serum P R L level (126.9 ng/ml) and ir-PRL in the t u m o r tissue was demonstrated.
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FIG. 3. Cytoplasmic dot hybridization demonstration of prolactin messenger ribonucleic acid (mRNA) in growth hormone-secreting adenomas. Prolactin mRNA was detected in Cases 1, 3, 5, 7, 8, 9, 10, 11, 13, 14, 15, 17, and 18. Although in some cases the signals are faint in the photograph, they are clearly present in the original autoradiogram. L = liver RNA; F = fetal pituitary tissue.
When the distribution o f the cells with positive P R L i m m u n o r e a c t i v i t y in the a d e n o m a tissue was examined, five samples (from Cases 9, 10, 13, 15, and 18) showed a single PRL-positive cell s u r r o u n d e d by i r - G H cells (Fig. 1 left). In three t u m o r samples (from Cases 7, 14, and 17), a cluster o f P R L cells was surrounded by irG H cells (Fig. 1 center). In the t u m o r tissue from Case 8, a mosaic pattern o f P R L - a n d GH-positive cells was seen (Fig. 1 right). Serum P R L levels in all patients with the two types illustrated in Fig. 1 left and center were within n o r m a l limits. A high P R L level was observed only in Case 8.
Cytoplasmic Dot Hybridization Growth h o r m o n e m R N A was detected in the adenohaa tissues f r o m all patients (Fig. 2). Prolactin m R N A was present in 13 (72%) o f the 18 cases (Fig. 3). It should be noted that P R L m R N A was demonstrated in all patients with positive P R L i m m u n o r e a c t i v i t y in the t u m o r tissues. In four instances (Cases 1, 3, 5, and 11), positive P R L m R N A was d e m o n s t r a t e d in the t u m o r tissue without P R L i m m u n o r e a c t i v i t y . In Case 6, a high serum P R L level was not associated with either i m m u noreactive P R L or P R L m R N A in the a d e n o m a tissue. Discussion A m o n g 18 cases o f GH-secreting a d e n o m a s studied, hyperprolactinemia was observed in three (17%), P R L i m m u n o r e a c t i v i t y in the t u m o r tissue was d e m o n strated in nine (50%), a n d P R L m R N A in 13 cases 881
T. Nagaya, et al. (72%). The frequency of detection of P R L m R N A in GH-secreting adenomas was high, as mentioned by Lloyd, et al. 7 Prolactin m R N A was demonstrated in all cases with positive P R L cells in the t u m o r tissues, indicating that the presence o f P R L immunoreactivity in the a d e n o m a tissue was due to P R L synthesis in the tissues. Hyperprolactinemia was not observed in patients whose t u m o r tissue showed a single or a few PRLpositive cells surrounded by GH-positive ceils. However, P R L m R N A was demonstrated in all o f these cases. Thus, some GH-secreting adenomas without hyperprolactinemia can also synthesize PRL in the tissue. The absence of hyperprolactinemia in these cases could be due to the small n u m b e r o f PRL-synthesizing cells in the adenoma. In only one patient (Case 8) was hyperprolactinemia associated with positive P R L cells and P R L m R N A in the t u m o r tissue. In Case 6, despite a high serum P R L level, both irP R L and P R L m R N A investigations were negative in the a d e n o m a tissue. It was suggested that the high serum P R L level in this patient was not caused by PRL production in the a d e n o m a tissue but by hypothalamic dysfunction due to the t u m o r mass (the disturbance of PRL-inhibitory factor). Thus, the detection o f PRL immunoreactivity and P R L m R N A in the adenoma tissue makes it possible to differentiate the causes of hyperprolactinemia in patients with GH-secreting adenoma. In this study, the presence o f P R L m R N A without P R L immunoreactivity in the t u m o r tissue was demonstrated in four cases. There are three possibilities for the absence o f immunoreactive P R L in those tissues: 1) cytoplasmic dot hybridization is more sensitive than immunohistochemistry; 2) sparse distribution of PRL cells in the a d e n o m a makes it difficult for these cells to be included in sections obtained for immunohistochemical study; and 3) P R L synthesized by the t u m o r cells has no immunoreactivity. Acknowledgments
We are indebted to Dr. J. A. Martial (University of Liege) for providing human GH and human PRL cDNA. We are also grateful to the National Institute of Diabetes, Digestive and Kidney Diseases for supplying the antisera of human PRL and human GH used in the immunohistochemistry. References
1. Bassetti M, Spada A, Arosio M, et al: Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J Cliu Endocrinol Metab 62:1093-1100, 1986 2. Cooke NE, Coit D, Shine J, et al: Human prolactin. cDNA structural analysis and evolutionary comparisons.
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J Biol Chem 256:4007-4016, 1981 3. Hardy J, Vezina JL: Transsphenoidal neurosurgery of intracranial neoplasm, in Thompson RA, Green JR (eds): Neoplasia in the Central Nervous System. Advances in Neurology, Vol 15. New York: Raven Press, 1976, pp 261-274 4. Kanie N, Kageyama N, Kuwayama A, et al: Pituitary adenomas in acromegalic patients: an immunohistochemical and endocrinological study with special reference to prolactin secreting adenoma. J Clin Endocrinol Metab 57:1093-1100, 1983 5. Kovacs K, Horvath E: Adenomas with GH and PRL production, in Kovacs K, Horvath E (eds): Tumors of the Pituitary Gland. Atlas of Tumor Pathology, Series 2, Fascicle 21. Washington, DC: Armed Forces Institute of Pathology, 1986, pp 116-133 6. Lamberts SWJ, Liuzzi A, Chiodini PG, et al: The value of plasma prolactin levels in the prediction of the responsiveness of growth hormone secretion to bromocriptine and TRH in acromegaly. Eur J Clin Invest 12:151-155, 1982 7. Lloyd RV, Cano M, Chandler WF, et al: Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization. Am J Pathol 134: 605-613, 1989 8. Martial JA, Hallewell RA, Baxter JD, et al: Human growth hormone. Complementary DNA cloning and expression in bacteria. Science 205:602-607, 1979 9. Moriondo P, Travaglini P, Rondena M, et al: Prolactin secretion in acromegaly, in Faglia G, Giovanelli MA, MacLeod RM (eds): Pituitary Mieroadenomas. London: Academic Press, 1980, pp 247-256 10. Nakane PK, Pierce GB Jr: Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histoehem Cytochem 14:929-931, 1966 (Letter) 11. Rigby PWJ, Dieckmann M, Rhodes C, et al: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237-251, 1977 12. Sakurai T, Seo H, Yamamoto N, et al: Detection of mRNA of prolactin and ACTH in clinically nonfunctioning pituitary adenomas. J Neurosurg 69:653-659, 1988 13. Trouillas J, Girod C, Sassolas G, et al: Immunocytochemistry, What does it add to clinical management?, in Landolt AM, Heitz PU, Zapf J, et al (eds): Advances in Pituitary Adenoma Research. Oxford: Pergamon Press, 1988, pp 11-20 14. White BA, Bancroft FC: Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem 257:8569-8572, 1982 Manuscript received August 3, 1989. Accepted in final form November 22, 1989. This work was supported in part by Grant-in-Aid for Scientific Research 61570695 from the Japanese Ministry of Education, and by a Research Grant for Intractable Disease from the Japanese Ministry of Health and Welfare. Address reprint requests to: Hisao Seo, M.D., Department of Endocrinology and Metabolism, The Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464, Japan.
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