Regulatory Peptides, 31 (1990) 33-40
33
Elsevier
REGPEP 00963
Isolation and characterization of human thyrotropin-releasing hormone (TRH) from an endocrine pancreatic tumor* Olli Vuolteenaho 1, Juhani Lepp/iluoto 1, Shao-Yao Ying 2 and Naguib A. Samaan 3 1Department of Physiology, University of Oulu, Oulu (Finland), 2Laborutoriesfor Neuroendocrinology, Salk Institute for Biological Studies, La Jolla, CA and 3The University of Texas, M.D. Anderson Hospital Cancer Center, Texas Medical Center, Houston, TX (U.S,A.) (Received 1 February 1990; revised version received 26 June 1990; accepted 17 July 1990)
Key words: Carcinoid; Ectopic hormone production; HPLC
Summary An extract of a pancreatic carcinoid tumor obtained at autopsy from a patient who had suffered from Cushing's syndrome was found to have the ability to release thyrotropin (but not any other pituitary hormones) from cultured rat anterior pituitary cells, and to bind to a specific thyrotropin-releasing hormone (TRH) antiserum. The tumor contained 2.2 and 3.9 nmol/g of TRH bit- and immunoreactivity, respectively. The active material was purified and its amino acid composition and chromatographic properties were found to be identical with those of synthetic ovine/porcine TRH. This represents the first isolation of human TRH and the first established case of a 'TRHoma', a TRH-producing tumor.
Introduction
It is well known that non-endocrine and neoplastic tissues can produce peptide hormones [ 1-3 ]. Growth hormone-releasing hormone (GRH) was originally isolated from pancreatic tumors [4-6], and its biological properties and structure were subsequently * Some of the data in this paper were presented at the 70th Annual Meeting of the Endocrine Society, June 10-14 1988, New Orleans, LA, U.S.A. (Abstract No. 1012). Correspondence: O. Vuolteenaho, Department of Physiology, University of Oulu, 90220 Oulu, Finland. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
34 found to be the same in normal and neoplastic tissues. Tumor-derived G R H has clinical significance in the pathophysiology of growth hormone hypersecretion states [2]. For this reason we have studied the presence of hypothalamic releasing factors in tumors available to us. We report here the isolation and characterization of thyrotropin-releasing hormone (TRH) from a carcinoid tumor located in the pancreas.
Materials and Methods
Case report A 22-year-old woman sought medical advice from the Endocrine Section at the University of Texas M.D. Anderson Cancer Center. She was found to have marked cushingoid features. Her fasting plasma cortisol was higher than 400 ng/ml (normal range 80-150) with no diurnal variation and no suppression in the standard low- or high-dose dexamethasone tests. The basal plasma ACTH on four separate days was 42, 67, 52, and 123 pg/ml (normal range 0-100). She was clinically euthyroid. The serum TSH was 3.1 #U/ml (normal range 0-10), the s e r u m T 4 71 ng/ml (normal range 45-120) and the T 3 resin uptake 24.8~o (normal range 24-34). The serum prolactin ranged between 11 and 20 ng/ml (normal lower than 20). The pituitary CT scan was normal, but both adrenal glands were enlarged. A laparotomy was performed and an unresectable pancreatic tumor and multiple liver metastases were found. Biopsies from both organs were taken. Both adrenals were about four times larger than normal and they were removed. After the operation the patient was put on cortisol replacement therapy. She lost her Cushingoid features over a period of 2-6 weeks. The pancreatic tumor was found to be of islet cell origin by microscopy. Grimelius stain was positive indicating that the tumor was a carcinoid. Electron microscopy exhibited neurosecretory granules which is consistent with the diagnosis of a neuroendocrine tumor. No immunostaining studies for TRH were performed. The tumor was unresponsive to conventional chemotherapy and the patient died four years after the laparotomy. The autopsy was performed 9 1/2 h after death. It showed a massive tumor in the pancreas with metastases in the liver and the abdominal lymph nodes. The pancreatic tumor and metastases were removed in dry ice and stored at - 20 °C. Peptide extraction A tumor sample (22 g) was extracted in eight volumes (w/v) of 0.3 M HCI containing the enzyme inhibitors pepstatin A and phenylmethylsufonyl fluoride (10 mg/1). The homogenate was centrifuged at 10 000 g for 30 min and the pellet was reextracted and centrifuged as above. The combined supernatants were defatted 3 times with 2 vol. of petroleum ether/ether (2/1, vol/vol) and the pH of the aqueous phase was adjusted to 7.45 with sodium hydroxide. Because the tumor was originally part of a G R H screening project, the extract was passed through a G R H immunoaffinity column as previously described [6]. Peptide purification Two 20 ml aliquots of the flow-through of the G R H immunoaffinity column were lyophilized and processed independently through the purification procedure below. The
35 lyophilized extracts were each dissolved in 6 ml of 30% acetic acid and chromatographed in a 1.5 x 118 cm column of Sephadex G-25F (Pharmacia) eluted with the same solvent at + 4 °C. Recoveries of the immunoreactivity for this step were 72% and 77% for samples I and II, respectively. The fractions containing TRH-like immunoreactivity were applied without concentration into an SP-Sephadex C-25 (Pharmacia) cation-exchange column (1.5 x 18 cm) eluted at room temperature with a linear 250 ml gradient from 0.05 to 0.4 mol/1 ammonium acetate pH 4.5. The recoveries of immunoreactivities were 92% and 100~o for the two samples. The fractions with TRH-like immunoreactivity were pooled, concentrated in a Savant Speed Vac and injected into a Techopak C 18 reverse phase column (0.39 x 30 cm) connected to a Knauer 97.00 UV spectrophotometer and a Shimadzu C-R3A integrator/plotter. The HPLC instrument was a Varian Model 5020 liquid chromatograph. The column was first eluted at 1 ml/min for 10 min with 0.1% aqueous heptafluorobutyric acid (HFBA) followed by a 24 min linear gradient to 18 % acetonitrile in 0.1% HFBA. Fractions were collected manually on the basis of the 220 nm UV-trace and subjected to radioimmunoassay and amino acid analysis (see below). The recoveries of the immunoreactivity for this step were 112% and 93.5% for the two samples.
Bioassay and radioimmunoassays Pituitary cells of immature female rats were enzymaticaUy dispersed and used for the monolayer culture assay as described [7]. TSH, FSH and LH in the incubation fluid were measured with radioimmunoassay materials and protocols provided by Dr. A. F. Parlow and the National Hormone Agency, National Institute of Diabetes and Digestive and Kidney Diseases. ACTH was measured by a radioimmunoassay using an ACTH antiserum generously supplied by Dr. David Orth [8]. TRH radioimmunoassays were performed as described previously [ 9,10] using the antiserum' 185' specific for the pyroGlu- and ProNH2-moieties of TRH.
Amino acid analysis Aliquots (100 pmol) of the HPLC column fractions containing TRH immunoreactivity were dried in a Savant Speed Vac, hydrolyzed in 25 #1 of 6 mol/l HC1 (for 20 h at + 105 °C under N2) and the amino acid compositions were determined as described previously [ 11 ] using quantitative precolumn dansylation [ 12] and reverse phase H PLC for the separation of the dansyl amino acids (Spherisorb ODS2, 5/~m, 0.46 x 25 cm column), Varian Fluorichrom for detection and Shimadzu C-R3A for data analysis.
Results
The carcinoid tumor extract was found to contain 2.2 nmol/g and 3.9 nmol/g TRHlike bio- and immunoactivity, respectively. The slopes of the dose-response curves in both assays were similar to those produced by synthetic TRH (Fig. 1, radioimmunoassay data not shown). The extract had no effect on the release of GH, ACTH, FSH or LH (data not shown). The concentration of immunoreactive TRH in an abdominal metastasis of the tumor was 40 times lower than in the primary tumor (data not shown).
36 700-
"~
m
eoo
~
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/~"
500-
/~/
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/ ~3.125
~°°-10"'*
'
......
.......... e.~5 12~5 2's ~ 5o Tumor Extract (/zl/ml) ;~"
'
......
1~"
'
......
;~"
TRH(M) Fig. 1. TSH-releasing effect of an extract of the human pancreatic tumor (interrupted line). Note that the dose-response curve is parallel to that produced by synthetic TRH (solid line). Each symbol represents the mean of three incubations. Two 20 ml samples of the extract each representing ca. 2 g of the original tumor were independently subjected to identical steps o f purification, first by gel t'titration (Fig. 2A) followed by ion exchange chromatography (Fig. 2B). The immunoreactive TRH-like material was finally subjected to reverse phase H P L C which yielded an immunoreactive peptide corresponding to a distinct UV-absorbing peak (Fig. 2C). The tumor TRH-like peptide and synthetic T R H eluted identically in all steps o f purification. The isolation yields (as immunoreactive T R H ) were 5.8 and 5.6 nmol from the two 20 ml extracts, - 1.5
0.6-
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o
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....
~II
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4'0 . . . . . . . . .
50~ .
.
.
.
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.
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.
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60~ . . . . . .
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Fraction Number Fig. 2A. Fig. 2. Isolation of TRH from the human pancreatic tumor. (A) Gel filtration of the tumor extract. Immunoreactive TRH is represented by shaded columns. (B)Cation-exchange chromatography of the immunoreactive fractions from gel filtration. (C)Reverse phase HPLC of the immunoreactive fractions from cation-exchange chromatography. For details, see Materials and Methods.
37
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60
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. . . . .
70
Fig. 2B.
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38 TABLE I Amino acid composition of the tumor-derived TRH-immunoreactivepeptide Amino acid
Batch 1
Batch 2
His Glx Pro Glyb
1.0 + 0.2 a 0.9 ± 0.1 1.1 _+0.1 0.2 _+0.0
0.2 0.9 + 0.0 1.2 + 0.2 0.3 + 0.1 0.9 +
a Means _+S.D. b Similar amounts of Gly were present in buffer blank hydrolyzates. All other amino acids less than 0.1.
corresponding to overall recoveries of 74~o and 72~o, respectively, for the chromatographic steps. The amino acid compositions of the peptides isolated from the two batches of the tumor extract were identical to those of synthetic T R H (Table I).
Discussion
T R H has previously been shown to be present in the nervous system, gastrointestinal tract and pancreas [ 10,13-15]. In addition TRH-like immunoreactivity has previously been detected in human lung, breast and bladder carcinomas as well as in a leiomyosarcoma in extremely low concentrations (0.52 pmol/g or less) [3]. In the present study we have isolated from a carcinoid tumor of the pancreas a peptide with T R H bio- and immunoactivity, and with amino acid composition and chromatographic properties identical to those of ovine/porcine T R H , pyroGlu-His-ProNH 2 [16,17]. To our knowledge this represents the fn'st isolation and chemical characterization at the amino acid level of a peptide with T R H activity from human tissues. This paper also documents the first established case of a ' T R H o m a ' , a tumor which produces biologically active T R H . The level of T R H in the tumor we described here was remarkably high (3900 pmol/g immunoreactive TRH), approximately 10 times higher than that found in the hypothalamus [13]. The plasma level of T R H of the patient was not determined because the production of T R H was found only after the death of the patient. Therefore we do not know whether the tumor secreted T R H into the blood circulation. The patient did not show any noticeable signs of thyroid hyperfunction, and her serum T4, T3 resin uptake as well as T S H and prolactin levels were normal. The significance of these results is difficult to know because there is no agreement as to whether a chronic elevation of T R H levels would lead to pituitary thyrotroph/lactotroph and thyroid hyperfunction [ 18-21 ]. The patient described here had also suffered from Cushing's syndrome and she had had her adrenals removed. Whether there was an association with the adrenal hyperfunction and the pancreatic tumor is not known. The tumor extract did not contain any corticotropin-releasing hormone activity. Other more subtle clinical effects of the tumorderived peptide might have gone unnoticed. Because T R H has a multitude of biological effects [22] the production of T R H by tumors, if it will not prove to be a rarity, might
39 b e r e s p o n s i b l e at least in p a r t to s o m e o f the v a r i e d clinical s y n d r o m e s a s s o c i a t e d with n e u r o e n d o c r i n e t u m o r s , in a n a l o g y to g r o w t h h o r m o n e - r e l e a s i n g h o r m o n e ( G R H ) [23].
Acknowledgment T h i s s t u d y w a s s u p p o r t e d in part b y a g r a n t f r o m the Sigrid Juselius F o u n d a t i o n .
References 1 Jones, J.E., Shane, S.R., Gilbert, E. and Fluik, E.B., Cushing's syndrome induced by the ectopic production of ACTH by a bronchial carcinoid, J. Clin. Endoerinol. Metab., 29 (1969) 1-5. 2 Frohman, L.A., Szabo, M., Berelowitz, M. and Stachura, M.E., Partial purification and characterization of a peptide with growth hormone-releasing activity from extrapituitary tumors in patients with acromegaly, J. Clin. Invest., 65 (1980) 43-54. 3 Wilber, J.F.,and Spinella, P., Identificationofimmunoreactive thyrotropin-releasing hormone in human neoplasia, J. Clin. Endocrinol. Metab., 59 (1984) 432-435. 4 Guillemin, R., Brazeau, P., BShln, P., Esch, F., Ling, N. and Wehrenberg, W.B., Growth hormonereleasing factor from a human pancreatic tumor that caused acromegaly, Science, 218 (1982) 585-587. 5 Rivier, J., Spiess, J., Thorner, M. and Vale, W., Characterization of a growth hormone-releasing factor from a human pancreatic islet tumor, Nature, 300 (1982) 276-278. 6 Ling, N., Esch, F., B6hlen, P., Brazeau, P., Wehrenberg, W.B. and Guillemin, R., Isolation, primary structure and synthesis of human hypothalamic somatocrinin: Growth hormone-releasing factor. Proc. Natl. Acad. Sci. USA, 81 (1984) 4302-4306. 7 Vale, W., Grant, G., Amos, M., Blackwell, R. and Guillemin, R., Culture of enzymatically dispersed anterior pituitary cells: Functional validation of the method, Endocrinology, 91 (1972) 562-572. 8 Nicholson, W.E. and Orth, D.N., Rapid radioimmunoassay for corticotropin in unextracted human plasma, Clin. Chem. 30 (1984) 259-263. 9 Ling, N., Lepp~iluoto, J. and Vale, W., Chemical, biological and immunologicalcharacterization ofmonoand diiodothyrotropin releasing factor, Anal. Biochem., 76 (1976) 125-133. 10 Lepp~Uuoto, J., Vuolteenaho, O. and Koivusalo F., Thyrotropin-releasing factor: Radioimmunoassay and distribution in biological fluids and tissues, Med. Biol., 59 (1981) 85-91. 11 Vuolteenaho, O., Beta-endorphin-related peptides in the human pituitary, Acta Physiol. Stand., Suppl 531 (1984) 1-84. 12 Tapuhi, Y., Schmidt, D.E., Lindner, W. and Karger B.L., Dansylation of amino acids for highperformance liquid chromatography analysis, Anal. Biochem., 115 (1981) 123-129. 13 Jackson, I. M. D. and Reichlin, S., Thyrotropin-releasinghormone (TRH): Distribution in hypothalamic and extrahypothalamic brain tissue of mammalian and submammalian chordates, Endocrinology, 95 (1974) 854-862. 14 Vuolteenaho, O. and Lepp~iluoto J., Extrahypothalamic thyrotropin-releasing hormone (TRH). In W. S. Hancock, CRC Handbook of HPLC for the separation of amino acids, peptides and proteins, vol. 2, CRC Press Boca Raton, Florida 1984, pp. 205-212. 15 Lepp~iluoto, J., Koivusalo, F. and Kraama, R., Thyrotropin-releasing factor: Distribution in neural and gastrointestinal tissues, Acta Physiol. Scand., 104 (1978) 175-179. 16 Burgus, R., Dunn, T. F., Desiderio, D., and Guillemin,R., Structure moleculaire du facteur hypothalamique hypophysiotrope TRF d'origine ovine: Mise en evidence par spectrometric de masse de la sequence, C.R. Acad. Sci. Paris, 269 (1969) 1870-1873. 17 Boler, J., Enzmann, F., Folkers, K., Bowers, C.Y. and Schally, A.V., The identity of chemical and hormonal properties of the thyrotropin-releasing hormone and pyroGlutamyl-Histidyl-Prolineamide, Biochem. Biophys. Res. Commun. 37 (1969) 705-710. 18 Tyson, J.E., Perez, A. and Zanartu, J., Human lactational response to oral thyrotropin-releasing hormone, J. Clin. Endocrinol. Metab. 43 (1976) 760-768.
40 19 Reichlin, S., Neuroendocrine control ofthyrotropin secretion. In S. H. Ingbar and L. E. Braverman, The Thyroid, 5th Edition, Lippincott Philadelphia, 1986, p. 254. 20 Mitsuma, T., Nogimori, T., Chaya, M. and Nakao, N., Influences of TRH treatment on hormone secretion in SCD. In Sobue, I. (Ed.), TRH & Spinocerebellar degeneration, Elsevier Amsterdam, 1986, pp. 197-202. 21 Kaplan, M. M., Taft, J., Reichlin, S. and Munsat, T. L., Sustained rises in serum thyrotropin, thyroxine, and triiodothyronine during long term, continuous thyrotropin-releasing hormone treatment in patients with amyotrophie lateral sclerosis, J. Clin. Endocrinol. Metabl. 63 (1986) 808-814. 22 Horita, A., Carino, A.M. and Lai, H., Pharmacology of thyrotropin-releasing hormone, Annual Rev. Pharmacol. Toxicol., 26 (1986) 311-332. 23 Guillemin, R., Brazeau, P., B6hlen, P., Esch, F., Ling, N., Wehrenberg, W.B., Block, B., Mougin, C., Zeytin, F. and Baird, A., Somatocrinin, the growth hormone releasing factor, Recent Progr. Horm. Res., 40 (1984) 233-299.
33
Elsevier
REGPEP 00963
Isolation and characterization of human thyrotropin-releasing hormone (TRH) from an endocrine pancreatic tumor* Olli Vuolteenaho 1, Juhani Lepp/iluoto 1, Shao-Yao Ying 2 and Naguib A. Samaan 3 1Department of Physiology, University of Oulu, Oulu (Finland), 2Laborutoriesfor Neuroendocrinology, Salk Institute for Biological Studies, La Jolla, CA and 3The University of Texas, M.D. Anderson Hospital Cancer Center, Texas Medical Center, Houston, TX (U.S,A.) (Received 1 February 1990; revised version received 26 June 1990; accepted 17 July 1990)
Key words: Carcinoid; Ectopic hormone production; HPLC
Summary An extract of a pancreatic carcinoid tumor obtained at autopsy from a patient who had suffered from Cushing's syndrome was found to have the ability to release thyrotropin (but not any other pituitary hormones) from cultured rat anterior pituitary cells, and to bind to a specific thyrotropin-releasing hormone (TRH) antiserum. The tumor contained 2.2 and 3.9 nmol/g of TRH bit- and immunoreactivity, respectively. The active material was purified and its amino acid composition and chromatographic properties were found to be identical with those of synthetic ovine/porcine TRH. This represents the first isolation of human TRH and the first established case of a 'TRHoma', a TRH-producing tumor.
Introduction
It is well known that non-endocrine and neoplastic tissues can produce peptide hormones [ 1-3 ]. Growth hormone-releasing hormone (GRH) was originally isolated from pancreatic tumors [4-6], and its biological properties and structure were subsequently * Some of the data in this paper were presented at the 70th Annual Meeting of the Endocrine Society, June 10-14 1988, New Orleans, LA, U.S.A. (Abstract No. 1012). Correspondence: O. Vuolteenaho, Department of Physiology, University of Oulu, 90220 Oulu, Finland. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
34 found to be the same in normal and neoplastic tissues. Tumor-derived G R H has clinical significance in the pathophysiology of growth hormone hypersecretion states [2]. For this reason we have studied the presence of hypothalamic releasing factors in tumors available to us. We report here the isolation and characterization of thyrotropin-releasing hormone (TRH) from a carcinoid tumor located in the pancreas.
Materials and Methods
Case report A 22-year-old woman sought medical advice from the Endocrine Section at the University of Texas M.D. Anderson Cancer Center. She was found to have marked cushingoid features. Her fasting plasma cortisol was higher than 400 ng/ml (normal range 80-150) with no diurnal variation and no suppression in the standard low- or high-dose dexamethasone tests. The basal plasma ACTH on four separate days was 42, 67, 52, and 123 pg/ml (normal range 0-100). She was clinically euthyroid. The serum TSH was 3.1 #U/ml (normal range 0-10), the s e r u m T 4 71 ng/ml (normal range 45-120) and the T 3 resin uptake 24.8~o (normal range 24-34). The serum prolactin ranged between 11 and 20 ng/ml (normal lower than 20). The pituitary CT scan was normal, but both adrenal glands were enlarged. A laparotomy was performed and an unresectable pancreatic tumor and multiple liver metastases were found. Biopsies from both organs were taken. Both adrenals were about four times larger than normal and they were removed. After the operation the patient was put on cortisol replacement therapy. She lost her Cushingoid features over a period of 2-6 weeks. The pancreatic tumor was found to be of islet cell origin by microscopy. Grimelius stain was positive indicating that the tumor was a carcinoid. Electron microscopy exhibited neurosecretory granules which is consistent with the diagnosis of a neuroendocrine tumor. No immunostaining studies for TRH were performed. The tumor was unresponsive to conventional chemotherapy and the patient died four years after the laparotomy. The autopsy was performed 9 1/2 h after death. It showed a massive tumor in the pancreas with metastases in the liver and the abdominal lymph nodes. The pancreatic tumor and metastases were removed in dry ice and stored at - 20 °C. Peptide extraction A tumor sample (22 g) was extracted in eight volumes (w/v) of 0.3 M HCI containing the enzyme inhibitors pepstatin A and phenylmethylsufonyl fluoride (10 mg/1). The homogenate was centrifuged at 10 000 g for 30 min and the pellet was reextracted and centrifuged as above. The combined supernatants were defatted 3 times with 2 vol. of petroleum ether/ether (2/1, vol/vol) and the pH of the aqueous phase was adjusted to 7.45 with sodium hydroxide. Because the tumor was originally part of a G R H screening project, the extract was passed through a G R H immunoaffinity column as previously described [6]. Peptide purification Two 20 ml aliquots of the flow-through of the G R H immunoaffinity column were lyophilized and processed independently through the purification procedure below. The
35 lyophilized extracts were each dissolved in 6 ml of 30% acetic acid and chromatographed in a 1.5 x 118 cm column of Sephadex G-25F (Pharmacia) eluted with the same solvent at + 4 °C. Recoveries of the immunoreactivity for this step were 72% and 77% for samples I and II, respectively. The fractions containing TRH-like immunoreactivity were applied without concentration into an SP-Sephadex C-25 (Pharmacia) cation-exchange column (1.5 x 18 cm) eluted at room temperature with a linear 250 ml gradient from 0.05 to 0.4 mol/1 ammonium acetate pH 4.5. The recoveries of immunoreactivities were 92% and 100~o for the two samples. The fractions with TRH-like immunoreactivity were pooled, concentrated in a Savant Speed Vac and injected into a Techopak C 18 reverse phase column (0.39 x 30 cm) connected to a Knauer 97.00 UV spectrophotometer and a Shimadzu C-R3A integrator/plotter. The HPLC instrument was a Varian Model 5020 liquid chromatograph. The column was first eluted at 1 ml/min for 10 min with 0.1% aqueous heptafluorobutyric acid (HFBA) followed by a 24 min linear gradient to 18 % acetonitrile in 0.1% HFBA. Fractions were collected manually on the basis of the 220 nm UV-trace and subjected to radioimmunoassay and amino acid analysis (see below). The recoveries of the immunoreactivity for this step were 112% and 93.5% for the two samples.
Bioassay and radioimmunoassays Pituitary cells of immature female rats were enzymaticaUy dispersed and used for the monolayer culture assay as described [7]. TSH, FSH and LH in the incubation fluid were measured with radioimmunoassay materials and protocols provided by Dr. A. F. Parlow and the National Hormone Agency, National Institute of Diabetes and Digestive and Kidney Diseases. ACTH was measured by a radioimmunoassay using an ACTH antiserum generously supplied by Dr. David Orth [8]. TRH radioimmunoassays were performed as described previously [ 9,10] using the antiserum' 185' specific for the pyroGlu- and ProNH2-moieties of TRH.
Amino acid analysis Aliquots (100 pmol) of the HPLC column fractions containing TRH immunoreactivity were dried in a Savant Speed Vac, hydrolyzed in 25 #1 of 6 mol/l HC1 (for 20 h at + 105 °C under N2) and the amino acid compositions were determined as described previously [ 11 ] using quantitative precolumn dansylation [ 12] and reverse phase H PLC for the separation of the dansyl amino acids (Spherisorb ODS2, 5/~m, 0.46 x 25 cm column), Varian Fluorichrom for detection and Shimadzu C-R3A for data analysis.
Results
The carcinoid tumor extract was found to contain 2.2 nmol/g and 3.9 nmol/g TRHlike bio- and immunoactivity, respectively. The slopes of the dose-response curves in both assays were similar to those produced by synthetic TRH (Fig. 1, radioimmunoassay data not shown). The extract had no effect on the release of GH, ACTH, FSH or LH (data not shown). The concentration of immunoreactive TRH in an abdominal metastasis of the tumor was 40 times lower than in the primary tumor (data not shown).
36 700-
"~
m
eoo
~
"•
/~"
500-
/~/
~4oo "1"
300-
/ ~3.125
~°°-10"'*
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......
.......... e.~5 12~5 2's ~ 5o Tumor Extract (/zl/ml) ;~"
'
......
1~"
'
......
;~"
TRH(M) Fig. 1. TSH-releasing effect of an extract of the human pancreatic tumor (interrupted line). Note that the dose-response curve is parallel to that produced by synthetic TRH (solid line). Each symbol represents the mean of three incubations. Two 20 ml samples of the extract each representing ca. 2 g of the original tumor were independently subjected to identical steps o f purification, first by gel t'titration (Fig. 2A) followed by ion exchange chromatography (Fig. 2B). The immunoreactive TRH-like material was finally subjected to reverse phase H P L C which yielded an immunoreactive peptide corresponding to a distinct UV-absorbing peak (Fig. 2C). The tumor TRH-like peptide and synthetic T R H eluted identically in all steps o f purification. The isolation yields (as immunoreactive T R H ) were 5.8 and 5.6 nmol from the two 20 ml extracts, - 1.5
0.6-
0 ".P (.) 0.4-
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.
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Fraction Number Fig. 2A. Fig. 2. Isolation of TRH from the human pancreatic tumor. (A) Gel filtration of the tumor extract. Immunoreactive TRH is represented by shaded columns. (B)Cation-exchange chromatography of the immunoreactive fractions from gel filtration. (C)Reverse phase HPLC of the immunoreactive fractions from cation-exchange chromatography. For details, see Materials and Methods.
37
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Fig. 2B.
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.
.
m
40
.
.
.
.
.
.
.
.
.
m
50
1
1
T
IO cq
38 TABLE I Amino acid composition of the tumor-derived TRH-immunoreactivepeptide Amino acid
Batch 1
Batch 2
His Glx Pro Glyb
1.0 + 0.2 a 0.9 ± 0.1 1.1 _+0.1 0.2 _+0.0
0.2 0.9 + 0.0 1.2 + 0.2 0.3 + 0.1 0.9 +
a Means _+S.D. b Similar amounts of Gly were present in buffer blank hydrolyzates. All other amino acids less than 0.1.
corresponding to overall recoveries of 74~o and 72~o, respectively, for the chromatographic steps. The amino acid compositions of the peptides isolated from the two batches of the tumor extract were identical to those of synthetic T R H (Table I).
Discussion
T R H has previously been shown to be present in the nervous system, gastrointestinal tract and pancreas [ 10,13-15]. In addition TRH-like immunoreactivity has previously been detected in human lung, breast and bladder carcinomas as well as in a leiomyosarcoma in extremely low concentrations (0.52 pmol/g or less) [3]. In the present study we have isolated from a carcinoid tumor of the pancreas a peptide with T R H bio- and immunoactivity, and with amino acid composition and chromatographic properties identical to those of ovine/porcine T R H , pyroGlu-His-ProNH 2 [16,17]. To our knowledge this represents the fn'st isolation and chemical characterization at the amino acid level of a peptide with T R H activity from human tissues. This paper also documents the first established case of a ' T R H o m a ' , a tumor which produces biologically active T R H . The level of T R H in the tumor we described here was remarkably high (3900 pmol/g immunoreactive TRH), approximately 10 times higher than that found in the hypothalamus [13]. The plasma level of T R H of the patient was not determined because the production of T R H was found only after the death of the patient. Therefore we do not know whether the tumor secreted T R H into the blood circulation. The patient did not show any noticeable signs of thyroid hyperfunction, and her serum T4, T3 resin uptake as well as T S H and prolactin levels were normal. The significance of these results is difficult to know because there is no agreement as to whether a chronic elevation of T R H levels would lead to pituitary thyrotroph/lactotroph and thyroid hyperfunction [ 18-21 ]. The patient described here had also suffered from Cushing's syndrome and she had had her adrenals removed. Whether there was an association with the adrenal hyperfunction and the pancreatic tumor is not known. The tumor extract did not contain any corticotropin-releasing hormone activity. Other more subtle clinical effects of the tumorderived peptide might have gone unnoticed. Because T R H has a multitude of biological effects [22] the production of T R H by tumors, if it will not prove to be a rarity, might
39 b e r e s p o n s i b l e at least in p a r t to s o m e o f the v a r i e d clinical s y n d r o m e s a s s o c i a t e d with n e u r o e n d o c r i n e t u m o r s , in a n a l o g y to g r o w t h h o r m o n e - r e l e a s i n g h o r m o n e ( G R H ) [23].
Acknowledgment T h i s s t u d y w a s s u p p o r t e d in part b y a g r a n t f r o m the Sigrid Juselius F o u n d a t i o n .
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40 19 Reichlin, S., Neuroendocrine control ofthyrotropin secretion. In S. H. Ingbar and L. E. Braverman, The Thyroid, 5th Edition, Lippincott Philadelphia, 1986, p. 254. 20 Mitsuma, T., Nogimori, T., Chaya, M. and Nakao, N., Influences of TRH treatment on hormone secretion in SCD. In Sobue, I. (Ed.), TRH & Spinocerebellar degeneration, Elsevier Amsterdam, 1986, pp. 197-202. 21 Kaplan, M. M., Taft, J., Reichlin, S. and Munsat, T. L., Sustained rises in serum thyrotropin, thyroxine, and triiodothyronine during long term, continuous thyrotropin-releasing hormone treatment in patients with amyotrophie lateral sclerosis, J. Clin. Endocrinol. Metabl. 63 (1986) 808-814. 22 Horita, A., Carino, A.M. and Lai, H., Pharmacology of thyrotropin-releasing hormone, Annual Rev. Pharmacol. Toxicol., 26 (1986) 311-332. 23 Guillemin, R., Brazeau, P., B6hlen, P., Esch, F., Ling, N., Wehrenberg, W.B., Block, B., Mougin, C., Zeytin, F. and Baird, A., Somatocrinin, the growth hormone releasing factor, Recent Progr. Horm. Res., 40 (1984) 233-299.
