A New Bioactive Form of Human Calcitonin1

[CANCER RESEARCH 43, 3793-3799, August 1983] A New Bioactive Form of Human Calcitonin1

Paul H. Tobler,2 Maximilian A. Dambacher, Walter Born, Philipp U. Heitz, RenÃ©Maier, and Jan A. Fischer3

Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery (Balgrist) and Medicine, University of Zurich, 8008 Zurich, Switzerland [P. H. T., M. A. D., W. B., J. A. F.]; and Department of Pathology, University of Basel [P. U. H.] and Pharmaceuticals Division, Ciba-Geigy, Ltd., 4002 Basel, Switzerland [R. M.]

ABSTRACT cannot be identified. With the recent advent of HPLC analysis, hCT-(1-32) and its Distinct immunoreactive forms of calcitonin (CT), extracted sulfoxide form have been recognized in thyroid extracts and in with 2 M acetic acid from two pancreatic tumors, were charac the plasma of normal subjects and of patients with MTC (17,35, terized and identified by gel permeation chromatography and by 36). Moreover, large molecular weight (12 to 25 k dalton) com reverse-phase high-performance liquid chromatography. The ex ponents of CT have been detected in cell extracts and in the tracted CT forms were compared to CT obtained from medullary incubation medium of a CT-producing epidermoid bronchial car thyroid carcinoma and from normal thyroid glands, and were, cinoma cell line (21, 24). furthermore, analyzed in a rat hypocalcÃ©miebioassay. On gel In the present report, we have for the first time recognized filtration analysis, two broad peaks coeluting with synthetic different CT forms in tissue extracts of patients with endocrine human CT-(1-32) and extracted dimeric CT, respectively, were pancreatic tumors and MTC by HPLC analysis, and have, fur found in variable amounts. An acetonitrile gradient high-perform thermore, detected a new biologically active CT-like peptide. ance liquid chromatography system revealed two to three pre dominant CT peaks. Biologically active monomeric and dimeric MATERIALS AND METHODS CT and the biologically inactive sulfoxide form of human CT-(1 - 32) have been identified. Moreover, we have detected for the Patients. Two female patients with endocrine pancreatic tumors se first time a new biologically active CT-like component which was creting CT ectopically, a 52-year-old with a benign tumor and a 74-year- most prominently recognized in a benign pancreatic tumor. old with a malignant tumor, were compared to 2 patients (a 32-year-old female and a 38-year-old male) with CT producing MTC. Plasma CT levels were raised in the patients with pancreatic tumors (27.0 and 97.4 INTRODUCTION ng equivalent/ml; normal range, <0.05 ng equivalent/ml) and with MTC hCT4 is a 32-amino acid polypeptide hormone (M, 3418) of the (2.0 and 2000 ng equivalent/ml). In the pancreatic tumor patients, plasma levels of calcium were increased (3.44 and 2.89 mmol/liter; normal range, thyroid gland that causes hypocalcemia by inhibition of the 2.07 to 2.42 mmol/liter) and those of parathyroid hormone, 5 and 18 ng release of calcium from bone (3, 23). Increased production of CT equivalent/ml, were in the normal range (6 to 40 ng equivalent/ml). The by medullary carcinoma of the thyroid has been amply docu benign pancreatic tumor was surgically removed, plasma levels of CT mented. In 1973, Sizemore ef al. (31) detected raised concentra became undetectable, and plasma calcium was normalized. The patient tions of immunoreactive CT in the plasma of 6 of 8 patients with with the malignant pancreatic tumor died of pulmonary embolism, and the Zollinger-Ellison syndrome and mentioned the possibility of the diagnosis was established at autopsy. ectopie CT secretion from non-0-islet cell tumors of the pancreas. In one of the MTC patients, plasma calcium (2.37 mmol/liter) was in the upper normal range, and parathyroid hormone (62 ng equivalent/ml) One of their patients showed no postmortem evidence of med was increased. This patient belonged to a kindred with multiple endocrine ullary carcinoma of the thyroid. Schwartz ef a/. (30) have found adenomatosis type 2 (32) and, moreover, presented hyperplastic para elevated plasma levels of CT in 42% of the patients with pan thyroid glands and pheochromocytomas. The other MTC patient was creatic tumors. Immunocytochemical examination of endocrine inoperable because of generalized mÃ©tastasesas confirmed at autopsy. pancreatic tumors revealed the presence of CT among other Peptides. Synthetic hCT-(1-32) (monomeric form), extracted purified polypeptide hormones such as adrenocorticotropic hormone, dimeric hCT from MTC, and synthetic human parathyroid hormone-(1- gastrin, /3-endorphin, and somatostatin (25). The findings have 34) have been donated by W. Rittel, Ciba-Geigy AG, Basel, Switzerland; [3H]hCT-(1-32), by R. Wade, Ciba-Geigy, Ltd., Horsham, Great Britain been confirmed by other groups of investigators (2, 8,9,16,19). (6); and extracted bovine parathyroid hormone-(1-84), by the Medical Abe ef al. (1) demonstrated superimposable Â¡mmunodilution curves of extracts of pancreatic tumors and of synthetic hCT-(1- Research Council, Great Britain. Bovine serum albumin was purchased from Sigma Chemical Co., St. Louis, Mo., and human serum albumin 32) suggesting immunological similarities. On gel permeation was from Behring Werke, Hoechst Pharma AG, Zurich, Switzerland. chromatography of tumor extracts (15) and plasma (10, 26, 29) Preparation of Tissues and Extraction. Tumor tissue obtained at of patients with pancreatic tumors, several immunoreactive CT surgery was, after determination of wet weight, snap-frozen in liquid components have been recognized. In view of the limited reso nitrogen shortly after excision and stored at -70Â° until extraction. The lution of peptides on gel filtration analysis, different CT forms frozen tissues were homogenized in 2 M acetic acid, and the clear supernatants of the centrifuged homogenates were extracted by adsorp tion of the peptides on octadecasilyl silica (C,e-Sep-Pak cartridges; ' This work was supported by the Swiss National Science Foundation Grants 3.941-0.78 and 3.813-0.81, the State of Zurich, and the Schweizerische Verein Waters Assoc., Milford, Mass.) according to a method described previ Balgrist. ously (36). Lyophilized extracts were dissolved in 1 ml 0.1 M acetic acid 2 In partial fulfillment of the requirements for a Dr.sc.nat. degree at the Federal for further analysis. In 5 individual extractions, the overall recovery of Institute of Technology, Zurich, Switzerland. [3H]hCT-(1-32) initially added to the frozen tissue amounted to 73.1 Â± 3 To whom requests for reprints should be addressed, at Klinik Balgrist, Forch trasse 340, CH-8008 Zurich, Switzerland. 4.3% (S.E.) with a range of 62 to 84%. 'The abbreviations used are: hCT, human calcitonin; CT, calcitonin; HPLC, Gel Filtration. For gel filtration of tissue extracts, columns of Bio-Gel high-performance liquid chromatography; MTC, medullary thyroid carcinoma. P-150 (100 to 200 mesh; Bio-Rad Laboratories, Richmond Calif.), 1.6 x Received July 16, 1982; accepted May 9.1983. 100 cm, have been used by ascending flow (flow rate, 3.8 to 5.2 ml/hr)

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. P. H. Tabler et al. at 4Â°in0.2 MammoniumacÃ©tate,pH4.7, containing 0.5 g bovine sÃ©rum 4.6 mm; Macherey-NagelGmbH, Duren, West Germany)columns under albumin per liter, and fractions were collected in 1.9- to 2.6-ml volumes. either isocratic conditions or by gradient elution as described previously The void volume(V0)was estimated by the elution position of the largest- (13, 36). Extracts containing 150 ng to 10 ÃŸQimmunoreactiveCT and molecular-weightproteins determined spectrophotometrically at 280 nm tracer amounts of [3H]hCT-(1-32)and its sulfoxide (30,000 to 60,000 and the salt volume (Vs)was estimated with Na31l.The elution position dpm) not interfering in the radioimmunologicaldeterminations were in of CT components, designated ka,is defined as: jected in a volume of 0.5 ml. Fractionswere collected in siliconizedtubes; 0.2 to 0.3-ml aliquots were analyzed for 3Hradioactivity by liquid scintil (elution volume of the substance - V0) lation spectroscopy (Model MR 300; Kontron AG) in Rotiszint 22 (Carl (V, - Vo) Roth KG, Karlsruhe, West Germany). The remaining fractions were analyzed radioimmunologically.Peak fractions of several extractions of Tracer amounts of radioiodinatedhuman serum albumin,bovine parathy all numbered CT components (Charts 2 and 3) were pooled and recir- roid hormone-(1-84),humanparathyroidhormone-(1-34),hCT-(1-32),and culated in the same systems. The purified CT peptides were analyzedin Na'3'l were added to each extract as calibrating substances. Radioactiv the hypocalcÃ©mieratbioassay. The recoveries of immunoreactive hCT ity was measured in an automatic -y-wellspectrometer (Model MR 252; on HPLC were 63.7 Â±7.2% (range, 36.7 to 105.0%) under isocratic Â«ontrÃ³nAG, Zurich, Switzerland). Recovery of immunoreactive CT conditionsand 80.9 Â±4.2%(range,62.3 to 95.0%) in the gradient elution ranged from 60 to 80%. system. HPLC. The HPLC systemconsistedof a programmer(Model420; Radioimmunoassays.CT was determinedina homologoushCT-(1- Altex, Berkeley, Calif.)and 2 pumps (Model 110 A; Altex). Sampleswere 32) assay described previously (12, 13). The antibodies (goat-6A ob injected via a septumlessvalve(Model 7125; Rheodyne,Berkeley,Calif.) tained on Day 143) were used at a dilution of 1/100,000. They are fitted with a 0.5-ml injector loop. Acetonitrile (HPLC-gradeS), methanol predominantly directed to determinants located in the COOH-terminal (HPLC-grade),and trifluoroacetic and heptafluorobutyric acid (both se parts of the hCT-(1-32)molecule. The specificity of the antibodies used quencer grade) were obtained from Rathburn Chemicals (Walkerburn, has been studied previously(13). The CT-like structure of the corticotro- Great Britain). Doubly glass-distilledwater from the deionized laboratory pin-fi-lipotropin precursor (13, 22) and salmon CT-(1-32) (18) were not water supply was pumped through a 0.22-jim filter (Millipore/Continental recognized radioimmunologically. Of several polypeptide hormones Water Systems, Bedford, Mass.)and subsequentlythrough a Cis-Radial- tested, only the corticotropins, -(1-13), -(1-24),and -(1-39), and 0-endor- PAK cartridge (Waters Assoc.) to remove traces of organic impurities. phin, oxytocin, and vasopressin inhibited the immunological reaction in Reverse-phase HPLC was performed on Cie-Nucleosil (10 urn, 250 x at least 105-foldhigher concentrations than those of hCT-(1-32); all of

hCT-D hCT-(1-321 nCT-D hCT-(1-32l t I t "DhPTH r^IlhCT Ife |'Â»1HSA ["IIWTM ('"UhPTM l'"jhCT 11-341 (1-321 I1-Ã•4I (1-341 11-32] l

Chart 1. Fractionation of immunoreactive hCT of tissue extracts by gel permeation chromatography on Bio-Gel P-150. Radioiodinated human serum albumin (['J5I]H&4), bovine para

thyroid hormone - (1 - 84) [|13'l]bPTH - (1-84)], t l synthetic human parathyroid hormone-(1-34) |['25llhPTH-<1-34)], I3'l-hCT-(1-32), and Na'3'l t t Â» were added as calibrating substances to ex tracts containing 150 ng to 10 ng immunoreac tive CT. Arrows, elution positions of dimeric hCT (hCJ-D) and of hCT-(1-32); 1.6 x 100-cm column; 0.2 M ammonium acetate (pH 4.7) and bovine serum albumin (0.5 mg/ml) as eluent; reversed flow (flow rate. 4.2 ml/hr; 2.1-ml frac tions). A, benign pancreatic tumor; B, malignant pancreatic tumor; C, medullary carcinoma of the thyroid; 0, lymph node metastasis of a medullary carcinoma of the thyroid; E, normal thyroid; F, extracted dimeric CT from MTC be t t fore (â€¢)and after (O) incubation with 1 M am monia at 45Â°for 1 hr. F l t

3794 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. New Bioactive Form of hCT these peptides had retention times of less than 4 min in the isocratic this peak was clearly recognized (Chart '\,B\o D). In extracts of HPLC system (Chart 3) used in the present study and, therefore, did not a normal human thyroid, dimeric CT was only visible as a interfere with our radioimmunological CT measurements. All of the CT shoulder (Chart 1Â£)(36). The peak coeluting with dimeric CT components analyzed and plasma showed parallel immunodilution from MTC was, in part, converted into the peak coeluting with curves to hCT-(1-32) (11) (not shown). monomeric hCT-(1-32) by incubation in 1 M ammonia for 1 hr at Immunoreactive parathyroid hormone was estimated as described 45Â°(Chart 1F) (28). Moreover, in the extract of the malignant previously (34). pancreatic tumor, a broad peak of nonidentified CT-like compo Immunohistochemistry. Tumor tissue was fixed in liquid formalde hyde or Bouin's fluid. Sections (5 /Â¿m)werestained with hematoxylin and nents having apparent molecular weights greater than that of eosin, van Gieson's stain, and the periodic acid-Schiff reaction. Samples dimeric CT was seen. of the tumors were immediately quenched in melting isopentane at HPLC. Analysis by reverse-phase HPLC with an acetonitrile -160Â°, lyophilized overnight in a thermoelectric freeze-dryer at -40Â°, gradient system revealed 2 to 3 predominant peaks of immuno vapor-fixed at 60Â°for 3 hr with formaldehyde vapor, and embedded in reactive CT (Chart 2). Peak 1 had the retention time of synthetic paraffin. Deparaffinized sections (5 um) were incubated with the antibod methionine8-hCT-(1-32) sulfoxide (Chart 3F). In our extractions, ies to hCT used in the radioimmunological measurements of CT, but at CT was almost fully recovered in peaks coeluting with the a dilution of 1/400 using the unlabeled antibody enzyme method (33). nonoxidized biologically active monomeric (Peak 2) and dimeric For immunocytochemical controls, adsorption of the primary antibody with 10~12to 10"4 M hCT-(1-32) was carried out at 4Â°for 24 hr. (Peak 3) CT forms. HPLC revealed a similar variation of the Bioassay. The hypocalcÃ©mie rat bioassay was performed according amounts of presumed dimeric CT in the different tumors exam to the method of Kumar ef al. (20) in female rats. ined as noted previously on gel filtration analyses (Charts 1 and 2). A shoulder designated as 2a eluting 2 min earlier than hCT- (1-32) was primarily visible in the extract of the benign pancreatic RESULTS tumor; Peak 2b represents a CT form coeluting with hCT-(1-32) with low biological activity (Chart 2A). An additional small peak, Gel Filtration. Analyses of tumor extracts by gel permeation noted in the malignant pancreatic tumor and in one of the MTC chromatography revealed a broad peak coeluting with synthetic extracts with a retention time of 2 to 3 min shorter than that of hCT-(1-32) (0.74 kj (Chart 1). 131l-labeled hCT-(1-32) always dimeric CT, was not identified (Chart 2, o C, and F). eluted 0.1 KÂ«units after the peak of immunoreactive synthetic The region between Peaks 1 and 3 was examined in more hCT-(1 -32). A second peak eluting in the position of dimeric CT detail by HPLC under isocratic conditions (Chart 3). Excellent (0.60 K/) was also seen. In the extract of the benign pancreatic resolution of 12 hCT analogues differing in only one or 2 amino tumor (Chart ~\A), the earlier eluting component was hardly acids from hCT-(1 -32) has been achieved (not shown). The 2 CT visible, whereas, in the malignant pancreatic tumor and MTC, forms, designated Peaks 2a and 2b, in 5 different extracts of the

|1H|hCT-|l-32ls(), I |JH]hCT-ll-32l , l3H)hCT-(l-32) I) 04, A 6-1 B

Chart 2. Reverse-phase HPLC profiles of hCT of tissue extracts. Column (C,8-Nucleosil) was eluted with a linear gradient of acetonitrile A containing heptafluorobutyric acid as hydrophobic anion-pairing reagent ( ) (for details, see "Materials and Methods"). Effluent frac tions were analyzed for immunoreactive CT (â€¢) and for 3H radioactivity (O and arrows). Arrows, i i elution positions of [3H]hCT-(1-32) sulfoxide IOVU1200800-400U [[3H]hCT-(32Ur; retention time, 39 to 40 min] and (3H]hCT-(1-12) (retention time, 48 to 50 21 ~_-j_60 min) added to the extracts as calibrating sub stances. Ciphers, immunoreactive CT forms >0 coeluting with hCT-(1-32) sulfoxide (Pea* 1), (Tl40 with monomeric hCT-(1-32) (Peak 2), and with 0L dimeric CT (Pea* 3). Shoulder Za and Peak 2b were examined in more detail under isocratic 5 conditions (see Chart 3). A, benign pancreatic tumor; 8, malignant pancreatic tumor; C, med ullary carcinoma of the thyroid; D, lymph node metastasis of a medullary carcinoma of the 0030 thyroid; E, normal thyroid; F, calibration with tritiated hCT sulfoxide (Peak 1) and monomeric 3 [3H]hCT-(1-32) (Pea* 2) (O), and immunoreac tive dimeric CT (Peak 3) (â€¢). OOI5 2

20 40 20 40 60 80

TIME , mm

AUGUST 1983 3795

l3H|hCT-(l-32lsox [3H]hCT-(l-32l [3H|hCT-(l-32)s

2b DIO,

Charts. Reverse-phase HPLC profiles of hCT of tissue extracts under isocratic condi tions. Column (Cia-Nucleosil) was used with methanol/water/trifluoroacetic acid (65/34/1, v/ v/v) at a flow rate of 1 ml/min. Effluent fractions were analyzed for immunoreactive CT (â€¢)and for 3H radioactivity (O and arrows). Arrows, elution positions of [3H]hCT-(1-32) sulfoxide [[3H]hCT-(1-32)Â«â€ž,retentiontime, 9 min; range, ISO 7.8 to 10.2 min] and (3HlhCT-(1-32) (retention 6- time, 21.2 min; range, 20 to 22.9 min). Ciphers, immunoreactive CT forms coeluting with hCT- IOO 4 (1-32) sulfoxide (Peak 1) and hCT-(1-32) (Pea* 2). Peak 2a, a new CT-like peptide. Peak 2b 2- 50 Ã©lÃ»tesinthe position of hCT-(1-32). A. benign pancreatic tumor; B, malignant pancreatic tu mor; C, medullary carcinoma of the thyroid; 0, 0 lymph node metastasis of a medullary carci noma of the thyroid; E, normal thyroid; F, cali bration with tritiated (O) and immunoreactive (â€¢)hCT-(1-32) and its sulfoxide form.

003

002

001-

o 20 30

TIME.min benign pancreatic tumor were now clearly separated (Chart 3A). In all of the other tumor extracts examined, Peak 2a was absent or visible as a shoulder only (Chart 3, B to D). In normal thyroid glands, a small peak with the same retention time was also noted -0.2- (Chart 3E). Tritiated and synthetic hCT-(1-32) and its sulfoxide form have been used as calibrating substances (Chart 3F). Peaks 1 and 2 correspond to hCT-(1-32) sulfoxide and to monomeric hCT-(1-32), respectively. Peaks 2a and 2b, and an immunoreac tive peak in the dead volume have not been identified. Bioassay. HypocalcÃ©mieactivityof the immunoreactive Peaks -0.6 2 and 3 extracted from MTC representing monomeric and dimeric CT was comparable to that of synthetic hCT-(1-32) (Chart 4). Peak 1, corresponding to the methionine8-sulfoxide form of hCT- -0.8 J (1-32), was biologically inactive in amounts as high as 100 ng. 50 100 250 Similar findings have been obtained in extracts of normal human hCT (ngl thyroid glands (36). Assuming that the affinity of the antibodies Chart 4. HypocalcÃ©mie activity measured 50 min after i.v. injection of tissue to Peaks 1, 2a, and 2b of the benign pancreatic tumor extract extracts additionally purified on reverse-phase HPLC (for details, see Charts 2 and and to hCT-(1-32) is the same, Peak 2a from the benign pan 3). V, A, A, immunoreactive CT Peaks 7, 2a, and 20, respectively, extracted from creatic tumor had twice the biological potency of synthetic hCT(1 - the benign pancreatic tumor; hCT-(1-32) sulfoxide (O; Peak 7), monomeric hCT-(1- 32) (D; Peak 2), and dimeric hCT (â€¢;Peafc3) have been extracted from medullary 32), and Peaks 1 and 2b had low biological activity. carcinoma of the thyroid. Each point represents the mean of 3 to 5 rats. â€¢,dose- Immunocytochemistry. CT-like immunoreactivity was de response line of hCT-(1-32). Each point represents the mean of 10 rats. Bars, S.E. tected in many but not all cells of the pancreatic tumors, and of the MTC and a metastasis thereof (Fig. 1). Staining was confined the pancreatic tumors and MTC after preadsorption of the hCT antiserum with 10~6 and 10~5 M hCT-(1-32) and equally blocked to the cytoplasm of the tumor cells with the nuclei remaining clear. The CT-specific immunoreactivity was similarly reduced in with10'4MhCT-(1-32).

3796

DISCUSSION tritiated hCT-(1-32) and of its sulfoxide form added to the frozen tissues prior to the extractions was identical to the nonlabeled Ectopie production of CT by tumors has been widely recog hormones. Moreover, we have detected for the first time a new nized (4, 14, 27). In the present report, we have characterized biologically active CT-like form. The question as to whether this different CT forms extracted from pancreatic tumors secreting CT-like component was formed pre- or posttranslationally re CT ectopically and from MTC. The fact that CT was localized mains to be elucidated. immunocytochemically in tumor cells suggests that hCT or CT- like components have been formed in the tumor tissue. More over, the pancreatic tumors produced hypercalcÃ©mie factors ACKNOWLEDGMENTS unrelated to parathyroid hormone secreted in primary hyperpar- We are indebted to Professor Christoph Hedinger (Department of Pathology, athyroidism. Demonstration of the specific mRNA coding for the University of Zurich) for MTC tissue. factors would have been required for more definitive evidence that indeed the biosynthesis took place in the tumors (7). Proof REFERENCES of the expression of the translation products, however, can only 1. Abe, K., Adachi, I., Miyakawa, S., Tanaka, M., Yamaguchi, K., Tanaka, N., be obtained by identification of the different CT forms generated Kameya, T., and Shimosato, Y. Production of calcitonin, adrenocorticotropic by the tumor cells. Sufficient amounts of tumor material have hormone, and /i-melanocyte-stimulating hormone in tumors derived from amine precursor uptake and decarboxylation cells. Cancer Res., 37: 4190-4194, not been available for an analysis of the sequence of the amino 1977. acid residues. Nonetheless, we and others have attempted to 2. Asa, S. L, Kovacs, K., Killinger, D. W., Maroon, N.. and Platts, M. Pancreatic identify different immunologically and biologically active CT forms islet cell carcinoma producing gastrin, ACTH, alpha-endorphin, somatostatin and calcitonin. Am. J. Gastroenterol., 74: 30-35, 1980. in crude extracts of pancreatic tumors (10, 15, 26, 29). We 3. Austin, L. A., and Heath, H., III. Calcitonin: physiology and pathophysiology. realize that CT-like peptides might cross-react in our systems. N. Engl. J. Med., 304: 269-278, 1981. Different CT forms have been characterized by gel permeation 4. Baylin, S. B., and Mendelson, G. Ectopie (inappropriate) hormone production by tumors: mechanisms involved and the biological and clinical implications. chromatography and reverse-phase HPLC. Peaks coeluting with Endocrine Rev., 1: 45-77, 1980. biologically active hCT-(1-32) and its biologically inert 5. Brot, N., Weissbach, L., Werth, J., and Weissbach, H. Enzymatic reduction of methionine8-hCT-(1 -32) sulfoxide form have been detected in 2 protein-bound methionine sulfoxide. Proc. Nati. Acad. Sei. U. S. A., 78: 2155- 2158,1981. HPLC systems. Moreover, the peak coeluting with synthetic 6. Brundish, D. E., and Wade, R. Tritiated peptides. Part 9. Synthesis of [3,5- methionine8-hCT-(1-32) sulfoxide was in part converted into the 3H2-Tyr12]-and [2,5-3H2-HisÃ®o]-humancalcitonin. J. Chem. Soc. Perkin Trans. 1,318-321,1981. nonoxidized monomeric hCT-(1-32) by a methionine sulfoxide 7. Craig, R. C., Hall, L., Edbrooke, M. R., Allison, J., and Maclntyre, I. Partial reducÃase obtained from Escherichia coli (5, 7, 35). A hypocal nucleotide sequence of human calcitonin precursor mRNA identifies flanking cryptic peptides. Nature (Lond.), 295: 345-347, 1982. cÃ©miepeak on HPLC with the retention time of dimeric hCT has 8. Deftos, L. J., and Burton, D. W. Immunohistological studies of non-thyroidal also been recognized; its conversion into the monomer during calcitonin-producing tumors. J. Clin. Endocrinol. Metab., 50. 1042-1045,1980. incubation with ammonia (28) is further evidence that the peak 9. Deftos, L. J., McMillan, P. J., Sartiano, G. P., Abuid, J., and Robinson, A. G. Simultaneous ectopie production of parathyroid hormone and calcitonin. Me probably corresponds to the dimeric form of the hormone. Simi tabolism (Baltimore), 25: 543-550, 1976. larly, monomeric hCT-(1 -32) and its sulfoxide form and dimeric 10. Deftos, L. J., Roos, B. A., Bronzert, D., and Parthemore, J. G. Immunochemical CT have been recognized in extracts of thyroid glands from heterogeneity of calcitonin in plasma. J. Clin. Endocrinol. Metab., 40: 409- 412,1975. normal subjects (36). 11. Dietrich, F. M., and Fischer, J. A. Immunological studies with synthetic human In some tissue extracts, a new peak (2a), eluting 3 min earlier calcitonin. In: I. Maclntyre (ed.). Human Calcitonin and Paget's Disease, pp. than hCT-(1-32), was, moreover, detected in our isocratic HPLC 179-194. Beni: Hans Huber Publishers, 1977. system. This CT-like form was primarily visible in the extract of 12. Dietrich, F. M., Hunziker, W. H., and Fischer, J. A. Synthetic human calcitonin: analysis of antibodies obtained from various animal species and determination the benign pancreatic tumor, and it was biologically active in the of immunoreactive hormone in human sera. Acta Endocrinol (Copenh.), 80: 465-486, 1975. rat hypocalcÃ©mieassay. Another peak (2b) of the same extract 13. Fischer, J. A., Tobler, P. H., Kaufmann, M., Bom, W., Henke, H., Cooper, P. coeluted with hCT-(1-32), but showed low biological activity; it E., Sagar, S. M., and Martin, J. B. Calcitonin: regional distribution of the probably does not correspond to the native hormone. On gel hormone and its binding sites in the human brain and pituitary. Proc. Nati. Acad. Sei. U. S. A., 78: 7801-7805, 1981. permeation chromatography, a single immunoreactive peak elut 14. Friesen, S. R. Tumors of the endocrine pancreas. N. Engl. J. Med., 306: 580- ing in the region of monomeric hCT-(1 -32) has been detected in 590,1982. extracts of the benign pancreatic tumor. In extracts of normal 15. Galmiche, J. P., Chayvialle, J. A., Dubois, P. M., David, F., Deseos, F., Paulin, C., Ducastelle, T., Colin, R., and Geffroy, Y. Calcitonin-producing pancreatic thyroid glands, gel permeation chromatography under nondena- somatostatinoma. Gastroenterology, 78:1577-1583,1980. turing (Chart 1) and denaturing conditions (using a column equi 16. Galmiche, J. P., Colin, R., Dubois, P. M., Chayvialle, J. A., Deseos, F., Paulin. librated with 6 M guanidine-HCI) yielded essentially the same C., and Geffroy, Y. Calcitonin secretion by a pancreatic somatostatinoma. N. Engl. J. Med., 299: 1252, 1978. results (not shown). The new CT-like form is related in molecular 17. Girgis, S. I., McMartin, C., and Maclntyre, I. Nature of normal human calcitonin weight to monomeric hCT-(1-32). The structure of this immuno in thÃ©circulation. J. Endocrinol., 83: 55P-56P, 1979. reactive CT-like component, which is also recognized in extracts 18. Huwyler, R., Bom, W., Ohnhaus, E. E., and Fischer, J. A. Plasma kinetics and urinary excretion of exogenous human and salmon calcitonin in man. Am. J. of normal thyroid glands and possibly in MTC tissue, remains to Physiol., 236: E15-E19,1979. be elucidated. 19. Krejs, G. J., Orci, L, Colon, J. M., Ravazzola, M., Davis, G. R., Raskin, P., Collins, S. M., McCarthy, D. M., Baetens, D., Rubenstein, A., Aldor, T. A. M., In conclusion, CT components extracted from pancreatic tu and Unger, R. H. Somatostatinoma syndrome. N. Engl. J. Med., 307: 285- mors, from MTC, and from normal thyroid glands have been 292,1979. characterized by gel permeation chromatography and by HPLC 20. Kumar, M. A., Slack, E., Edwards, A., Soliman, H. A., Baghdiantz, A., Foster, G. V., and Maclntyre, I. A biological assay for calcitonin. J. Endocrinol., 33: as well as in a hypocalcÃ©miebioassay. Three predominant com 469-475,1965. ponents, corresponding to biologically active dimeric and mono 21. Lumsden, J., Ham, J., and Ellison, M. L. Purification and partial characterization of high-molecular-weight forms of ectopie calcitonin from a human bronchial meric CT and to the biologically virtually inactive sulfoxide form carcinoma cell line. Biochem. J., 797: 239-246,1980. of hCT-(1-32) have been identified. The retention behavior of 22. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S.

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Fig. 1. Immunocytochemical localization of CT-like peptides in tumor cells, x 333. Unlabeled antibody enzyme method (for details, see "Materials and Methods" i.A. benignign pancreatic tumor; B, malignant pancreatic tumor; C, medullary carcinoma of the thyroid; D, bone marrow metastasis of a medullary carcinoma of the thyroid.

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Cancer Res 1983;43:3793-3799.

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