Releasing Peptide, in a Human Medullary Thyroid Carcinoma1

Home , Calcitonin, Neuromedin B, Peptide hormone, Signal peptide

[CANCER RESEARCH 48, 2412-2416, May 1, 1988] Molecular Forms of Katacalcin, Calcitonin Gene-related Peptide and Gastrin- releasing Peptide, in a Human Medullary Thyroid Carcinoma1

J. Michael Gonion,2 Gerard P. McGregor, GÃ¶ranWallin, Lars Grimelius, and Lars Thim

Clinical Research Group for Gastrointestinal Endocrinology of the Max-Planck-Gesellschaft at the University of GÃ²ttingen, D-3400 GÃ¶ttingen,Federal Republic of Germany [J. M. C., G. P. M.]; Department of Surgery, Karolinska Hospital, Stockholm, Sweden [G. W.]; Department of Pathology, University Hospital, Uppsala, Sweden [L. G.J; and Novo Research Institute, Bagsvaerd, Denmark [L. T.]

ABSTRACT amination of a fine needle biopsy showed medullary thyroid carcinoma. Subsequent examination of the patient revealed extensive mÃ©tastases Peptides synthesized by a human medullary thyroid carcinoma were purified to homogeneity by reverse-phase high performance liquid chro- in lymph nodes, in the neck, right supraclavicular fossa, mediastinum, and lungs. Total thyroidectomy was performed and a large tumor mass matography and structurally characterized by determination of amino growing upwards into the neck was removed. The patient had a serum acid composition, amino acid sequence, and fast atom bombardment mass calcitonin concentration of 40.8 ng/ml before operation and 20.8 ng/ spectra. The katacalcin-related material in the tumor extract was heter ml after tumor resection (healthy subjects, 8-40 pg/ml). There were no ogeneous. Katacalcin (1-21) represented the predominant molecular form obvious signs of thyroid disease or other endocrine disorders in the but metabolites, identified as katacalcin (1-20), (1-19), (1-15) and (1- patient's nearest relatives. 13), were also identified in high concentration. Calcitonin gene-related peptide-I was isolated from the tumor but calcitonin gene-related pept Â¡de Tumor Histopathology. Light microscopy showed an argyrophil tu ll was absent. A minor component of calcitonin gene-related peptide-like mor with polyglonal cells and a mixed growth pattern (solid and trabecular arrangement). Moderate nuclear atypia was seen. Mitoses immunoreactivity was of higher molecular weight and may represent an were fairly frequent. Immunostaining using the peroxidase-antiperoxi- incompletely processed form of the prohormone. Gastrin-releasing peptide (1-27) and gastrin-releasing peptide (18-27) (neuromedin C) were dase technique showed that the majority of tumor cells displayed isolated from the tumor but gastrin-releasing peptide (14-27) and bom- calcitonin and chromogranin A immunoreactivity. Groups of cells showing GRP, somatostatin, and neurotensin immunoreactivity were besin were absent. observed. An antiserum that reacts with bombesin but not GRP did not immunostain any tumor cells. Full details of the antisera used in INTRODUCTION Â¡ninninoliistochemistry are provided in Ref. 12. Medullary carcinomas of the thyroid are derived from para- Extraction of Tumor Tissue. The tumor was immersed in liquid follicular ("-cells and are frequently associated with the produc nitrogen immediately after resection and stored at â€”70Â°Cuntiltime of tion of multiple regulatory peptide products. Immunohisto- extraction. The tissue (2.81 g) was homogenized while still frozen with chemical and radioimmunoassay studies have identified calci ethanol/0.7 M HC1 (3:1 vol/vol; 25 ml) using an Ultraturrax blender. tonin (1), katacalcin (2), CGRP3 (3), gastrin-releasing peptide The homogenate was stirred at 4"C for 16 h and centrifuged (20,000 x (GRP) (4), somatostatin (5), pancreatic polypeptide (6), and g for 1 h). Ethanol was removed from the supernatant under reduced adrenocorticotropic hormone (7). Calcitonin (and its COOH- pressure and peptides were isolated using Sep-pak CIS cartridges terminal flanking peptide, katacalcin) and CGRP-I are products (Waters Associates, Milford, MA) as described (13). Bound material was eluted with acetonitrile (80% vol/vol) and lyophilized. Synthetic of the same gene but they arise from different mRNAs by katacalcin (1-21) (40 nmol) and gastrin-releasing peptide (20 nmol) alternative RNA processing events (8). More recently, a second were incubated with the original extract (0.5 ml) for 16 h at 4Â°Cand human gene has been identified that encodes the precursor of the peptides were isolated using a Sep-pak cartridge. CGRP-II (9). The human CGRP-II sequence differs from the Purification of Peptides. The tumor extract, after partial purification CGRP-I sequence by three amino acid residues and the peptide on Sep-pak cartridges, was redissolved in 0.1% vol/vol trifluoroacetic has been detected in nervous tissue and medullary thyroid acid (2 ml) and injected onto a reverse-phase Supelcosil LC-18-DB column (250 x 10 mm; Supelco Inc., BeliefÂ«mte, PA) equilibrated with carcinoma (10). A single gene encoding the sequence of human 0.1 % trifluoroacetic acid. The column was eluted at 30Â°Cand at a flow GRP directs the synthesis of three distinct mRNAs resulting in the production of three preproGRP peptides (11). These pre rate of 2 ml/min. The acetonitrile concentration in the eluting solvent cursors differ in structure in the region of the COOH-terminal was raised to 21% vol/vol over 10 min followed by a raise to 49% vol/ extension peptide and all contain the full sequence of GRP (I- vol over 80 min. Absorbance was measured at 214 and 280 nm and individual peaks were collected by hand. 27). In this study, peptides derived from procalcitonin, pro- Peaks 1-5 (Fig. 1) representing katacalcin and related metabolites CGRP, and proGRP were isolated in pure form from an extract were purified to homogeneity on a Supelcosil LC-3DP column (250 x of a human medullary thyroid carcinoma. Structural character 4.6 mm) equilibrated with 0.1% trifluoroacetic acid. The column was ization of these peptides has provided insight into the pathways eluted at 30Â°Cand at a flow rate of 1.5 ml/min. The concentration of of posttranslational processing of the prohormones in the tumor acetonitrile in the eluting solvent was raised to 21% vol/vol over 30 cells. min. Peak 6 (Fig. 1), representing GRP (18-27), was purified to homo MATERIALS AND METHODS geneity on a Supelcosil LC-18-DB column (250 x 4.6 mm) equilibrated with 0.1% trifluoroacetic acid. The column was eluted at 30Â°Cand at Patient Details. The patient, a 41-year-old male, was admitted to hospital because of a progressive growth of a goiter. Cytological ex- a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised from 14-35% vol/vol over 60 min. Received6/2/87; revised 10/30/87; accepted2/8/88. Peak 7 (Fig. 1), representing GRP (1-27), was purified under the The costs of publication of this article were defrayed in part by the payment same conditions as peak 6 except that the acetonitrile concentration of page charges. This article must therefore be hereby marked advertisement in was raised from 17.5-38.5% vol/vol over 60 min. The peptide was accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by the Stiftung Volkswagenwerk and the Swedish purified to homogeneity on a Supelcosil LC-3DP column (250 x 4.6 Medical Research Council (Project 102). mm) equilibrated with 0.1 % trifluoroacetic acid. The column was eluted 2To whom requests for reprints should be addressed, at Klinische Arbeits at 30Â°Cand at a flow rate of 1.5 ml/min. The concentration of gruppe der MPC, Gosslerstrasse 10D, D-3400 GÃ¶ttingen, Federal Republic of acetonitrile was raised from 17.5-38.5% vol/vol over 60 min. Germany. 3The abbreviations used are: CGRP, calcitonin gene-related peptide; GRP, Peak 8 (Fig. 1), representing CGRP-1, was purified to homogeneity gastrin-releasing peptide; cDNA, complementary DNA. on a Vydac 218TP54 column (250 x 4.6 mm; The Separations Group, 2412

AB GRP CORP

20 CD < 70 80 Time (min) 5 Fig. 1. Reverse phase high performance liquid chromatography on a semipre parative CIS column of an extract of a medullary thyroid carcinoma. Details of the elution conditions are given in the text. Arrows,retention times of neuromedin C (NMC) identical to GRP (18-27), GRP (14-27), bombesin (B), GRP (1-27), and human CGRP-I. O. fractions with CGRP-like immunoreactivity. The follow ing peptides were isolated: peak 1, katacalcin (1-13); peak 2, katacalcin (1-15); 20 30 40 50 peak 3, katacalcin (1-21); peak 4, katacalcin (1-19); peak 5, katacalcin (1-20); Time (min) peak 6, GRP (18-27); peak 7, GRP (1-27); peak Â«,CGRP-I; peak 9, a high molecular weight form of CGRP. AHSi,,, absorbance at 214 nm. Fig. 2. Purification of tumor katacalcin (1-21) (peak 3 in Fig. 1) by reverse- phase high performance liquid chromatography on an analytical column. , concentration of acetonitrile in the eluting solvent. Peak A represents katacalcin Hesperia, CA) equilibrated with 0.1% trifluoroacetic acid. The column (1-21) and peak B was not part of the procalcitonin sequence. AUS.,,,, absorbance was eluted at 30*C and at a flow rate of 1.5 ml/min. The concentration at 214 nm. of acetonitrile was raised from 21-48% vol/vol over 60 min. Structural Analysis. Amino acid compositions were determined using peptides were katacalcin (1-13) (peak 1) 4.7 nmol; katacalcin a Durrum DSOOautomatic analyzer as previously described (13). Tryp- (1-15) (peak 2) 3.9 nmol; katacalcin (1-19) (peak 4) 13.1 nmol, tophan and cysteine residues were not determined. Primary structures katacalcin (1-20) (peak 5) 5.8 nmol; katacalcin (1-21) (peak 3) were determined by automated Edman degradation using an Applied 27.6 nmol. Synthetic katacalcin (1-21), incubated with the Biosystems Model 470A gas phase sequencer (14). The detection limit original extract and subjected to the same isolation procedure, for phenylthiohydantoin amino acids was 0.5 pmol. Fast-atom bom was eluted from the reverse-phase high performance liquid bardment mass spectrometry was carried out by M-Scan, Ascot, United chromatography column as a single peak (recovery, 5*85%) with Kingdom, under conditions previously described (15). Radioimmunoassay Methods. GRP-like immunoreactivity was meas no evidence for conversion to metabolites. ured using an antiserum directed against the COOH-terminal region of Approximately 90% of the CGRP-like immunoreactivity in GRP (1-27) that reacts fully with GRP (18-27) (neuromedin C), GRP the tumor extract was eluted from the semipreparative column (14-27), and bombesin but shows <1% reactivity with neuromedin B with the same retention time as human CGRP-I (peak 8 in Fig. (16). CGRP-like immunoreactivity was measured using antiserum 1). A minor peak of immunoreactivity was eluted at a higher RAS6012 supplied by Peninsula Laboratories Europe, Ltd., ([2-'"I] concentration of acetonitrile (peak 9 in Fig. 1). Under these lodohistidyl1") human CGRP-I (specific activity, 2000 Ci/mmol; Amer- conditions of chromatography CGRP-I was not resolved from sham Buchler, Braunschweig, Federal Republic of Germany) was used CGRP-II. Peak 8 CGRP was eluted from an analytical reverse- as tracer and human CGRP-I (Peninsula Laboratories Europe) as phase column with a retention time of 29.2 min compared with standard. The antiserum shows full reactivity with human CGRP-II. a retention time of 29.1 min for synthetic CGRP-I. The yield The 50% inhibitory dose of the assay was 3.3 fmol/tube and the intra- and interassay coefficients of variation were 7 and 11%, respectively. of pure peptide was 6.3 nmol. Surprisingly, in view of the difference in primary structure, CGRP-I could not be resolved Other regulatory peptides were measured as described in Ref. 17. from CGRP-II on the analytical column. CGRP-II, but not CGRP-I, contains a methionine residue and treatment of RESULTS CGRP-II with hydrogen peroxide under conditions in which this residue is oxidized to methionine sulfoxide (18) resulted in Concentrations of Regulatory Peptides in Tumor Extract. The a reduction in the retention time of the peptide by 3.5 min. The concentration of CGRP-like immunoreactivity in the tumor retention time of CGRP-I did not change. Treatment of tumor extract was 4.97 nmol/g wet weight, GRP-like immunoreactiv CGRP with hydrogen peroxide did not alter its retention time ity was 556 pmol/g, and somatostatin-like immunoreactivity suggesting that the peptide did not contain a methionine resi was 22 pmol/g. Serial dilutions of these immunoreactivities in due. Gel permeation chromatography of peak 9 CGRP-like the extract gave rise to a series of lines whose slopes were immunoreactivity indicated that the peptide was of higher mo parallel to the corresponding synthetic standards in radio- lecular weight than CGRP-I. Peak 9 CGRP was also eluted immunoassay. Glucagon, vasoactive intestinal polypeptide, sub from the column with an elution volume less than that of stance P, and neurokinin A were not detected in the extract. synthetic CGRP-I when chromatography was carried out in the Purification of Tumor Peptides. The elution profile on a presence of the chaotropic agent, 6 M guanadine hydrochloride, semipreparative reverse-phase CIS column of the tumor ex demonstrating that the peptide was not a noncovalently bound tracts after concentration on Sep-pak cartridges, is shown in aggregate of CGRP-I (Fig. 3). Fig. 1. As a result of a screening of the amino acid composition The GRP-like immunoreactivity in the tumor extract was of the major peaks in the chromatogram, it was apparent that eluted from the semipreparative column as two peaks with the peaks 1-5 were associated with peptides structurally related to same approximate retention times as porcine GRP (1-27) (peak katacalcin. The purification of peak 3 peptide [katacalcin (1- 7 in Fig. 1) and GRP (18-27) (peak 6 in Fig. 1). No immuno 21)] is shown in Fig. 2. The approximate yields of the purified reactivity was detected at the retention times of GRP (14-27) 2413

Â«â€¢'I 1 2â€” 800-y 11 600-2

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200-tr0;hi\\Q.10

20 30 U) 50 6070Fraction Number K) 20 30 Â«> 50 Time (mm] Fig. 3. Gel permeation chromatography of a high molecular weight form of CGRP (peak 9 in Fig. 1) isolated from a medullary thyroid carcinoma. Chroma Fig. 5. Reverse-phase high performance liquid chromatography on a diphen tography was carried out on a Sephadex G-SO column (90 X 1.6 cm) equilibrated ylmethylsilylsilica column of tumor GRP (18-27) (neuromedin C NMC). Arrows, with 0.1 M ammonium acetate, pH 6.8, and 2.1-ml fractions were collected. retention times of neuromedin C and GRP (14-27); , concentration of Arrows, elution volumes of blue dextran. (Va), cytochrome e (C.C.; M, 12,000), acetonitrile in the eluting solvent. â€¢,onlypeak showing GRP-like immunoreac and potassium enrÃ³mate ( Vt).Rechromatography in the presence of 6 Mguanadine tivity. ABSn4, absorbance at 214 nm. hydrochloride demonstrated that the peptide was not an aggregate of CGRP.

Table 1 Amino acid compositions of katacalcin-relatedpeptides and CGRP-l isolated from a medullary thyroid carcinoma 0.2 amountI,-'"",,'^,-'".-'"â€ž-'-'J_ Relative

^*^^â€¢*'":--*'I1II1VV,ResidueAsxThr30

(3)Â°2.27 (3)2.93 (4)3.10(3)2.41(4)3.07 (4)3.06 SerGlxProGly20 (2)1.46(1)0.99(1)0.78(1)0.97(1)1.80(2)1.78(2)Katacalcin(1-15)3.05(3)1.41(1)0.98 (3)2.51 (3)2.43 (3)0.93(1)4.19(4)4.10(4)4.17(5)3.35 (2)1.94 (2)1.95(2)1.00(1)0.87(1)1.70(2)1.04(1)1.81(2)2.09 (1)0.79(1)0.79(1)0.99(1)1-71(2)0.97(1)1.76 (2)1.04(1)0.97 0.1 m gAlaÂ£ ,-1 Val<-> (1)1.96(2)1.02(1)1.95(2)2.00 MetJ! (2)1.06(1)1.98(2)2.05 LeuPheHisLysArgKatacalcin(1-13)3.00 (3)1.98(2)1.03(1)2.00

(2)1.70(2)Katacalcin(1-19)3.91(2)1.83(2)Katacalcin(1-21)5.10(5)3.04 (2)2.03 rÃ¨ S\f*^_J\*. (2)" (2)Katacalcin(1-20)3.95 (2)proposed theso Numbers in parentheses, number of residues predicted from

20 30 Â«1Â¿0> structuresTimÂ«60 (min )CGRP-I4.15(4)3.95 Fig. 4. Reverse-phase high performance liquid chromatography on an analyt ical diphenylmethylsilylsilica column of tumor GRP (1-27). j. retention time of Table 2 Automated Edman degradation ofkatacalcin (1-21), GRP, and porcine GRP (1-27); , concentration of acetonitrile in the eluting solvent. neuromedin C from a medullary thyroid carcinoma Only the peak denoted by â€¢showedGRP-like immunoreactivity and was purified The sequence determination of GRP was stopped after 22 cycles. The average to homogeneity on an analytical CIS column, f/i.s.,,,. absorbance at 214 nm. repetitive yields during operation of the sequencer were katacalcin (1-21) (92%), gastrin-releasing peptide (93%), and neuromedin C (76%). or bombesin. The total GRP-like immunoreactivity in peak 6 (1-21)CycleKatacalcin CPTHamino was 590 and 910 pmol in peak 7. Tumor GRP (1-27) was peptidePTHamino eluted from an analytical reverse-phase column with a retention time of 40.9 min compared with a retention time of 40.0 min no.1234567891011121314IS16171819202122PTHÂ°aminoacidAspMetSerSerAspLeuGtaArgAspHisArgProHisValSerMetProGinAsnAlaAsnYield(nmol)5.512.94.74.52.75.74.20.62.51.50.63.21.53.41.13.02.01.51.51.50.7Gastrin-releasingacidValProLeuProAUGlyCryCryThrValLeuThrLysMetTyrProArgGlyAsnHisTrpAlaYield(pmol)6035857634677744414694592055203941322223952982471042001435927105NeuromedinacidGlyAsnHisTrpAlaValGlyHisLeuMetYield(pmol)406221908813385742411Trace for porcine GRP (1-27) (Fig. 4). The peptide was purified to homogeneity on a diphenylmethylsilylsilica column and the final yield was 810 pmol. Tumor GRP (18-27) was eluted from an analytical CIS reverse-phase column with a retention time of 32.4 min compared with a retention time of 32.2 min for synthetic GRP (18-27) (Fig. 5). The final yield of the peptide was 480 pmol. Structural Characterization of Tumor Peptides. The amino acid composition data of the purified peptides from peaks 1-5 indicated that peak 3 represented intact katacalcin (1-21) and peaks 1, 2, 4, and 5 represented the (1-13), (1-15), (1-19), and (1-20) metabolites (Table 1). The primary structure ofkatacal cin (1-21) was established by automated Edman degradation (Table 2). The amino acid sequence of the peptide was identical to that predicted from the nucleotide sequence of the corre sponding cDNA (25). It is not possible to differentiate on the basis of composition between the (1-20) metabolite (des Asn ' PTH, phenylthiohydantoin. 2414

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. REGULATORY PEPTIDES FROM MEDULLARY THYROID CARCINOMA katacalcin) and the (2-21) metabolite (des Asp katacalcin) and peptide represents the COOH-terminal 21-amino acid flanking so the peptide was subjected to fast-atom bombardment mass region separated from the calcitonin sequence by a Gly-Lys- spectroscopy. The observed quasimolecular ion (M + H+) at Lys-Arg processing site. Katacalcin-like immunoreactivity was OT/Z2320 was indicative of the (1-20) metabolite. The identi observed by immunohistochemistry in 23 of 25 medullary thy fication of several fragment ions in the spectrum [e.g., m/z 1388 roid carcinomas (2) and the value of measurements of circulat corresponding to katacalcin (9-20)] allowed confirmation of ing concentrations of the peptide in diagnosis has been assessed the structure. The identities of katacalcin (1-13), (1-19), and (22). The katacalcin isolated from the tumor investigated in (1-21) were also confirmed by mass spectrometry (Table 3) and this study was characterized by heterogeneity in the COOH- the spectra indicated that no further posttranslational modifi terminal region. Because the tumor was frozen in liquid nitro cations to individual amino acids, e.g., phosphorylation or gen immediately after resection, stored at â€”70Â°C,andextracted glycosylation had taken place. under conditions in which activation of a protease is unlikely, The assertion made on the basis of Chromatographie prop it is improbable that this heterogeneity is an artifact of the erties that tumor CGRP was predominantly, if not exclusively, experimental procedure. This conclusion was supported by the CGRP-I was confirmed by amino acid analysis (Table 1). The fact that synthetic katacalcin (1-21) was not proteolytically data showed no trace of a methionine residue in the peptide degraded during an incubation with the original extract. Further and the presence of three seryl residues is indicative of CGRP- work is required, however, using noncancerous thyroid tissue I. The low value for the relative amount of valine in the tumor to determine whether the formation of katacalcin metabolites peptide is a consequence of the known resistance to hydrolysis is associated only with tumor cells. The isolation of the (1-19), of the sterically hindered Val22-Val23bond (19). Before deter (1-17) and (1-13) fragments is suggestive of the action of a mination of the mass spectra of tumor CGRP, the cysteine peptidyl dipeptidase. For example, cathepsin B digests glucagon residues were derivatized with 4-vinylpyridine (20). A quasi- by sequential cleavage of dipeptides from the COOH-terminus molecular ion (M + H)* was observed at m/z 4001.8 Â±1. The of the molecule (23) and peptidyl dipeptidase A (angiotensin- theoretical mass of derivatized CGRP-I is 4001.2, thereby converting enzyme) will remove COOH-terminal dipeptides confirming identity with the tumor peptide. from a variety of peptide substrates (24). The absence of metab There was insufficient material for a reliable determination olites arising from cleavages in the NH2-terminal region of the of amino acid composition of tumor GRP (1-27) and GRP molecule indicates that proteolysis was not random but the (18-27) and so all material was used for sequence analysis. The isolation of the (1-20) fragment is indicative of the action of a results of automated Edman degradation of tumor GRP ( 18- carboxypeptidase. 27) are shown in Table 3. The primary structure was established The present tumor is unusual in expressing only the calci as a decapeptide identical to porcine GRP (18-27) (neuromedin tonin CGRP-I (CALC-1) gene and not the calcitonin CGRP-II C). Although the phenylthiohydantoin derivative of the COOH- (CALC-2) gene (9). Previous studies have identified both terminal methionine residue was detected only in trace amount, CGRP-I and CGRP-II in human medullary thyroid carcinomas the strong reactivity of the tumor peptide with a COOH- (9,10,25). If CGRP-II is present in the tumor, its concentration terminally directed antiserum and the identical Chromato is less than 1% of the CGRP-I concentration. The nucleotide graphie properties of the tumor and synthetic peptides strongly sequence of a cDNA complementary to human preproCGRP-I suggests the methionine residue is a-amidated. The Edman rnRNA predicts that the precursor is of 128 amino acids. degradation of tumor GRP (1-27) was terminated in error after Consequently, the high molecular weight component of CGRP- 22 cycles (Table 2). The amino acid sequence of residues (1- like immunoreactivity identified in this study (peak 9) may 22) is identical to that of the corresponding region of human represent proCGRP that is not processed further after removal GRP predicted from the nucleotide sequence of the preproGRP of the signal peptide. Further work is required, however, to gene (11). Again, the strong reactivity of tumor GRP with a substantiate this hypothesis. COOH-terminally directed antiserum suggests that residues Synthesis and storage of peptides structurally related to GRP (23-27) of the peptides are the same as in porcine GRP and is a common feature of pulmonary carcinoid tumors and small neuromedin C. cell carcinomas of the lung (26, 27). Analysis of lung tumor extracts by gel permeation chromatography has shown that proGRP is processed to a peptide the same size as porcine GRP DISCUSSION (1-27) and to a smaller molecular form (26, 27). The smaller component was originally regarded as identical to amphibian The isolation of peptides arising from the calcitonin/CGRP- bombesin (28) but subsequent work has suggested that it may I gene and the GRP gene from an extract of a medullary thyroid represent GRP (14-27) (29), GRP (18-27) (30), or GRP (19- carcinoid has provided insight into the pathway of posttrans 27) (31). GRP (18-27) has been purified from a lung tumor lational processing of the corresponding prohormones in the (30) and from porcine spinal cord (32) and is also termed tumor cells. The existence of katacalcin, also referred to as neuromedin C. This study has shown that the smaller form of peptide-aspartic acid-asparagine, was predicted from the nu GRP-like immunoreactivity in a thyroid tumor is GRP (18-27) cleotide sequence of a human procalcitonin cDNA (21). The and that bombesin and other metabolites of GRP are absent. This peptide probably did not represent an artifact of the Table 3 Masses of peptides isolated from a medullary thyroid carcinoma determined by positive-ion fast-atom bombardment mass spectrometry isolation procedure because synthetic GRP (1-27) was not converted to GRP (18-27) in vitro during an incubation with mass mass PeptideKatacalcin (M +H)*1594 (M)1593 the tumor extract. Generation of this peptide requires cleavage (1-1 3) at the site of a single arginyl residue. In human preproGRP, Katacalcin (1-19) 2250 2249 the GRP (1-27) sequence is flanked by the signal peptide and Katacalcin (1-20) 2321 2320 Katacalcin (1-21) 2435 2434 by a Gly-Lys-Lys processing site (11). Although the full se CGRP-I'Observed 4001.8 Â±1Theoretical 4001.2 quence of human GRP (1-27) was not unequivocally estab *After derivatization of the cysteine residues with 4-vinylpyridine. lished, determination of the NH2-terminal sequence has dem- 2415

gene in human small cell lung cancer. Evidence for alternative processing onstrated that the site of cleavage of the signal peptide is at the resulting in three distinct mRNAs. J. Biol. Chem., 261: 2451-2457, 1986. Ala25-Val24bond of the precursor. The pathways of posttrans- 12. Abrahamsson, P.-A., WaldstrÃ¶m, L. B., Alumets, J., Falkmer, S., and Gri- lational processing of procalcitonin, proCGRP, and proGRP melius, L. Peptide-hormone and serotonin-immunoreactivity in normal and hyperplastic prostate glands. Pathol. Res. Pract., 181:675-683, 1986. identified in this study were determined in a single tumor. (, Conlon, J. M., and McCarthy. D. Fragments of prosomatostatin isolated Clearly, further work is required with more tumor samples to from a human pancreatic tumour. Mol. Cell. Endocrinol., .IK:81-86, 1984. ascertain whether these pathways are generally followed in 14-Thim. L., Hansen, M. T., and Sorensen, A. R. Secretion of human insulin by a transformed yeast cell. FEBS Lett., 2/2: 307-312, 1987. medullary thyroid carcinomas. ,5 Conlon, J. M., Falkmer, S., and Thim, L. Primary structures of three fragments of proglucagon from the pancreatic islets of the daddy sculpin (Cottus scorpius). Eur. J. Biochem., 164:117-122, 1987. ACKNOWLEDGMENTS 16. Shaw, C., Thim, L., and Conlon, J. M. Primary structure and tissue distri bution of guinea pig gastrin-releasing peptide. J. Neurochem., 49: 1348- The authors thank Professor N. Hilschmann, Max-PIanck-Institut 1354, 1987. fÃ¼rExperimentelle Medizin, GÃ¶ttingen, for providing facilities for '?â€¢Conlon, J. M., Deacon, C. F., Bailey, C. J., and Flatt, P. R. Effects of a transplantable insulinoma upon regulatory peptide concentrations in the amino acid analysis. gastrointestinal tract of the rat. Diabetologia, 29: 334-338, 1986. 18. Conlon, J. M., Deacon, C. F., Richter, G., Schmidt, W. E., StÃ¶ckmann, F., and Creutzfeldt, W. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1988 American Association for Cancer Research. Molecular Forms of Katacalcin, Calcitonin Gene-related Peptide and Gastrin-releasing Peptide, in a Human Medullary Thyroid Carcinoma

J. Michael Conlon, Gerard P. McGregor, Göran Wallin, et al.

Cancer Res 1988;48:2412-2416.

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