Unraveling the Amyloid Associated with Human Medullary Thyroid Carcinoma

RITU KHURANA, AMIT AGARWAL, VIRENDRA K. BAJPAI, NIDHI VERMA, ASHOK K. SHARMA, RAM P. GUPTA, AND KUNNATH P. MADHUSUDAN Molecular and Structural Biology Division (R.K., N.V.), Electron Microscopy Unit (V.K.B.), Sophisticated Analytical Instrument Facility (K.P.M.), Central Drug Research Institute, Chattar Manzil Building, Lucknow 226001; and Department Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 of Endocrine Surgery (A.A.), Sanjay Gandhi Post Graduate Institute, Lucknow 226014; and Central Electronics and Engineering Research Institute (A.K.S., R.P.G.), Pilani 333031, India

Medullary thyroid carcinoma (MTC) is associated with amy- extracts revealed typical amyloid fibrils. Matrix-assisted laser loid deposition in the surrounding tissues. MTC-positive tu- desorption ionization-time of flight mass spectrometric anal- mor thyroid tissues surgically removed from patients were ysis demonstrated full-length calcitonin as the constituent of used in our study to extract amyloid. We tested the MTC ex- the MTC amyloid from seven patients. Our results unequivo- tracts for the presence of amyloid by measuring fold enhance- cally demonstrated that full-length calcitonin is the sole con- ment of thioflavin T fluorescence. Transmission electron mi- stituent of amyloid in MTC. (Endocrinology 145: 5465–5470, croscopic study and atomic force microscopy of MTC patient 2004)

MYLOID IS A SELF-AGGREGATED protein/polypep- with pheocytochromas or adrenal medullary hyperplasia A tide forming long insoluble fibrils that are extracel- where many endocrine glands of a patient such as thyroid, lularly deposited (1) in many protein-folding disorders such adrenals and parathyroid are affected simultaneously and is as prion disease, Alzheimer’s, and various systemic amy- a prime example of hereditary malignancy (10, 11). Vassar loidosis due to overexpression, proteolytic digestion, or mu- and Culling (12) demonstrated the presence of amyloid in the tations in protein. In addition to the primary and secondary stroma and colloid of thyroidal acini within and adjacent to amyloidosis that are associated with Ig light chains and se- the MTC. rum amyloid A proteins, respectively, there are many other In this manuscript, we report identification of the com- systemic amyloidosis associated with transthyretin, gelsolin, ponent of amyloid from human MTC. Matrix-assisted laser cystatin C, apolipoprotein A1, and lysozyme, etc. In addition desorption ionization-time of flight mass spectrometry to typical amyloid diseases, many common diseases such as (MALDI-TOFMS) analysis has been used after denaturation type II diabetes, myocardial infarctions, and cancers are of amyloid to completely unravel the MTC amyloid. Asso- found to be associated with amyloid involving different ciation of calcitonin with MTC has been confirmed by im- polypeptides. Among tumor-associated amyloids, many munoelectron microscopic studies using antibodies against nonneoplastic and malignant tumors of the breast and squa- calcitonin conjugated to colloidal gold particles (13). Sletton mous cell carcinoma of the lungs are associated with amyloid et al. (14) suggested involvement of an alternately processed (2). Several endocrine tumors have been demonstrated to be prohormone of calcitonin in the amyloid in MTC. Our data associated with amyloid wherein tumors secreting calcito- directly test the component of MTC after denaturation of nin, insulin, and GH very often produce amyloid, and those amyloid and MALDI-TOFMS analysis and challenges the secreting gastrin, ACTH, and prolactin also sometimes dem- suggestion made by Sletton et al. (14). onstrate amyloid (3). Medullary thyroid carcinoma (MTC) (4, 5) has been dem- Materials and Methods onstrated to be associated with amyloid fibrils deposited in MTC tissues thyroid and adjacent enlarged lymph nodes (6). MTC is MTC tissues from patients were frozen soon after surgery and stored caused by transformation of the parafollicular C cells (7) and in Ϫ70 C freezer at Sanjay Gandhi Post Graduate Institute (Lucknow, several thousand-fold increase in the blood calcitonin levels India). No ethical committee consent was required for this study in our also resulting in amyloid deposition (8, 9). MTC is a feature institutions. The tissues were transported in liquid nitrogen cryo canister. of multiple endocrine neoplasia type 2a, which occurs along Amyloid extraction from thyroid tissues Abbreviations: AFM, Atomic force microscopy; MTC, medullary thy- Extraction of amyloid was performed using the method described by roid carcinoma; MALDI-TOF, matrix-assisted laser desorption ioniza- Pras et al. (15), and the presence of amyloid in the extracts was monitored tion-time of flight; MS, mass spectrometry; SDS, sodium dodecyl sulfate; by enhanced thioflavin T fluorescence emission (16). Tissue (0.2–0.5 g) TEM, transmission electron microscopy. was thawed at 37 C for 20 min and then cut into small pieces with the Endocrinology is published monthly by The Endocrine Society (http:// help of a scalpel. The tissue pieces were washed three times for 20 min www.endo-society.org), the foremost professional society serving the each with Tris-buffered saline (pH 7.4) in a 50-ml tube, and the tube was endocrine community. kept in a tube rotator to remove trapped blood cells. The buffer was

5465 5466 Endocrinology, December 2004, 145(12):5465–5470 Khurana et al. • Brief Communications

decanted carefully. Tissue was then transferred into a glass homoge- examined using FEI-Philips Technai-12 (Eindoven, The Netherlands) at nizer, and 20 ml fresh cold Tris-buffered saline (pH 7.4) were added and 120 kV filament. homogenized using a mechanized Teflon plunger in ice bath until no visible tissue pieces were observed. The homogenate was transferred to Amyloid sample preparation for MALDI and a centrifuge tube and centrifuged at 20,000 ϫ g for 30 min at 5 C, and supernatant was separated. This process was repeated six times or until PAGE analysis the absorbance of supernatant measured at 280 nm was less than 0.1. The Amyloid samples were denatured in 7 m guanidine hydrochloride pellet was then stored at4Cin2mltriple-distilled water. Water ex- with 0.1% trifluoroacetic acid overnight. The denatured amyloid sam- traction was performed five times by homogenization in 5 ml cold water ples were bound to C-18 Zip-tip (Millipore, Bedford, MA) for concen- ϫ and centrifugation at 10,000 g for 1 h. The supernatant was saved in trating the sample and removal of denaturant by pipetting every 10-␮l screw cap glass vials at 4 C. sample five times to ensure complete binding. After binding of the sample, the C-18 Zip-tip was extensively washed with 0.1% trifluoro- Amyloid formation from synthetic calcitonin peptides acetic acid 50 times for complete removal of all denaturant molecules. The sample was then eluted using 75% acetonitrile and 0.1% trifluoro- Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 Synthetic human and salmon calcitonin peptides were purchased acetic acid just before analysis using MALDI-TOF and SDS-PAGE. from Calbiochem (San Diego, CA) and set up to form amyloid under varying conditions in small screw cap glass vials and incubated at either SDS-PAGE 28 C and 37 C for human and salmon calcitonin peptides, respectively. Amyloid formation was monitored by fold enhancement of thioflavin T High-density (20% polyacrylamide gel containing 30% polyethylene fluorescence emission at 482 nm upon excitation at 450 nm. We used the glycol) polyacrylamide gel SDS-PAGE was performed using Phast gel water extracts directly in our study without the ultracentrifugation step system (Amersham Biosciences, Uppsala, Sweden). The samples of MTC to concentrate the fibrils because we were unable to pellet all the amyloid amyloid were prepared by denaturing the amyloid using 7 m GdnHCl fibrils in the extract by ultracentrifugation as described by Pras et al. (15). and using the C-18 zip-tips to remove denaturant molecules eluting the protein and adding sodium dodecyl sulfate (SDS)-sample buffer before Thioflavin T fluorescence measurement loading the gel. The gels were either silver stained or stained with Coomassie brilliant blue. A stock thioflavin T solution of 1 mm was prepared and concentration Ϫ1 Ϫ1 measured using extinction coefficient of 266, 20 m cm at 416 nm. For MALDI-TOFMS both the Tris-buffered saline supernatants and water extracts, 20 ␮m thioflavin T (final concentration) was added to 0.5 ml supernatant, and One microliter of the sample eluted from C-18 Zip-Tip with75% fluorescence emission spectrum between 465 and 565 nm was measured acetonitrile and 0.1% trifluoroacetic acid was mixed with 1 ␮lof␣-cyano- upon excitation at 450 nm with excitation slit width at 5 nm and emission 4-hydroxycinnamic acid (1:1 methanol:acetonitrile) or sinapinic acid (in slit width at 10 nm and scan speed of 100 nm/min using PerkinElmer 60:40 0.1% TFA in water: acetonitrile) matrices. Analyses by MALDI- (Foster City, CA) spectrofluorimeter. Fluorescence emission at 482 nm TOFMS were performed in the positive ion mode on a Micromass was divided by the 20 mm thioflavin T fluorescence emission at 482 nm TofSpec 2E mass spectrometer (Micromass, Manchester, UK) equipped in TBS or water to calculate fold enhancement as the case may be. with a 337 nm nitrogen laser (4-nsec pulse) and time lag focusing. Analyses were carried out in the mass range 500–10000 with an accel- Detection of extracted amyloid using enhanced thioflavin erating voltage of 20 kV in both reflectron and linear modes. The data were accumulated over 40–50 laser shots. The instrument was calibrated T fluorescence with a mixture of angiotensin I, renin substrate, and ACTH (18–39 clip) Extraction of the amyloid from MTC tissues was performed using the mixed with appropriate matrix. method described by Pras et al. (15). Amyloid in the buffer and water extracts was tested by the fluorescence emission enhancement of thio- Results flavin T dye (see Materials and Methods). Thioflavin T dye was initially Selection of MTC tissues identified by Vassar and Culling (16) to be very specific for amyloid in tissue sections, later Naiki et al. (17) and LeVine (18) designed a spec- We used seven MTC tissues that were surgically removed trofluorimetric assay for amyloid detection in solution using fluores- by performing complete thyroidectomy in the endocrine sur- cence excitation at 450 nm and emission at 482 nm and monitored enhanced fluorescence emission in the presence of amyloid. gery department in the Sanjay Gandhi Post-Graduate Re- search Institute as a part of the clinical procedure for patients. Atomic force microscopy (AFM) Pathological tests such as Congo red birefringence and immunohistochemistry using antibodies against calcitonin The MTC extracts were examined using AFM performed in contact were performed on formalinized tissue sections to confirm mode using Nanoscope II (Digital Instruments, Santa Barbara, CA). that the tumor was MTC. All seven cases are listed in Table Imaging was done in air using 0.7 ␮m AFM head. The sample is placed on xyz-piezo-translator and scanned by using a sharp diamond tip 1 with their lab numbers and the results of diagnostic patho- ␮ mounted on a gold-coated 200 m triangular Si3N4 microfabricated logical tests. For one patient, a portion of thyroid tissue that cantilever (force constant ϭ 0.6 N/m). The force between the tip and was not affected by tumor was included in our study as Ϫ7 Ϫ9 sample usually ranges from 10 to 10 n. Images consists of 400 scan normal control and is also listed in Table 1. of 400 pixels each. Typical image acquisition time was 150–200 sec/scan. Samples were prepared by placing a drop of fibril solution (ϳ0.5 mg/ml) on freshly cleaved mica and dried. Samples containing buffers and salts Detection of amyloid in tissue extracts by thioflavin T dye were dried on mica and washed with water to remove salts. Buffer extracts did not show significant thioflavin T fluorescence fold enhancement in all tissue extracts. Varying Transmission electron microscopy (TEM) amounts of amyloid was detected in the water extracts in A drop of aqueous solution of amyloid fibrils extracted from tissues most patients as judged by fold enhancement in thioflavin T was placed on the Parafilm (Greenwich, CT), and polystyrene-coated fluorescence as summarized in Table 1. At least a 3-fold copper grid was floated film side down for 1–3 min and excess solution was absorbed using Whatman (Kent, UK) filter paper. Subsequently, increase in thioflavin T fluorescence in presence of amyloid sample contrast was enhanced by placing the grid film side down on a extracts was considered significant. Thioflavin T fluores- drop of the 1–2% uranyl acetate solution for 1–3 min. The grid was cence emission fold enhancement varied from 5-fold to Khurana et al. • Brief Communications Endocrinology, December 2004, 145(12):5465–5470 5467

TABLE 1. A list of seven MTC thyroid tissues and one normal tissue used in our study

Patient tissue Congo Thioflavin T fluorescence Fibrils detected MALDI-TOFMS Sample no. Lab no. designation red/Immunohistochemistry emission (fold enhancement) by EM/AFM calcitonin peaka 1 1 MTC 245 Positive Ͼ5 ϩ/ϩϩ 2 2 MTC 415 Positive Ͼ15 ϩ/ϩϩ 3 3 MTC 787 Negative/ϩϾ10 ϩ/ϩϩ 4 4 MTC 795 Positive Ͼ20 ϩ/ϩϩϩ 5 5 MTC 799 Positive Ͼ5 ϩ/ϩϩϩ 6 6 MTC 913 Negative/b Ͼ10 ϩ/ϩϩ 7 7 MTC 770 Positive Ͼ50 c/ϩ ϩϩ 8 8 Normal 463 Negative Ͻ2 Ϫ/c Ϫ The results of histopathological test to confirm MTC on tissue sections and thioflavin T fluroescence fold enhancement and detection of typical fibrils by TEM or AFM and calcitonin peak observed by MALDI-TOFMS analysis on water extracts are summarized below. Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 a ϩ, Indicative of less than 100 counts; ϩϩ, more than 200 counts in MALDI-TOFMS peak; and Ϫ, indicative of no significant MALDI-TOF peak observed. b Immunohistochemistry not conclusive. c Sample not imaged by TEM or AFM. greater than 50-fold in different MTC patient water extracts, whereas it was less than 2-fold for normal thyroid extract (Table 1). A comparison of amyloid extracted from normal area of thyroid from a patient did not reveal amyloid, by either histopathological observations or by fluorescence emission fold enhancement of thioflavin T, or electron microscopy of water extracts (Table 1). One patient, 6 MTC, who was not conclusively proved MTC by histopathology, demonstrated significant amyloid by thioflavin T fold enhancement of the water extract.

In vitro amyloid formation from synthetic calcitonin peptides Human calcitonin peptide readily formed amyloid with in a few hours when a 5 mg/ml solution was incubated at 28 C in PBS. A 5 mg/ml solution of salmon calcitonin peptide was incubated for 3–4 wk at 37 C before fibrils were observed by enhanced thioflavin T fluorescence or electron microscopy. In vitro amyloid formation from synthetic human and salmon calcitonin peptides has also been shown earlier by various groups (19–21).

Ultrastructural analysis of amyloid FIG. 1. TEM images of MTC and synthetic calcitonin amyloid. TEM images of MTC extracted amyloid fibrils of patient 4 MTC (A) and 1 TEM of amyloid fibrils extracted from tissues was per- MTC (B), human calcitonin amyloid (C), and salmon calcitonin amy- formed by negative staining using uranyl acetate stain on loid (D) observed after loading the samples on polystyrene-coated polystyrene-coated copper grids and typical fibrils were ob- copper grids and uranyl acetate staining. served in six MTC extracts tested and not in the normal thyroid extract (Table 1). The diameters of MTC extracted AFM analysis of patient amyloid samples dried on freshly amyloid fibrils ranged from 2–8 nm. The variation in the cleaved mica was also performed for all seven patient- diameters of fibrils arises due to the distance measurement extracted MTC samples, and all patient extracts revealed on manually drawn lines on the computer monitor (using the amyloid fibrils (Table 1). AFM images show amyloid fibrils software provided by FEI-Philips) across the fibrils on TEM of MTC water extracts placed on flat mica surface from two images introducing various error factors. Figure 1, A and B, different patients 3 MTC and 1 MTC are shown in Fig. 2, A shows fibrils from two different patients 4 MTC and 1 MTC, and B, respectively. The dark background is the mica surface respectively. Synthetic calcitonin fibrils were observed as and the lighter colored image is the fibril. The color grade in negatively stained amyloid fibrils of 4–8 nm diameter by the z-axis is the height of the fibril scanned above the mica TEM as shown in Fig. 1C for human calcitonin fibrils and Fig. surface. The heights of MTC amyloid fibrils ranged from 2–4 1D for salmon calcitonin amyloid. The amyloid fibrils shown nm corresponding to the diameter of amyloid fibrils. The in Fig. 1, A and B, are characteristic intertwined mature height measured in AFM is translated from the z direction amyloid fibrils also observed for several other protein and piezo movement and provides an accurate measurement of peptide amyloid (22) that we believe are formed from thin- the diameter of amyloid fibrils compared with the diameters ner protofilaments and protofibrils intertwining together measured by electron microscopy. AFM images of MTC (23–25). amyloid fibrils after high temperature (Fig. 2C) and synthetic 5468 Endocrinology, December 2004, 145(12):5465–5470 Khurana et al. • Brief Communications

bands for all the patient samples. Because SDS-PAGE gels bands were not always consistent, we decided to perform mass spectrometric analysis on these samples as native gels or other methods would not allow us determine the exact size of the constituent polypeptide of the MTC amyloid.

Calcitonin peaks observed in the MTC amyloid extracts using MALDI-TOFMS MALDI-TOFMS analysis of MTC amyloid was performed by first denaturing the amyloid in 7 m guanidine hydrochloride followed by binding of peptides/proteins to C-18 zip- tips and removal of denaturant molecules. A major peak of Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 mass 3419, corresponding to the mass of calcitonin, was observed in all seven patient MTC extracts and was not detected in the normal thyroid extract (Table 1). A major calcitonin peak and its dimer peak from three patients extracts (5 MTC, 4 MTC, and 7 MTC) were observed by linear

FIG. 2. AFM images of MTC amyloid. The AFM pictures show amyloid fibrils imaged on the flat surface of mica. The dark background is the mica surface, and the lighter-colored image is the fibril. The color grade is the height of the fibril above the surface of the mica. The fibril height measured by AFM very accurately is indicative of the diameter of amyloid fibrils. Contact mode AFM images of 3 MTC (A) and 1 MTC (B) patient MTC amyloid with height measured between 2–4 nm. Patient 4 MTC treated to 80 C temperature (C) and synthetic human calcitonin treated with Proteinase K at 55 C (D), demonstrat- ing no changes in the morphology of amyloid fibrils both by high temperature and proteinase K. human calcitonin amyloid after proteinase K treatment (Fig. 2D) showed no detectable morphological differences compared with the control fibrils (Fig. 2, A and B), indicating that both these treatments do not denature or unfold the amyloid conformation. Because 6 MTC patient water extract demonstrated amyloid fibrils by thioflavin T fluorescence and TEM and AFM (Table 1), we included it in our study, believing that this patient may well be MTC at a molecular level even though it was not conclusively proved MTC by histopathology.

SDS-PAGE analysis of MTC extracted amyloid Analyses of the MTC amyloid extract and synthetic calcitonin amyloid was performed on 20% polyacrylamide high-density gels obtained from Amersham Biosciences and especially designed to separate peptides ranging from 1–50 kDa. For synthetic calcitonin amyloid, we did not observe any band until the MTC amyloid was denatured. However, upon denaturation of synthetic calcitonin amyloid with 7 m GdnHCl and denaturant removed using C-18 Zip-tips we observed higher oligomeric bands corresponding to 6-, 12-, and 15-kDa bands under varying concentrations of SDS in the sample buffer (Fig. 3A, lane 3, shows a 15-kDa band). At higher concentrations of freshly prepared synthetic human calcitonin, we also observed a major band at 3 kDa and a 15-kDa band on SDS-PAGE with 2% SDS in the sample buffer FIG. 3. SDS-PAGE analysis of MTC extracted amyloid. A, Peptide (Fig. 3B, lane 2). All of this led us to conclude that disag- markers—lane 1, salmon calcitonin; lane 2, MTC extracted amyloid gregated calcitonin monomer from MTC extract, and freshly from 4 MTC patient in lane 3; lane 4, low molecular weight protein markers and insulin on 20% high-density SDS-PAGE phast gel and prepared synthetic calcitonin can form SDS stable oligomers stained with Coomassie brilliant blue. B, Peptide markers (lane 1), in the presence of SDS that shows up as oligomeric bands in synthetic human calcitonin in 2% SDS sample buffer (lane 2) and was 20% high-density SDS-PAGE. SDS-PAGE did not show silver stained. Khurana et al. • Brief Communications Endocrinology, December 2004, 145(12):5465–5470 5469 mode upon mixing with ␣-matrix are shown in Fig. 4, A–C, removal of denaturants using C-18 zip-tips has been reported respectively. The data observed for remaining patients had here for the first time. This method can be applied to amyloid counts less than hundred for calcitonin peak and no calci- extracted from other tissues as well and is far superior to tonin peak was observed for the normal thyroid tissue extract using SDS-PAGE because amyloid may not denature com- (Table 1). No calcitonin peaks were observed in any of the pletely in SDS and also forms SDS stable oligomers. Obser- extracts until the amyloid was denatured indicating that the vation of calcitonin peak in all patient amyloid extracts after denaturation step was absolutely essential for detection of denaturation and MALDI-TOFMS analysis led us to con- the component peaks from amyloid. Even when we used half clude that calcitonin forms amyloid in MTC. It would be a gram of tissue, we sometimes barely got enough material much better to perform MS/MS analysis to say with com- to perform MALDI analyses and ultrastructural studies. plete authority that calcitonin is involved in amyloid forma- We also mixed the MTC extracts with sinapinic acid matrix tion in MTC tissue, but our attempts to perform MS/MS and explored the possibility of observing serum amyloid P analysis did not give us satisfactory results most likely due Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 protein (26) or other high molecular weight proteins possibly to insufficient sample available. Because MALDI-TOFMS associated with amyloid, but were unable to detect any sig- analysis gave the exact calcitonin size peaks for all seven nificant high molecular weight peaks. In addition no signif- patients in our hands and many other groups including icant high molecular weight bands were observed in 8–25% Bulter and Khan (13) have clearly demonstrated that amyloid SDS-PAGE gels (data not shown). This led us to conclude that associated with MTC is recognized by the antibodies against amyloid-associated proteins are possibly separated during calcitonin hormone, we conclude that it is calcitonin that is the extraction procedure and maybe found in salt extracts as involved in amyloid formation in MTC. suggested by Pras et al. (15) and demonstrated clearly by Scrapie prion conformation is also insoluble in detergents Sletton et al. (14) for MTC tissue extracts. (28) but can be denatured by guanidine hydrochloride (29). This matches with our results for calcitonin amyloid, which Discussion is not soluble in SDS but can be denatured in guanidine Amyloid extraction procedure that we used to extract hydrochloride. It is possible that Sletton et al. (14) observed amyloid from MTC was described by Pras et al. in 1968 and a band of approximately 6000 Daltons from the MTC extract has been used by many groups since then successfully. Amy- on SDS-PAGE corresponding to a SDS stable dimer of cal- loid is intact as shown by both the TEM analysis and AFM citonin. Because our results also demonstrated a major 3.42- images in Figs. 1 and 2, respectively. Westermark (27) also kDa peak with MALDI and either 6-, 12-, or 15-kDa bands on used the same method with slight modifications. SDS-PAGE, we concluded that it is the full-length calcitonin Our method of detecting component peaks of amyloid that is the constituent of amyloid in MTC that has the ten- extracted from tissues by MALDI-TOFMS followed by de- dency to form SDS stable oligomers. naturing the amyloid (in 7 m guanidine hydrochloride) and Sletton et al. (14) concluded that it is the alternately pro-

FIG. 4. MALDI-TOF spectra of denatured amyloid from MTC tissues. MALDI- TOF spectra of MTC extracts from three representative patients 5 MTC (A), 4 MTC (B), and 7 MTC (C) showing major peaks of mass 3420 (corresponding to mono- meric calcitonin MHϩ) and a minor peak of 6839 (corresponding to dimeric calcitonin 2MHϩ) using linear mode with ␣- matrix after denaturation of amyloid in 7 M guanidine and removal of denaturant using C-18 Zip-tips. 5470 Endocrinology, December 2004, 145(12):5465–5470 Khurana et al. • Brief Communications cessed prohormone of calcitonin that is involved in amyloid 3. Westermark P, Grimelius L, Polak JM, Larson L-I, van Noorden S, Wilander E, Pearse AGE 1977 Amyloid in polypeptide hormone-producing tumors. Lab formation in MTC (14, 27). The results in the study conducted Invest 37:212–215 by Sletton et al. (14) were based on the observations of a major 4. Hazard JB, Hawk WA, Crille G 1959 Medullary (solid) carcinoma of the band corresponding to approximately 6000 Daltons from the thyroid—a clinicopathologic entity. J Clin Endocrinol 19:152–161 5. Williams ED 1966 Histogenesis of medullary carcinoma of the thyroid. J Clin extracted MTC amyloid on the SDS-PAGE. The actual pro- Pathol 19:114–118 hormone of calcitonin is 9.9 kDa, and the alternately pro- 6. Scopsi L, Sampietro G, Boracchi P, DelBo R, Gullo M, Placucci M, Pilotti S cessed prohormone of calcitonin implicated in amyloid for- 1996 Multivariate analysis of prognostic factors in sporadic medullary carcinoma of the thyroid: a retrospective study of 109 consecutive patients. Cancer mation in MTC by Sletton et al. is 5.68 kDa. Because none of 78:2173–2183 these are secreted extracellularly, they are very unlikely to be 7. Williams ED, Brown CL, Doniach I 1966 Pathological and clinical findings in a series of 67 cases of medullary carcinoma of the thyroid. J Clin Pathol involved in the amyloid formation in MTC. In addition, 19:103–113 Sletton et al. (14) attempted N-terminal sequencing of the 8. Huang SN, McLeish WA 1968 Pheochromocytoma and medullary carcinoma of thyroid. Cancer 21:302–311

MTC extracted amyloid, but no N-terminal sequence was Downloaded from https://academic.oup.com/endo/article/145/12/5465/2499752 by guest on 29 September 2021 9. Meyer JJ 1968 Fine structure of two amyloid-forming medullary carcinomas detected most likely due to the N-terminal disulfide loop of thyroid. Cancer 21:406–425 formed between cysteine 1 and cysteine 7 in calcitonin hor- 10. Shapiro MJ 1977 Medullary carcinoma of the thyroid gland. Am Surg 43: mone. The amino acid composition analysis performed by 601–609 11. Sizemore GW, Health 3rd H, Carney JA 1980 Multiple endocrine neoplasia Sletton et al. (14) of MTC amyloid was interpreted as a 53- type 2. Clin Endocrinol Metab 9:299–315 amino acid polypeptide that was suggested to be an alter- 12. Vassar PS, Culling CF 1961 The significance of amyloid in carcinoma of the thyroid gland. Am J Clin Pathol 36:244–247 nately processed prohormone of calcitonin of size 5680 Dal- 13. Butler M, Khan S 1986 Immunoreactive calcitonin in amyloid fibrils of med- tons. Because the actual prohormone of calcitonin has a size ullary carcinoma of the thyroid gland. Arch Pathol Lab Med 110:647–649 of 9908 Daltons. An alternate processing would require a 14. Sletton K, Westermark P, Natwig JB 1976 Characterization of amyloid fibril proteins from medullary carcinoma of thyroid. J Exp Med 143:993–998 different mechanism for processing of the calcitonin hor- 15. Pras MD, Schubert M, Zucker-Franklin D 1968 The characterization of soluble mone in the transformed C cells that has not been reported amyloid prepared in water. J Clin Invest 47:924–933 so far. The data obtained by cyanogen bromide cleavage 16. Vassar PS, Culling CF 1959 Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 68:487–494 followed by N-terminal sequencing of the MTC amyloid by 17. Naiki H, Higuchi K, Hosokawa M, Takeda T 1989 Fluorimetric determination Sletton et al. (14) revealed region 9–19 amino acids of human of amyloid fibrils in vitro using fluorescent dye, thioflavin T. Anal Biochem 177:244–249 calcitonin indicating involvement of calcitonin in MTC, but 18. LeVine III H 1993 Thioflavine T interaction with synthetic Alzheimer’s disease the authors (14) suggested an alternately processed prohor- ␤-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci mone of calcitonin. 2:404–410 19. Arvinte T, Cudd A, Drake AF 1993 The structure and mechanism of formation Clear calcitonin peaks obtained by us using MALDI- of human calcitonin fibrils. J Biol Chem 268:6415–6422 TOFMS after denaturation of amyloid from MTC tissue ex- 20. Bauer HH, Aebi U, Ha¨ner M, Hermann R, Mu¨ ller M, Arvinte T, Merkle HP tracts led us to conclude that it is the full-length calcitonin 1995 Architecture and polymorphism of fibrillar supramolecular assemblies by in vitro aggregation of human calcitonin. J Struct Biol 115:1–15 hormone and not an alternately processed prohormone of 21. Gilchrist PJ, Bradshaw JP 1993 Amyloid formation by salmon calcitonin. calcitonin that forms amyloid in MTC. Biochim Biophs Acta 1182:111–114 22. Chamberlain AK, MacPhee CE, Zurdo J, Morozova-Roche LA, Hill HAO, Dobson CM, Davis JJ 2000 Ultrastructural organization of amyloid fibrils by Acknowledgments atomic force microscopy. Biophys J 79:3282–3293 23. Ionescu-Zanetti C, Khurana R, Gillespie JR, Petrick JS, Trabachino LC, We acknowledge the help and support provided by Dr. Uma Roy Minert LJ, Carter SA, Fink AL 1999 Monitoring the assembly of Ig light chain throughout the writing and completion of this work. Mr. Rit Vatsayan amyloid fibrils by atomic force microscopy. Proc Natl Acad Sci USA 96:13175– and Dr. Uma Roy from the Central Drug Research Institute (Lucknow, 13179 India) also helped with silver staining of SDS-PAGE. Ms. Madhuli and 24. Kad NM, Myers SL, Smith DP, Smith DA, Radford SE, Thomson NH 2003 Ms. Abha are fondly acknowledged for their help in the electron mi- Hierarchical assembly of ␤2 microglobulin amyloid in vitro revealed by atomic croscopy laboratory at Central Drug Research Institute, Lucknow, India. force microscopy. J Mol Biol 333:785–797 25. Khurana R, Ionescu-Zanetti C, Pope M, Neilson L, Li J, Ramirez-Alvarado M, Regan L, Fink AL, Carter SA 2003 A general model for amyloid fibril Received June 22, 2004. Accepted September 24, 2004. assembly based on morphological studies using atomic force microscopy. Address all correspondence and requests for reprints to: Dr. Ritu Biophys J 85:1135–1144 Khurana, Centre for Cellular and Molecular Biology, Uppal Road, Hy- 26. Pepys MB, Rademacher TW, Amatayakul-Chantler S, Williams P, Noble GE, derabad 500007, India. E-mail: [email protected]. 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