Synthesis of Parathyroid Hormone-Like Peptides by a Human Squamous Cell Carcinoma JAMES W. HAMILTON, CHARLES R. HARTMAN, DOUGLAS H. MCGREGOR, AND DAVID V. COHN Calcium Research Laboratory, Veterans Administration Hospital, Kansas City, Missouri 64128, University of Kansas School of Medicine, Kansas City, Kansas 66103 umn eluate with antisera specific for either the amino- or carboxylregion of parathyroid hormone revealed 2 closely associated peaks of immunoreactivity in the parathyroid region. Each of these corresponded approximately to the elution position of a peak of radioactivity. The fractions comprising these 2 peaks of immunoreactivity were pooled separately and subjected to CM-cellulose chromatography together with [14C]bovine parathyroid hormone as marker. Each sample yielded 3 radioactive components upon ion exchange chromatography—one of which co-migrated with the bovine parathyroid hormone marker. Since human parathyroid hormone elutes slightly earlier than the bovine hormone on CM-cellulose, this suggests that the co-migrating tumor peptide is more basic than the glandular human parathyroid hormone. On the basis of the known elution position of proparathyroid hormone from CM-cellulose, it did not appear that the tumor was producing proparathyroid hormone. We conclude that one of the tumors studied synthesized a peptide or peptides similar to but not necessarily identical with glandular parathyroid hormone. (J Clin Endocrinol Metab 45: 1023, 1977)

ABSTRACT. Hypercalcemia associated with various types of cancer has been reported by several investigators. In most cases, the postulated cause of the hypercalcemia, ectopic production of parathormone, has been identified only from immunologic measurement of serum or tumor extracts. We have studied the capacity of the malignant tissue from 6 patients with non-parathyroid cancer and hypercalcemia to synthesize parathyroid hormone in vitro. One of these, an esophageal tumor from a 51 year old man which was identified histopathologically as a squamous cell carcinoma, appeared to synthesize peptides(s) with properties similar to parathyroid hormone. Ultrastructural examination of the carcinoma cells revealed the presence of secretory granule-like structures. A sample of tumor tissue slices was incubated with [3H]lysine in a balanced salt solution for 3 h. The tissue was processed by standard procedures for isolation of parathyroid hormone. The resultant tissue extract was gel filtered on G-100 Sephadex. The elution profile contained 5 peaks of radioactivity, one of which was in the region where authentic parathyroid hormone and proparathyroid hormone would elute. Radioimmunoassay of the col-

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YPERCALCEMIA associated with malignancy not involving bone or parathyroid glands has been recognized for many years, but the underlying cause of the elevated calcium has not been explained. Albright (1) originally suggested that it together with the associated hypophosphatemia might result from the tumor producing a parathyroid hormone-like substance. Other more recent studies indicate that the hypercalcemia may be due to the elaboration by the tumor of agents such as prostaglandins (2-4), or osteoclastic activation factor (OAF) (5). Thus, many investigators currently feel that there may be multiple causes for the hypercalcemia. A number of investigators have attempted Received March 22, 1976. Presented in part at the 57th Annual Meeting of the Endocrine Society, New York City, June 1975.

to demonstrate that above-normal levels of parathyroid hormone exist when hypercalcemia and malignancy are associated. Berson and Yalow (6) and Riggs et al. (7) reported that serum immunoreactive parathyroid hormone levels of some hypercalcemic tumor patients without bone involvement were elevated. Tashjian et al. (8) and Sherwood et al. (9) obtained immunological evidence that parathyroid hormone is present in some tumor extracts but not in others. Benson et al. (10) reported the detection of more than one form of serum immunoreactive parathyroid hormone in cancer patients with hypercalcemia. Although these results suggest that some tumors produce and secrete parathyroid hormone, the only direct evidence to this effect is the recent work of Greenberge£ al. (11) in which cell cultured from a renal carcinoma were shown to produce parathyroid hormone. This conclu-

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HAMILTON ET AL.

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sion was based on the incorporation of labeled amino acid and the subsequent identification of some newly synthesized peptide as PTH, using immunoassay and gel electrophoresis. In an attempt to provide additional direct evidence we have examined the capacity of tumors from patients exhibiting hypercalcemia to specifically synthesize parathyroid hormone. In this report we present data which indicate that one such tumor synthesized two peptides which physically and chemically resemble authentic parathyroid hormone. Patients and Methods Patient A The patient was a 51 year old Caucasian male first seen in July 1974 with symptoms of dysphagia and multiple subcutaneous nodules. Esophagoscopy revealed a mass lesion of the middle third of the esophagus. One of the nodules upon surgical removal was found to be a metastatic moderately differentiated squamous cell carcinoma. Serum calcium concentration was 12.9 mg/100 ml (normal 8.5 to 10.5), inorganic PO 4 ,3 mg/100 ml (normal 2.5 to 4.5), alkaline phosphatase, 125 mU/ml (normal 30 to 85) and total protein, 7.6 g/100 ml. There was no evidence of skeletal involvement, based on bone scan using technetium-labeled phosphate or by skeletal xray. Patient B The patient was a 53 year old Caucasian male first seen in April 1975 with symptoms of a dull ache in his right shoulder and a mass in the right supraclavicular space. He was subsequently found on bronchoscopy to have a mass lesion in the right upper lobe bronchus. Upon removal, the tissue was histologically diagnosed as moderately to poorly differentiated squamous cell carcinoma. Serum calcium was 11.5 mg/100 ml, inorganic PO4, 2.9 mg/100 ml, alkaline phosphatase, 75 mU/ ml and total proteins, 5 g/100 ml. A radioactive bone scan was normal. Postmortem examination, which was limited to the neck and anterior chest, demonstrated invasion of the chest wall and normal parathyroid glands.

Additional

JCE & M • 1977 Vol 45 • No 5

patients

Tumors from other patients with hypercalcemia were also studied. Two of these were nasopharyngeal and the other three were bronchogenie carcinomas of the lung. Since the results obtained were similar to those of Patient B above, these data will not be presented in detail. Methods Immediately upon removal of the tissue a portion was placed on ice and transported to the laboratory. No more than 1 h elapsed from excision to initiation of in vitro incubation. Each tumor was sliced with a Stadie-Riggs tissue slicer into pieces about 2.5 mm thick. The tissue was incubated with shaking in 10 ml (per g of tissue) of bicarbonate buffer (12), pH 7.4 containing 5 /LtC per ml of [3H]lysine or [3H]leucine, or both. The temperature was 37 C and the gas phase was air. After either 60 or 120 min, the reactions were terminated by rapidly freezing the flask contents in a dry ice-alcohol bath. The frozen reaction mixture (tissue plus medium) was processed to a trichloroacetic acid powder (TCA-powder) as previously described for the isolation of parathyroid hormone from bovine (12) and human (12) parathyroid glands. This procedure essentially involves homogenation in urea-HCl, followed by solvent and salt precipitation and precipitation with trichloroacetic acid. This procedure with bovine tissue normally yields about 55% of the total tissue content of hormone. It is established that this procedure yields all of the biologically active peptide in the TCA precipitate; other fractions obtained during the preparation have been found to be biologically inactive. It is assumed that the tumor tissue would behave similarly. The resultant TCApowder was dissolved in 0.14M ammonium acetate, pH 4.8, and subjected to gel filtration on Sephadex G-100, superfine, in a 2.5 x 45 cm column with pH 4.8 ammonium acetate buffer. Calibration of column location of the elution position of parathyroid hormone has been previously described (13). 1.0 ml fractions were collected unless specified otherwise, pooled as indicated below and lyophilized. These pooled samples were dissolved in starting buffer and further fractionated by ion exchange chromatography on 0.5 x 5 cm columns of carboxymethylcellulose (CM-52, Whatman).

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1025

PARATHYROID HORMONE IN CARCINOMA

Results

Squamous cell carcinoma of patient A 2.4 g of the tissue were incubated with [3H]lysine for 2 h and subsequently prepared as described in Patients and Methods. The gelfiltrationelution profile of the tumor TCA-powder is portrayed in Fig. 1. A substantial amount of the radioactivity was incorporated into the large molecular weight proteins that eluted in the void volume. The region where authentic parathyroid hormone elutes under these chromatographic conditions (tubes 45 to 55) contained a broad peak of radioactivity. The profiles of immunoreactivity shown in Fig. 1 were similar for the carboxy- and amino-terminal directed antisera. Two immunoreactive peaks were

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The columns were eluted with a linear gradient of ammonium acetate from 0.01M, pH 5, to 0.33M, pH 7. Two ml fractions were collected. Conductivities of eluant were measured on every fifth tube at room temperature. Measurements of radioactivity were made in a Packard liquid scintillation spectrometer on aliquots from the column fractions using a Triton X-100-toluene based fluor. Bovine [14C]parathyroid hormone marker was prepared synthetically in the in vitro system as previously described (12). Radioimmunoassay was performed as described earlier by employing guinea pig anti-bovine parathyroid hormone antisera (13). Two antisera were used, one specific for the amino-terminal region of bovine parathyroid hormone, the other which recognizes primarily the carboxy-terminal region of the bovine hormone (14). 125Ilabeled bovine parathyroid hormone was used as tracer. Nonradioactive bovine parathyroid hormone prepared in this laboratory was used for the standard curve. [3H]Leucine (specific activity, 30-50 Ci/mmol) and [3H]lysine (specific activity, 20-40 Ci/mmol) were purchased from New England Nuclear, Boston, Massachusetts. Nonradioactive chemicals were analytical grade. Bovine parathyroid glands were kindly provided by Wilson Packing Company, Kansas City, Missouri and Dugdale Packing Company, St. Joseph, Missouri.

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FIG 1. Gel filtration of TCA powder of tumor patient A. The sample was dissolved in 0.14 M ammonium acetate, pH 4.8 and applied to a Sephadex G-100 superfine (2.5 x 40 cm) in a volume of 3.0 ml. Fractions of 2.3 ml were collected. Radioimmunoassay was performed on aliquots from every other tube. The solid line represents 3 H. The dashed line, C-terminal immunoreactivity and the heavy solid line, N-terminal immunoreactivity. iPTH indicates immunoreactive PTH. The peaks labeled I and II were pooled separately for subsequent ion exchange chromatography.

detected. One of these, Tumor Peak I, eluted in the region in which authentic parathyroid hormone would migrate; the other peak, Tumor Peak II, eluted somewhat later. These data suggest that the region of the chromatogram corresponding to Peaks I and II contained peptides similar to parathyroid hormone or to fragments derived from the parathyroid hormone molecule. Tumor Peak I was mixed with authentic bovine [14C] parathyroid hormone and was chromatographed on CM-cellulose. Since labeled protein eluted slightly earlier than did the 14C-marker parathyroid hormone (Fig. 2A), this minor difference in elution was equivalent to that previously reported by us when authentic human parathyroid hormone was chromatographed with bovine hormone (12). Thus Tumor Peak I appeared

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1026

HAMILTON ET AL.

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to be similar to human parathyroid hormone, in terms of shape, charge and immunoreactivity. When Tumor Peak II was similarly chromatographed (Fig. 2B), a significant amount of the 3H-radioactivity eluted in the region of the 14C-marker parathyroid hormone. But in contrast with the results with Tumor Peak I (Fig. 2A), it eluted coincident with the marker parathyroid hormone (that is, later than authentic human parathyroid hormone would elute). During the ion-exchange chromatography of Tumor Peak I or II there was no indication of proparathyroid hormonelike peptide. The material eluting at ap-

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FIG. 2A. Ion exchange chromatography of Sephadex fractions derived from patient A. A. Peak I from the Sephadex column was lyophilized and redissolved in 0.01M ammonium acetate, pH 5.3. To this was added 7,500 dpm of 14C-parathyroid hormone. The sample was then applied to a carboxymethylcellulose column 0.5 x 5 cm which eluted with a linear gradient from 0.01M ammonium acetate, pH 5.3, to 0.33M ammonium acetate, pH 7.0. Fractions of 2.0 ml were collected. Aliquots were removed for radioactivity measurement. The solid line is 3H and the dashed line is 14C.

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proximately tube 20 observed in both chromatograms was not identified. Squamous cell carcinoma from patient B When tumor slices from this patient were incubated with [ 3 H]leucine, little incorporation of radioactive amino acid was observed. The specific activity in the TCA-powder was 5,000 dpm per mg protein, compared to about 70,000 dpm per mg for Patient A. The gel filtration elution profile of the TCA-powder contained no peak of radioactivity in the region where authentic parathyroid hormone would elute. The column eluate,

FIG. 2B. Peak II from the Sephadex column was lyophilized, redissolved in 0.01M ammonium acetate, pH 5.3, and mixed with 7,500 dpm of 14C-parathyroid hormone. Elution was as for A above. See legend to Fig. 2A.

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PARATHYROID HORMONE IN CARCINOMA moreover, contained no immunoreactive species. Thus it is unlikely that this tumor manufactured or secreted the normal 84 amino acid parathyroid hormone molecule. It is not possible to exclude the possibility that not previously described parathyroid hormone-like molecules were made by this tissue. Similarly, none of the TCA extracts of the other tumors we studied contained significant radioactivity in the parathyroid hormone (Fig. 1, Peak I) region of the Sephadex profile even though the incorporation of radioactivity in proteins of the extracts was often equivalent or higher than that from the tumor of patient A.

1027

Electron microscopic study Representative sections of the tumor of patient A were examined in the electron microscope. The neoplasm was composed of atypical epithelial cells with desmosomes, tonofibrils and other features consistent with squamous cell carcinoma (Fig. 3A-B). The cytoplasm contained scattered mitochondria, many ribosomes, a moderately-developed rough endoplasmic reticulum and prominent Golgi zones. Many cells contained a varied population of single membrane-limited granules (Fig. 3A-B) which were homogeneous in content and had the appearance of secretory granules.

FIG. 3A. Electron micrographs of cells in the squamous carcinoma tissue from patient A; fixed in glutaraldehyde solution (pH 7.2) shortly after removal at surgency, postfixed in 1.33% sym-collidine buffered osmium tetroxide (pH 7.4), dehydrated and embedded in an eponaraldite mixture. Low power view of two cells demonstrating occasional desmosome and occasional tonofibril formation, consistent with squamous cell type. Cytoplasm of the cell at the top has many polyribosomes, scattered rough endoplasmic reticulum and several coarse membrane-limited granules. Cytoplasm of the cell at the bottom has fewer polyribosomes, slightly more prominent scattered rough endoplasmic reticulum, and a number of somewhat smaller, finer, more electron-dense membrane-limited granules, x 9,500.

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JCE & M • 1977 Vol 45 • No 5

FIG. 3B. Higher power of portions of two other cells in the carcinomatous tissue. Cytoplasm has a few mitochondria, scattered rough endoplasniic reticulum and interspersed fine filaments. Located near the prominent Golgi zone in the cell on the left, there are many round to oval membrane-limited, fine, homogeneously dense granules, suggestive of secretory granules, x 22,400. See legend to Fig. 3A.

Electron microscopic examination of the tumor from patient B revealed an epithelial neoplasm with a cellular morphology similar to that of patient A (Fig. 4), except for having less prominent Golgi zones, no identifiable secretory granules, and non-specific degenerative changes. Discussion Our data show that a tumor from a patient exhibiting hypercalcemia synthesized a material, Tumor Peak I, which was similar to human parathyroid hormone. This conclusion is based on a) the ability to isolate the peptide by the same chemical methods required to isolate and purify authentic human parathyroid hormone, b) by its immunoreactivity towards amino- and carboxyl-terminaldirected antisera to parathyroid hormone, and c) the similarity of its size and ionic

charge to parathyroid hormone as determined by gel filtration and ion exchange chromatography. The nature of Tumor Peak II is less clear. It might represent a partially degraded product of Tumor Peak I which retains sufficient structure to react with antisera directed against both the amino- and carboxyl-terminal regions of parathyroid hormone. In this regard, Murray et al. (15) reported finding in bovine parathyroid extracts a biologically active fragment of parathyroid hormone consisting of residues 1 to 65. Whether or not this was an artifact of the preparation or produced by the gland in vivo could not be deduced. Although the late elution position of Tumor Peak II during gel filtration could mean that it is smaller than Tumor Peak I, this is not necessarily the case, since we showed recently that parathyroid hor-

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PARATHYROID HORMONE IN CARCINOMA

1029

FlC. 4. Low power electron micrograph of the squamous cell carcinoma from patient B; fixed in glutaraldehyde from 2 h after death and subsequently processed in same manner as tissue Figs. 3A-B. Tonofibrils and rare desmosome formation are consistent with squamous cell type. Extracellular collagen and cellular degenerative changes are present. Rough endoplasniic reticulum and free ribosomes are few in number, Golgi zones are not identified, and no structures suggestive of secretory granules are seen, x 7,800.

mone migrates anomalously in gel filtration due to its high degree of molecular asymmetry (16).1 Thus a small degree of degradation could conceivably result in a major conformational change leading to a more compact molecule and its late-eluting position. Alternatively, Tumor Peak II might be a second type of parathyroid hormone, perhaps unique to the tumor tissue. This would imply a variation in amino acid composition and consequently a different charge and apparent size. On the basis of the CM-cellulose profile it does not behave as prohormone since this precursor peptide from a number of animal species is known to elute at a con1

Specifically, at the pH of the elutions used in the present study, parathyroid hormone elutes from a Sephadex gel filtration column at a position equivalent to a spherical protein of molecular weight of about 26,000.

ductivity about twice that of parathyroid hormone. Our data do not allow us to conclude that the secretion by the tumor of either or both of Tumor Peaks I and II accounted for the hypercalcemia. Since patient A died shortly after the biopsy and before the main tumor was removed, we were unable to follow the course of the hypercalcemia. Moreover, there was insufficient tissue to test it in an in vitro bioassay system for bone resorptive capacity. On the other hand, the absence of bone involvement, the positive identification of Tumor Peak I with parathyroid hormone and the presence of secretory granule-like bodies in the tumor make it consistent with the notion that the hypercalcemia was in fact due to a secretion of Tumor Peak I and/or II.

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Although proparathyroid hormone is routinely observed in normal parathyroid glands of many species (17-20), we detected no peptide resembling the prohormone in the present study. This could mean that the cleavage mechanisms responsible for conversion of the precursor to the hormone in the tumor tissue of patient A were so active that little prohormone was present at any given time. Alternatively, the fundamental mechanisms of synthesis in the tumor might be substantially different than in the parathyroid gland such that synthesis of parathyroid hormone does not proceed through a prohormone stage. Acknowledgments We wish to express our appreciation for the excellent technical assistance of Sylvia Dillon and William Bopp.

References 1. Case Records of the Massachusetts General Hospital (Case 27461), New EnglJ Med 225: 789, 1941. 2. Tashjian, A. H., Jr., E. F. Voelkel, L. Levine, and P. Goldhaber, Evidence that the bone resorptionstimulating factor produced by mouse fibrosarcoma cells is prostaglandin E: A new model for the hypercalcemia of cancer, 7 Exp Med 136: 1329, 1972. 3. Brereton, H. D., P. V. Halushka, R. W. Alexander, D. M. Mason, H. R. Keiser, and V. T. De Vita, Jr., Indomethacin-responsive hypercalcemia in a patient with renal cell adenocarcinoma, N Engl J Med 291: 83, 1974. 4. Hannsjverg, W. S., G. V. Segre, J. L. Morgan, B. J. Sweetman, J. T. Potts, Jr., and J. A. Oates, Prostaglandins as mediators of hypercalcemia associated with certain types of cancer, N Engl J Med 293: 1278, 1975. 5. Mundy, G. R., L. G. Raisz, R. A. Cooper, G. P. Schechter, and S. E. Salmon, Evidence for the secretion of an osteoclast stimulating factor in myeloma, N EnglJ Med 291: 1041, 1974. 6. Berson, S. A., and R. S. Yalow, Parathyroid hormone in plasma in adenomatous hyperparathyroidism, uremia, and bronchogenic carcinoma, Science 154: 907, 1966. 7. Riggs, B. L., C. D. Arnaud, J. C. Reynolds, and L. H. Smith, Immunologic differentiation of primary hyperparathyroidism from hyperparathyroidism due to nonparathyroid cancer, J Clin Invest 50: 2079, 1971.

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8. Tashjian, A. H., Jr., L. Levine, and P. L. Munson, Immunochemical indentification of parathyroid hormone in nonparathyroid neoplasms associated with hypercalcemia, 7 Exp Med 119: 467, 1964. 9. Sherwood, L. M., J. H. L. O'Riordan, G. D. Aurbach, and J. T. Potts, Jr., Production of parathyroid hormone by non-parathyroid tumorsj Clin Endocrinol Metab 27: 140, 1967. 10. Benson, R. C , Jr., B. L. Riggs, B. M. Pickard, and C. D. Arnaud, Immunoreactive forms of circulating parathyroid hormone in primary and ectopic hyperparathyroidism^ Clin Invest 54: 175, 1974. 11. Greenberg, P. B., T. J. Martin, and H. S. Sutcliffe, Synthesis and release of parathyroid hormone by a renal carcinoma in cell culture, Clin Sci Mol Med 45: 183, 1973. 12. Hamilton, J. W., F. W. Spierto, R. R. MacGregor, and D. V. Cohn, Studies on the biosynthesis in vitro of parathyroid hormone isolated from bovine parathyroid tissue and incubation medium,7 Biol Chem 246: 3224, 1971. 13. Chu, L. L. H., R. R. MacGregor, P. I. Liu, J. W. Hamilton, and D. V. Cohn, Biosynthesis of proparathyroid hormone and parathyroid hormone by human parathyroid glands, 7 Clin Invest 52: 3089, 1973. 14. Chu, L. L. H., L. Forte, C. S. Anast, and D. V. Cohn, Interaction of parathyroid hormone with membranes of kidney cortex: Degradation of the hormone and activation of adenylate cyclase, Endocrinology 97: 986, 1975. 15. Murray, T. M., S. A. Muzaffar, J. A. Parsons, and H. T. Keutmann, A biologically active hormonal fragment isolated from bovine parathyroid glands (BPTH 1-65), Biochemistry 14: 2705, 1975. 16. Cohn, D. V., R. R. MacGregor, D. Sinha, W. Y. Huang, H. Edelhoch, and J. Hamilton, The migration behavior of proparathyroid hormone, parathyroid hormone, and their peptide fragments during gel filtration, Arch Biochem Biophys 164: 669, 1974. 17. Cohn, D. V., R. R. MacGregor, L. L. H. Chu, J. R. Kummel, and J. W. Hamilton, Calcemic fraction-A: Biosynthetic peptide precursor of parathyroid hormone, Proc Natl Acad Sci USA 69: 1521, 1972. 18. Chu, L. L. H., R. R. MacGregor, C. S. Anast, J. W. Hamilton, and D. V. Cohn, Studies on the biosynthesis of rat parathyroid gland to dietary restriction of calcium, Endocrinology 93: 915, 1973. 19. MacGregor, R. R., L. L. H. Chu, J. W. Hamilton, and D. V. Cohn, Partial purification of parathyroid hormone from chicken parathyroid glands, Endocrinology 92: 1312, 1973. 20. Chu, L. L. H., W. Y. Huang, E. T. Littledike, J. W. Hamilton, and D. V. Cohn, Porcine proparathyroid hormone. Identification, biosynthesis, and partial amino acid sequence, Biochemistry 14: 3631,1975.

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