0013-7227/90/1271-0305$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 1 Printed in U.S.A.
Tumor Resection and Antibodies to Parathyroid Hormone-Related Protein Cause Similar Changes on Bone Histomorphometry in Hypercalcemia of Cancer* S. C. KUKREJA, T. J. ROSOL, S. A. WIMBISCUS, D. H. SHEVRIN, V. GRILL, E. I. BARENGOLTS, AND T. J. MARTIN Departments of Medicine and Nuclear Medicine, Veterans Administration West Side and University of Illinois Medical Centers (S.C.K., S.A.W., D.H.S., E.I.B.), Chicago, Illinois 60612; the Department of Veterinary Pathobiology, Ohio State University (T.J.R.), Columbus, Ohio 43210; and St. Vincent Institute of Medical Research (V.G., T.J.M.), Melbourne 3065, Australia
ABSTRACT. Bone resorption is increased in both humoral hypercalcemia of malignancy (HHM) and primary hyperparathyroidism. On the other hand, bone formation parameters are increased in primary hyperparathyroidism and decreased in HHM. Recently, a PTH-related protein (PTHrP) has been shown to be responsible for the hypercalcemia in the syndrome of HHM. In the present study we evaluated the effects of a neutralizing antiserum to PTHrP on bone histomorphometric parameters in hypercalcemic athymic mice bearing a human
squamous cell lung cancer. These effects were compared to those of tumor resection. Similar to the effects of tumor resection, the antiserum to PTHrP resulted in a decrease in serum Ca levels, a decrease in bone resorption, and an increase in bone formation parameters. The studies, therefore, indicate that PTHrP is the major factor responsible for all of the features, including the decreased bone formation seen in HHM. {Endocrinology 127: 305-310,1990)
H
syndrome of HHM (4, 5). Recently, a PTH-related protein (PTHrP) has been isolated from tumors associated with HHM (11-17). It is not yet firmly established whether PTHrP is responsible for all of the manifestations of HHM or whether other factors, such as transforming growth factor-a (TGFa) (1), interleukin-1 (18), or tumor necrosis factor (19), are also involved in its pathogenesis. We have previously shown that in tumorbearing hypercalcemic athymic mice, injection of a neutralizing antiserum to PTHrP reversed the hypercalcemia (20). In the present study we compared the effects of tumor resection with those of injection of a neutralizing antiserum to PTHrP on quantitative bone histomorphometry in these animals.
UMORAL hypercalcemia of malignancy (HHM) and primary hyperparathyroidism (PHPT) share several common features, such as increased nephrogenous cAMP, increased renal calcium reabsorption, and increased bone resorption (1-3). One major difference between the two syndromes in humans is that bone formation is normal or increased in PHPT, whereas it is diminished in patients with HHM (4-6). In some of the animal models of HHM, bone resorption as well as bone formation parameters are increased (7, 8). We have previously described an animal model of HHM in which a squamous cell lung cancer has been serially carried in athymic mice (9). The mice develop hypercalcemia and elevated urinary cAMP (9), and by quantitative histomorphometry bone resorption is increased and bone formation is decreased (10). The latter observation of decreased bone formation is characteristic of the human
Animal model
Received January 11,1990. Address all correspondence and requests for reprints to: Subhash C. Kukreja, M.D., Endocrinology Section and Nuclear Medicine Service, Veterans Administration West Side Medical Center (M.P. 115), 820 South Damen Avenue, Chicago, Illinois 60612. * A portion of this work was presented at the National Meeting of the American Federation for Clinical Research, Washington D.C., April 28-May 1,1989. This work was supported by the V.A. Medical Research Service and the National Health and Medical Research Council of Australia.
The animal model has been previously described in detail (9). In this model, a squamous cell lung cancer derived from a hypercalcemic patient has been serially carried in athymic mice. For propagation, 1- to 2-mm3 pieces of tumor tissue are implanted sc. Two to 3 weeks later, the mice become hypercalcemic and hypophosphatemic at a time when the tumor size is approximately 1 cm3. The tumor-bearing animals have elevated urinary cAMP levels (9) and relative hypocalciuria (21). In these animals bone resorption parameters are increased and
Materials and Methods
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bone formation parameters decreased compared to those in normal mice (10). Antiserum
(mg/dl
BONE CHANGES IN HYPERCALCEMIA OF CANCER
306
16-i
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The antiserum used for these studies was generated against PTHrP-(l-16) in New Zealand White rabbits. PTHrP-(l-16) was conjugated to soybean trypsin inhibitor and then injected with Freund's adjuvant into the rabbits. In the osteosarcoma cell cultures, this antiserum has been previously shown to neutralize the adenylate cyclase-stimulating activity of PTHrP (20, 22). Animal protocols Resection experiment. Four-week-old BALB/c athymic mice were implanted with the human squamous cell lung tumor. Three weeks later, the mice were bled by tail vein, and serum was analyzed for calcium. Hypercalcemic mice (serum Ca > 11.0 mg/dl) were selected and divided into six groups (n = 5 each). Tumors were removed in three groups of mice and left intact in the other three groups. Twenty-four, 48, and 96 h after the resection of tumor, groups of mice were killed, and lumbar vertebrae were removed for quantitative histomorphometry. At each time, a control group of mice, in which the tumor had not been removed, was also killed, and lumbar vertebrae removed. The animals that were killed 96 h after the resection of tumor and the corresponding control group received two injections of calcein ip (0.375 mg calcein with 2 mg NaHCO3 in 0.1 ml normal saline). One injection was given immediately after removal of tumor, and the other 72 h later. Antiserum experiment. Two groups of hypercalcemic tumorbearing mice were selected as described above. Animals in one group were injected (via tail vein) with 0.1 ml neutralizing antiserum at 0 and 48 h. In our previous study a single dose of this antiserum decreased serum calcium levels in the hypercalcemic tumor-bearing mice for 24-48 h. Therefore, with the second dose at 48 h, we hoped to maintain the neutralization of PTHrP for 96 h. The second group of mice received 0.1 ml normal rabbit serum at each of the two time periods. Ninetysix hours after the first injection of normal rabbit serum or antiserum, the animals were killed, and lumbar vertebrae removed. These two groups of mice also received two injections of calcein (0.375 mg each) at 0 and 72 h. Bone histomorphometry The vertebrae were fixed for at least 48 h in cold (4 C) 95% ethanol, infiltrated in JB4 methyl methacrylate (PolyScience, Inc., Warrington, PA) for 6 days, and embedded at 4 C. Midline sections (5 ^m thick) were cut on a model K sledge microtome, stained for tartrate-resistant acid phosphatase (no. 386, Sigma Diagnostics, St. Louis, MO), and counterstained with hematoxylin. The sections were evaluated by one of us (T.J.R.) without knowledge of the groups. The total endosteal perimeter of second or third lumbar vertebra was measured using a Zeiss Interactive Digital Analysis System (ZIDAS; Zeiss, New York, NY) at a magnification of X200. The perimeter lined by acid phosphatase-positive cells and the number of these cells
A
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Endo • 1990 Vol 127 • No 1
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Post Excision FIG. 2. Percentage of trabecular perimeter lined by osteoclasts (% OC, A) or osteoblasts (% OB, B) in various groups (mean ± SE). The data are presented for groups in which tumors were excised 24, 48, or 96 h earlier (•). D, The corresponding control animals, in which tumors were left intact. Ab, Animals that received normal rabbit serum (•) or the neutralizing antiserum (•). *, Significant difference (P < 0.05) compared to the corresponding control group.
PTHrP has been shown to have biological effects similar to those of PTH on adenylate cyclase and bone resorption activities and in its ability to produce hypercalcemia in vivo (17, 23-25). Increases in serum Ca, decreases in urinary Ca, and elevation of urinary cAMP
Endo • 1990 Vol 127 • No 1
are clinical features common to both HHM and PHPT (1-3). Bone resorption is also increased in both syndromes (1-5). One major difference between the two syndromes in humans is that in patients with PHPT, bone formation is increased, whereas it is decreased in patients with HHM (4, 5). Infusion of PTH into animals results in increases in both bone resorption as well as formation parameters (26). Similarly, in our previous study (27) infusion of PTHrP-(l-40) resulted in an increase in bone formation parameters. In this study the effects of PTHrP infusion were not directly compared to those of PTH infusion. In a recent study, Rizzoli et al. (28) compared the effects of bovine PTH-(l-34) and human PTHrP-(1-34) infusions on bone histomorphometry in thyroparathyroidectomized rats. Doses of the two peptides that caused a similar degree of hypercalcemia resulted in different effects on bone histomorphometry. Both PTH and PTHrP caused increases in bone resorption (slightly greater with PTH). PTH resulted in a marked stimulation of bone formation, whereas PTHrP caused only slight stimulation. In two models of HHM associated with excessive PTHrP production, implantation of tumors into athymic mice resulted in an increase in bone resorption as well as bone formation parameters (7, 8). The animal model used in the present study is unique, in that, similar to observations of the human syndrome of HHM, bone formation parameters are decreased (10). Therefore, it would appear logical that factors other than PTHrP are responsible for the inhibited bone formation seen in the human syndrome and in our animal model. On the other hand, in the present study infusion of antibodies directed against PTHrP resulted in a reversal of both the inhibited bone formation and the increased bone resorption. This would suggest that PTHrP was a prime contributor to both the increased bone resorption and the inhibited bone formation. Therefore, the effects of PTHrP on bone formation may be complex. PTHrP alone may stimulate bone formation (coupled to bone resorption), whereas in the presence of certain tumor-related factors that may be unique to certain tumors, bone formation may be
TABLE 2. Effect of tumor resection or antiserum injection on dynamic histomorphometric parameters
Group 96 h Control Resect Injection NRS
AS
Single labeled (%)
Double labeled (%)
21.7 ± 2.2 25.3 ± 1.8
9.7 ± 3.7 21.8 ± 3.0°
36.2 ± 1.9 31.1 ± 3.9
14.8 ± 5.2 24.3 ± 2.5°
Mineral app rate (fim/day)
Mineralizing perimeter (%)
BFR (xlO"3; mmVmmday)
1.8 ± 0.2 2.2 ± 0.1
20.5 ± 4.1 34.5 ± 2.5°
0.40 ± 0.11 0.75 ± 0.07°
1.3 ± 0.1 1.8 ± 0.02°
32.8 ± 4.9 39.8 ± 1.9
0.43 ± 0.08 0.72 ± 0.03°
See Table 1 for experimental design. Animals were injected with two doses of calcein (0.375 mg each) at 0 and 72 h. Mineral app rate, Mineral apposition rate; BFR, bone formation rate; Resect, resection; NRS, normal rabbit serum; AS, antiserum. Values are the mean ± SE. 0 P < 0.05 us. control.
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BONE CHANGES IN HYPERCALCEMIA OF CANCER inhibited. The nature of these other tumor-related factors is not clear from our studies. One such factor may be TGFa. TGFa has been shown to inhibit bone formation in vitro (29). We have previously shown that the tumor used for the present study contains a strong signal for the TGFa mRNA (30). An alternative explanation for the contradictory effects of PTHrP may be that the amino-terminal fragments of PTHrP stimulate, whereas the carboxy-terminal fragments inhibit bone formation. This is, however, an unlikely explanation, as we have recently shown that PTHrP-(l-34) and PTHrP-(l-Hl) have similar effects on bone resorption as well as formation paramaters (31). The results of the present studies demonstrate that tumor resection and passive immunization with PTHrP result in similar changes in serum calcium and bone histomorphometry in tumor-bearing mice. Therefore, PTHrP appears to be the prime factor responsible for all of the manifestations of HHM.
Acknowledgments We thank Darlene M. Castiglione for secretarial assistance and K. I. Gorny for technical assistance.
References 1. Mundy GR 1988 Hypercalcemia of malignancy revisited. J Clin Invest 82:1 2. Broadus AE, Mangin M, Ibeda K, Insogna KL, Weir EC, Burtis WJ, Stewart AF 1988 Humoral hypercalcemia of cancer. Identification of a novel parathyroid hormone-like peptide. N Engl J Med 319:556 3. Martin TJ 1988 Humoral hypercalcemia of malignancy. Bone Mineral 4:83 4. Sharp CF, Rude RK, Terry R, Singer FR 1982, Abnormal bone and parathyroid histology in carcinoma patients with pseudohyperparathyroidism. Cancer 49:1449 5. Stewart AF, Vignery A, Silvergate A, Ravin ND, LiVolsi V, Broadus AE, Baron R 1982, Quantitative bone histomorphometry in humoral hypercalcemia of malignancy: uncoupling of bone cell activity. J Clin Endocrinol Metab 55:219 6. McGuire JL, Mark Jr SC 1974 The effects of parathyroid hormone on bone cell structure and function. Clin Orthop Relat Res 100:392 7. Rosol TJ, Capen CC, Weisbrode SE, Horst RL 1986 Humoral hypercalcemia in nude mouse model of a canine adenocarcinoma derived from apocrine glands of the anal sac. Lab Invest 54:679 8. Strewler GJ, Wronski TJ, Halloran GP, Miller SC, Leung SC, Williams RD, Nissenson RA 1986 Pathogenesis of hypercalcemia in nude mice bearing a human renal carcinoma. Endocrinology 119:303 9. Abramson EC, Kukla LJ, Shevrin DH, Lad TE, McGuire WP, Kukreja SC 1984 A model for malignancy-associated humoral hypercalcemia. Calcif Tissue Int 36:563 10. Kukreja SC, Rosol TJ, Shevrin DH, York PA 1988 Quantitative bone histomorphometry in nude mice bearing a human squamous cell lung cancer. Bone Mineral Res 3:341 II. Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF 1987 Identification of a novel 17,000 dalton PTH-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 262:7151 12. Stewart AF, Wu T, Goumas D, Burtis WJ, Broadus AE 1987 NTerminal amino acid sequence of two novel tumor-derived aden-
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ylate cyclase-stimulating proteins: identification of parathyroid hormone-like and parathyroid hormone-unlike domains. Biochem Biophys Res Commun 146:672 Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, Rodda CP, Ebeling PR, Hudson PJ, Zajac JD, Martin TJ 1987 Parathyroid hormone-related protein purified from a human lung cancer line. Proc Natl Acad Sci USA 84:5048 Strewler GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC, Rosenblatt M, Nissenson RA 1987 Parathyroid hormone-like protein from human renal carcinoma cells: structural and functional homology with parathyroid hormone. J Clin Invest 80:1803 Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI 1987 A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 237:893 Mangin M, Webb AC, Dreyer BE, Possillico JT, Ikeda K, Weir EC, Stewart AF, Bander NH, Milstone L, Barton DE, Francke U, Broadus AE 1988 Identification of a complementary DNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy. Proc Natl Acad Sci USA 85:597 Kemp BE, Moseley JM, Rodda CP, Ebeling PR, Wettenhall REH, Stapleton D, Dieffenbach-Jagger H, Ure F, Michelangeli VP, Simmons HA, Raisz LG, Martin TJ 1987 Parathyroid hormone-related protein of malignancy: active synthetic fragments. Science 238:1568 Sato K, Fujii Y, Kasono K, Tsushima T, Shizume K 1988 Production of interleukin-la and a parathyroid hormone-like factor by a squamous cell carcinoma of esophagus (ECGI) derived from a patient with hypercalcemia. J Clin Endocrinol Metab 67:592 Johnson RA, Boyce BF, Mundy GR, Roodman GD 1989 Tumors producing human tumor necrosis factor induce hypercalcemia and osteoclastic bone resorption in nude mice. Endocrinology 124:1424 Kukreja SC, Shevrin DH, Wimbiscus SA, Eberling PR, Danks JA, Rodda CP, Wood WI, Martin TJ 1988 Antibodies to parathyroid hormone-related protein lower serum calcium in athymic mouse models of malignancy-associated hypercalcemia due to human tumors. J Clin Invest 82:1798 Shipley PJ, Shevrin DH, Kukreja SC 1988 Increased calcium reabsorption in an animal model of hypercalcemica of human malignancy. J Bone Mineral Res 3:555 Danks JA, Ebeling PR, Hayman J, Chou ST, Moseley JM, Dunlap J, Kemp BE, Martin TJ 1989 Parathyroid hormone-related protein: immunohistochemical localization in cancer and normal skin. J Bone Mineral Res 4:273 Horiuchi N, Caulfield MP, Fisher JE, Goldman ME, McKee RL, Reagan JE, Levy JL, Nutt RF, Rodan SB, Schofield TL, Clemens TL, Rosenblatt M 1987 Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 238:1566 Stewart AF, Mangin M, Wu T, Goumas D, Insogna KL, Burtis WJ, Broadus AE 1988 Synthetic human parathyroid hormone-like protein stimulates bone resorption and causes hypercalcemia in rats. J Clin Invest 81:596 Yates AJP, Guiterrez GE, Smolens P, Travis PS, Katz MS, Aufdemorte TB, Boyce BF, Hymer TK, Poser JW, Mundy GR 1988 Effects of a synthetic peptide of a parathyroid hormone-related protein on calcium homeostasis, renal tubular calcium reabsorption, and bone metabolism in vivo and in vitro in rodents. J Clin Invest 81:932 Tam CS, Heersche JNM, Murray TM, Parsons JA 1982 Parathyroid hormone stimulates the bone appositon rate independently of its resorptive action: differential effects of intermittent and continuous administration. Endocrinology 110:506 Rosol TJ, Capen CC, Horst RL 1988 Effect of infusion of human parathyroid hormone-related protein (1-40) in nude mice: histomorphometric and biochemical evaluation. J Bone Mineral Res 3:699 Rizzoli R, Arlot M, Caverzasio J, Meunier P, Bonjour JP 1989 Blunted stimulation of bone formation (BF) in parathyroid hormone-related protein (PTHrP)-infused thyroparathyroidectom-
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ized (TPTx) rats. J Bone Mineral Res 4:5265 (Abstract) 29. Ibbotson KJ, Harrod J, Gowen M, D'Souza S, Smith DD, Winkler M, Derynck R, Mundy GR 1986 Human recombinant transforming growth factor a stimulates bone resorption and inhibits bone formation in vitro. Proc Natl Acad Sci USA 83:2228 30. Mundy GR, Ibbotson KJ, D'Souza SM, Kukreja SC, Abramson EC, Kukla LJ, Carpenter G, Denynck R 1985 Evidence that transforming growth factor alpha production causes bone resorp-
Endo • 1990 Vol 127 • No 1
tion and hypercalcemia in squamous cell carcinoma of lung. Clin Res 33:573a (Abstract) 31. Kukreja SC, Rosol TJ, Wimbiscus SA, Shevrin DH, Hammonds RG, Martin TJ, Wood WI 1989 Comparison of the effects of parathyroid hormone related protein (PTHrP) (1-34) and PTHrP (1-141) on quantitative bone histomorphometry in mice. J Bone Mineral Res 4:S319 (Abstract).
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Vol. 127, No. 1 Printed in U.S.A.
Tumor Resection and Antibodies to Parathyroid Hormone-Related Protein Cause Similar Changes on Bone Histomorphometry in Hypercalcemia of Cancer* S. C. KUKREJA, T. J. ROSOL, S. A. WIMBISCUS, D. H. SHEVRIN, V. GRILL, E. I. BARENGOLTS, AND T. J. MARTIN Departments of Medicine and Nuclear Medicine, Veterans Administration West Side and University of Illinois Medical Centers (S.C.K., S.A.W., D.H.S., E.I.B.), Chicago, Illinois 60612; the Department of Veterinary Pathobiology, Ohio State University (T.J.R.), Columbus, Ohio 43210; and St. Vincent Institute of Medical Research (V.G., T.J.M.), Melbourne 3065, Australia
ABSTRACT. Bone resorption is increased in both humoral hypercalcemia of malignancy (HHM) and primary hyperparathyroidism. On the other hand, bone formation parameters are increased in primary hyperparathyroidism and decreased in HHM. Recently, a PTH-related protein (PTHrP) has been shown to be responsible for the hypercalcemia in the syndrome of HHM. In the present study we evaluated the effects of a neutralizing antiserum to PTHrP on bone histomorphometric parameters in hypercalcemic athymic mice bearing a human
squamous cell lung cancer. These effects were compared to those of tumor resection. Similar to the effects of tumor resection, the antiserum to PTHrP resulted in a decrease in serum Ca levels, a decrease in bone resorption, and an increase in bone formation parameters. The studies, therefore, indicate that PTHrP is the major factor responsible for all of the features, including the decreased bone formation seen in HHM. {Endocrinology 127: 305-310,1990)
H
syndrome of HHM (4, 5). Recently, a PTH-related protein (PTHrP) has been isolated from tumors associated with HHM (11-17). It is not yet firmly established whether PTHrP is responsible for all of the manifestations of HHM or whether other factors, such as transforming growth factor-a (TGFa) (1), interleukin-1 (18), or tumor necrosis factor (19), are also involved in its pathogenesis. We have previously shown that in tumorbearing hypercalcemic athymic mice, injection of a neutralizing antiserum to PTHrP reversed the hypercalcemia (20). In the present study we compared the effects of tumor resection with those of injection of a neutralizing antiserum to PTHrP on quantitative bone histomorphometry in these animals.
UMORAL hypercalcemia of malignancy (HHM) and primary hyperparathyroidism (PHPT) share several common features, such as increased nephrogenous cAMP, increased renal calcium reabsorption, and increased bone resorption (1-3). One major difference between the two syndromes in humans is that bone formation is normal or increased in PHPT, whereas it is diminished in patients with HHM (4-6). In some of the animal models of HHM, bone resorption as well as bone formation parameters are increased (7, 8). We have previously described an animal model of HHM in which a squamous cell lung cancer has been serially carried in athymic mice (9). The mice develop hypercalcemia and elevated urinary cAMP (9), and by quantitative histomorphometry bone resorption is increased and bone formation is decreased (10). The latter observation of decreased bone formation is characteristic of the human
Animal model
Received January 11,1990. Address all correspondence and requests for reprints to: Subhash C. Kukreja, M.D., Endocrinology Section and Nuclear Medicine Service, Veterans Administration West Side Medical Center (M.P. 115), 820 South Damen Avenue, Chicago, Illinois 60612. * A portion of this work was presented at the National Meeting of the American Federation for Clinical Research, Washington D.C., April 28-May 1,1989. This work was supported by the V.A. Medical Research Service and the National Health and Medical Research Council of Australia.
The animal model has been previously described in detail (9). In this model, a squamous cell lung cancer derived from a hypercalcemic patient has been serially carried in athymic mice. For propagation, 1- to 2-mm3 pieces of tumor tissue are implanted sc. Two to 3 weeks later, the mice become hypercalcemic and hypophosphatemic at a time when the tumor size is approximately 1 cm3. The tumor-bearing animals have elevated urinary cAMP levels (9) and relative hypocalciuria (21). In these animals bone resorption parameters are increased and
Materials and Methods
305
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bone formation parameters decreased compared to those in normal mice (10). Antiserum
(mg/dl
BONE CHANGES IN HYPERCALCEMIA OF CANCER
306
16-i
to
il:
The antiserum used for these studies was generated against PTHrP-(l-16) in New Zealand White rabbits. PTHrP-(l-16) was conjugated to soybean trypsin inhibitor and then injected with Freund's adjuvant into the rabbits. In the osteosarcoma cell cultures, this antiserum has been previously shown to neutralize the adenylate cyclase-stimulating activity of PTHrP (20, 22). Animal protocols Resection experiment. Four-week-old BALB/c athymic mice were implanted with the human squamous cell lung tumor. Three weeks later, the mice were bled by tail vein, and serum was analyzed for calcium. Hypercalcemic mice (serum Ca > 11.0 mg/dl) were selected and divided into six groups (n = 5 each). Tumors were removed in three groups of mice and left intact in the other three groups. Twenty-four, 48, and 96 h after the resection of tumor, groups of mice were killed, and lumbar vertebrae were removed for quantitative histomorphometry. At each time, a control group of mice, in which the tumor had not been removed, was also killed, and lumbar vertebrae removed. The animals that were killed 96 h after the resection of tumor and the corresponding control group received two injections of calcein ip (0.375 mg calcein with 2 mg NaHCO3 in 0.1 ml normal saline). One injection was given immediately after removal of tumor, and the other 72 h later. Antiserum experiment. Two groups of hypercalcemic tumorbearing mice were selected as described above. Animals in one group were injected (via tail vein) with 0.1 ml neutralizing antiserum at 0 and 48 h. In our previous study a single dose of this antiserum decreased serum calcium levels in the hypercalcemic tumor-bearing mice for 24-48 h. Therefore, with the second dose at 48 h, we hoped to maintain the neutralization of PTHrP for 96 h. The second group of mice received 0.1 ml normal rabbit serum at each of the two time periods. Ninetysix hours after the first injection of normal rabbit serum or antiserum, the animals were killed, and lumbar vertebrae removed. These two groups of mice also received two injections of calcein (0.375 mg each) at 0 and 72 h. Bone histomorphometry The vertebrae were fixed for at least 48 h in cold (4 C) 95% ethanol, infiltrated in JB4 methyl methacrylate (PolyScience, Inc., Warrington, PA) for 6 days, and embedded at 4 C. Midline sections (5 ^m thick) were cut on a model K sledge microtome, stained for tartrate-resistant acid phosphatase (no. 386, Sigma Diagnostics, St. Louis, MO), and counterstained with hematoxylin. The sections were evaluated by one of us (T.J.R.) without knowledge of the groups. The total endosteal perimeter of second or third lumbar vertebra was measured using a Zeiss Interactive Digital Analysis System (ZIDAS; Zeiss, New York, NY) at a magnification of X200. The perimeter lined by acid phosphatase-positive cells and the number of these cells
A
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Endo • 1990 Vol 127 • No 1
—-—f Control
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24
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9
72
Time (Hours)
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Post Excision FIG. 2. Percentage of trabecular perimeter lined by osteoclasts (% OC, A) or osteoblasts (% OB, B) in various groups (mean ± SE). The data are presented for groups in which tumors were excised 24, 48, or 96 h earlier (•). D, The corresponding control animals, in which tumors were left intact. Ab, Animals that received normal rabbit serum (•) or the neutralizing antiserum (•). *, Significant difference (P < 0.05) compared to the corresponding control group.
PTHrP has been shown to have biological effects similar to those of PTH on adenylate cyclase and bone resorption activities and in its ability to produce hypercalcemia in vivo (17, 23-25). Increases in serum Ca, decreases in urinary Ca, and elevation of urinary cAMP
Endo • 1990 Vol 127 • No 1
are clinical features common to both HHM and PHPT (1-3). Bone resorption is also increased in both syndromes (1-5). One major difference between the two syndromes in humans is that in patients with PHPT, bone formation is increased, whereas it is decreased in patients with HHM (4, 5). Infusion of PTH into animals results in increases in both bone resorption as well as formation parameters (26). Similarly, in our previous study (27) infusion of PTHrP-(l-40) resulted in an increase in bone formation parameters. In this study the effects of PTHrP infusion were not directly compared to those of PTH infusion. In a recent study, Rizzoli et al. (28) compared the effects of bovine PTH-(l-34) and human PTHrP-(1-34) infusions on bone histomorphometry in thyroparathyroidectomized rats. Doses of the two peptides that caused a similar degree of hypercalcemia resulted in different effects on bone histomorphometry. Both PTH and PTHrP caused increases in bone resorption (slightly greater with PTH). PTH resulted in a marked stimulation of bone formation, whereas PTHrP caused only slight stimulation. In two models of HHM associated with excessive PTHrP production, implantation of tumors into athymic mice resulted in an increase in bone resorption as well as bone formation parameters (7, 8). The animal model used in the present study is unique, in that, similar to observations of the human syndrome of HHM, bone formation parameters are decreased (10). Therefore, it would appear logical that factors other than PTHrP are responsible for the inhibited bone formation seen in the human syndrome and in our animal model. On the other hand, in the present study infusion of antibodies directed against PTHrP resulted in a reversal of both the inhibited bone formation and the increased bone resorption. This would suggest that PTHrP was a prime contributor to both the increased bone resorption and the inhibited bone formation. Therefore, the effects of PTHrP on bone formation may be complex. PTHrP alone may stimulate bone formation (coupled to bone resorption), whereas in the presence of certain tumor-related factors that may be unique to certain tumors, bone formation may be
TABLE 2. Effect of tumor resection or antiserum injection on dynamic histomorphometric parameters
Group 96 h Control Resect Injection NRS
AS
Single labeled (%)
Double labeled (%)
21.7 ± 2.2 25.3 ± 1.8
9.7 ± 3.7 21.8 ± 3.0°
36.2 ± 1.9 31.1 ± 3.9
14.8 ± 5.2 24.3 ± 2.5°
Mineral app rate (fim/day)
Mineralizing perimeter (%)
BFR (xlO"3; mmVmmday)
1.8 ± 0.2 2.2 ± 0.1
20.5 ± 4.1 34.5 ± 2.5°
0.40 ± 0.11 0.75 ± 0.07°
1.3 ± 0.1 1.8 ± 0.02°
32.8 ± 4.9 39.8 ± 1.9
0.43 ± 0.08 0.72 ± 0.03°
See Table 1 for experimental design. Animals were injected with two doses of calcein (0.375 mg each) at 0 and 72 h. Mineral app rate, Mineral apposition rate; BFR, bone formation rate; Resect, resection; NRS, normal rabbit serum; AS, antiserum. Values are the mean ± SE. 0 P < 0.05 us. control.
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BONE CHANGES IN HYPERCALCEMIA OF CANCER inhibited. The nature of these other tumor-related factors is not clear from our studies. One such factor may be TGFa. TGFa has been shown to inhibit bone formation in vitro (29). We have previously shown that the tumor used for the present study contains a strong signal for the TGFa mRNA (30). An alternative explanation for the contradictory effects of PTHrP may be that the amino-terminal fragments of PTHrP stimulate, whereas the carboxy-terminal fragments inhibit bone formation. This is, however, an unlikely explanation, as we have recently shown that PTHrP-(l-34) and PTHrP-(l-Hl) have similar effects on bone resorption as well as formation paramaters (31). The results of the present studies demonstrate that tumor resection and passive immunization with PTHrP result in similar changes in serum calcium and bone histomorphometry in tumor-bearing mice. Therefore, PTHrP appears to be the prime factor responsible for all of the manifestations of HHM.
Acknowledgments We thank Darlene M. Castiglione for secretarial assistance and K. I. Gorny for technical assistance.
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