0013-7227/90/1273-1444$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 3 Printed in U.S.A.
Measurement of Circulating Parathyroid HormoneRelated Protein in Rats with Humoral Hypercalcemia of Malignancy using a Two-Site Immunoradiometric Assay* GREGORY GAICH AND WILLIAM J. BURTIS Division of Endocrinology, West Haven Veterans Affairs Medical Center, West Haven, Connecticut 06516; and the Division of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06510
ABSTRACT. The Rice H500 rat Leydig cell tumor is a well characterized model of humoral hypercalcemia of malignancy (HHM). Circulating concentrations of PTH-related protein (PTHRP) have not been reported in this or any other animal model of HHM. Taking advantage of the marked N-terminal amino acid homology between rodent and human PTHRPs, we have adapted a sensitive two-site immunoradiometric assay developed for measurement of human PTHRP for use in measuring rat PTHRP. Circulating calcium and PTHRP concentrations were serially measured after sc passage of the Leydig cell tumor in rats. Significant hypercalcemia and elevation of PTHRP occurred on day 9 after tumor inoculation. When grouped by tumor size, both plasma calcium and PTHRP levels were significantly elevated in animals with tumor burdens greater than 10 cc. The PTHRP concentration was strongly correlated with both serum
H
UMORAL hypercalcemia of malignancy (HHM) is a syndrome most often caused by the production of PTH-related proteins (PTHRPs) by malignant neoplasms (1-3). The genes for rat and human peptides have recently been identified, their structure determined, and cDNA-predicted amino acid sequences synthesized and characterized (4-7). There is remarkable homology throughout a large N-terminal region among cDNApredicted PTHRPs derived from these two species. The Rice H500 Leydig cell tumor in rats is a well characterized animal model of HHM (8, 9). While it seems likely that rat PTHRPs are responsible for this syndrome, methods for measuring circulating PTHRP in rats have not been available. We have taken advantage of the rathuman N-terminal PTHRP homology in adapting a senReceived March 5, 1990. Address all correspondence and requests for reprints to: Gregory Gaich, M.D., c/o William J. Burtis, M.D., Ph.D., Research/151, West Haven VA Medical Center, 950 Campbell Avenue, West Haven, Connecticut 06516. * This work was supported by the Department of Veterans Affairs, West Haven, CT.
calcium (r = 0.88) and tumor size (r = 0.80). Circulating rat PTHRP averaged 12.8 pM on day 9 and 27.5 pM on day 10 or 11. PTHRP was undetectable in the plasma of 19 control rats. In 3 rats, plasma calcium returned to normal, and PTHRP became undetectable within 24 h after tumor excision. Rat milk displayed a PTHRP concentration of 2000 pM, while acid-urea extract of the rat tumor contained 0.32 pmol/mg protein. Dilutions of rat plasma, milk, and tumor extract displayed response curves that were parallel to the human PTHRP-(l-74) standard in the assay. This two-site immunoradiometric assay is a sensitive and easily performed means of measuring rat PTHRP. It should be useful in studying this animal model of HHM and the function of PTHRP in normal tissues. {Endocrinology 127: 1444-1449, 1990)
sitive human PTHRP two-site immunoradiometric assay (IRMA) for use in rat plasma and other tissues.
Materials and Methods Animals Male Fisher 344 rats (Harlan Sprague-Dawley, Indianapolis, IN), ranging in age from 8-12 weeks, were used in all studies involving the H500 Leydig cell tumor. Two lactating female Wistar rats (Harlan Sprague-Dawley) were used for the experiments involving rat milk. All animals were fed standard rodent chow and allowed access to water ad libitum. The Rice H500 Leydig cell tumor was passaged by serial sc transplantation in the flanks of anesthetized animals, as previously described (8), and allowed to grow for 9-14 days. Animals were typically moribund by days 10-14. Since the tumor masses were ovoid in shape, we estimated tumor volume using the formula V = 1/6 7r D2L, where D is the diameter, and L is the length of the tumor. Tumor volume was calculated in a blinded fashion before knowing the results of the plasma calcium or PTHRP measurements. Three of 12 hypercalcemic animals survived complete surgical excision of their tumors and had plasma
1444
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 11:17 For personal use only. No other uses without permission. . All rights reserved.
RAT PTHRP IRMA calcium and PTHRP measurements performed before and 24 h after surgery. Control rats were matched with respect to age and bled simultaneously with the experimental rats. Blood and milk sampling
All plasma samples (1.5 ml) were obtained in iced heparinized tubes containing 50 pi of a protease inhibitor mixture, including aprotinin (500 klU/ml), leupeptin (5 /ig/ml), pepstatin (5 ng/ ml), and EDTA (1 mM). Samples were immediately placed on ice, and the plasma was separated by centrifugation at 4 C and frozen at — 70 C. Plasma calcium measurements were performed by atomic absorption. In the time-course studies, 1.5 ml blood were obtained every 3 days by tail vein bleeds in anesthetized animals. At the end of the experiment, the rats were anesthetized, and the tumors were excised and frozen at —70 C. The rats were then exsanguinated by open cardiac puncture. Rat milk was manually expressed from anesthetized lactating rats (after separation from their pups for 2-8 h) and promptly frozen at -70 C. Tumor and control tissue extraction All extraction procedures were performed on ice, as detailed previously (10). Frozen Leydig cell tumor or normal rat testis was homogenized in a Waring blender in 0.1 M Tris-HCl, pH 7.4 (3 ml/g tissue), then centrifuged at 27,000 x g for 15 min. The resultant pellet was extracted in 8 M urea, 0.2 N HC1, and 0.1 M cysteine for 1 h at 0 C and centrifuged at 27,000 x g for 15 min. The supernatant was dialyzed against deionized water and then lyophilized. Protein was measured using the method of Lowry et al. (11), with BSA as standard. PTHRP-C1-74) IRMA The IRMA has been previously described in detail as it pertains to human plasma (12). The only modification was the use of rat plasma for the standard curve and sample diluent, as described below. Briefly, the two-site IRMA uses affinity-purified rabbit polyclonal anti-PTHRP-(37-74) antibody bound to a solid phase as the "capture" antibody, and iodinated affinity-purified rabbit polyclonal anti-PTHRP-(l-36) antibody as the "signal" antibody. Wells of polyvinyl chloride microtiter plates (Dynatech Laboratories, Chantilly, VA) were filled with 100 n\ anti-PTHRP-(37-74) (1:100) in borate buffer (100 mM boric acid, 25 mM sodium tetraborate, and 75 mM sodium chloride), pH 8.4, and incubated overnight at 4 C. The wells were washed with 0.1% Tween-20 (Sigma Chemical Co., St. Louis, MO) in 10 mM phosphate-buffered 150 mM sodium chloride, pH 7.4 (PBS), followed by three washes with PBS alone, blocked with 300 n\ 1% BSA in borate buffer for 2 h at 25 C, and then washed again as described above. Two hundredmicroliter plasma samples or standards were added to duplicate wells and incubated overnight at 4 C. After washing, 100 n\ radiolabeled anti-PTHRP-(l-36) antibody (-50,000 cpm/well) in PBT buffer (PBS, 0.1% BSA, and 0.1% Triton X-100) with 1% normal rabbit serum were added to each well and incubated overnight at 4 C. After the final wash, the wells were cut from the plate and counted individually. Synthetic human PTHRP(1-74) was employed as the assay standard. In the human
1445
PTHRP IRMA, normal human plasma is the standard and sample diluent. To determine whether normal rat plasma contained measurable PTHRP immunoreactivity, in an initial experiment normal rat plasma was stripped of endogenous Nterminal PTHRP using an anti-PTHRP-(l-36) antibody column, and this PTHRP-stripped plasma was used as diluent for the standard curve and as the assay zero standard. When the assay was thus configured, unstripped normal rat plasma displayed no detectable immunoreactivity. Normal rat plasma was accordingly used as a diluent for the subsequent assays. The detection limit for the assay was 1.4 pM PTHRP-(1-74). The antibodies used in the assay have been previously shown to be highly specific for PTHRP-(1-74), demonstrating no significant cross-reactivity with PTHRP-(l-36), PTHRP-(37-74), PTHRP-(109-138), or human PTH-(l-84) (12). The intra- and interassay variations for the rat averaged 3.4% and 8.9%, respectively. Statistics Statistical analysis was performed using Student's two-tailed t test, and simple regression using the StatView program on an Apple Macintosh computer.
Results Results using the PTHRP-(1-74) IRMA on serial dilutions of rat milk, H500 rat tumor extract, normal rat testis extract, and two representative plasma samples from hypercalcemic rats bearing the H500 tumor are compared with those using the PTHRP-(1-74) standard diluted in normal rat plasma in Fig. 1. The standard curve was linear over the range of 1.4-200 pM and exhibited saturation at 1000 pM. Rat milk examined at 100-, 400-, and 1600-fold dilutions displayed a response curve parallel to that of the PTHRP-(1-74) standard and contained 2000 pMeq PTHRP-(1-74). Rat Leydig cell tumor and normal rat testis extract were matched for total protein content and assayed at protein concentration of 4830, 483, 48.3, and 4.83 Mg/ml. As with milk, the Leydig cell tumor extract displayed parallelism with the PTHRP-(1-74) standard. Leydig cell tumor extract contained 0.32 pmol/mg protein or 3.1 pmol/gm tumor. In contrast, normal rat testis extract displayed marginal binding only at the highest concentration examined, yielding PTHRP concentrations some 100-fold lower than those observed in the Leydig cell tumor extract. Figure 2a shows the serum calcium concentration as a function of days of tumor growth. The shaded area represents the normal range, as defined by mean ± 2 SD of 44 samples from control rats (10.4 ± 0.8 mg/dl). The tumor-bearing rats achieved statistically significant hypercalcemia on day 9. Figure 2b displays immunoreactive PTHRP-(1-74) concentrations in tumor-bearing rats as a function of days after transplantation. Immunoreactive rat PTHRP was undetectable (
Vol. 127, No. 3 Printed in U.S.A.
Measurement of Circulating Parathyroid HormoneRelated Protein in Rats with Humoral Hypercalcemia of Malignancy using a Two-Site Immunoradiometric Assay* GREGORY GAICH AND WILLIAM J. BURTIS Division of Endocrinology, West Haven Veterans Affairs Medical Center, West Haven, Connecticut 06516; and the Division of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06510
ABSTRACT. The Rice H500 rat Leydig cell tumor is a well characterized model of humoral hypercalcemia of malignancy (HHM). Circulating concentrations of PTH-related protein (PTHRP) have not been reported in this or any other animal model of HHM. Taking advantage of the marked N-terminal amino acid homology between rodent and human PTHRPs, we have adapted a sensitive two-site immunoradiometric assay developed for measurement of human PTHRP for use in measuring rat PTHRP. Circulating calcium and PTHRP concentrations were serially measured after sc passage of the Leydig cell tumor in rats. Significant hypercalcemia and elevation of PTHRP occurred on day 9 after tumor inoculation. When grouped by tumor size, both plasma calcium and PTHRP levels were significantly elevated in animals with tumor burdens greater than 10 cc. The PTHRP concentration was strongly correlated with both serum
H
UMORAL hypercalcemia of malignancy (HHM) is a syndrome most often caused by the production of PTH-related proteins (PTHRPs) by malignant neoplasms (1-3). The genes for rat and human peptides have recently been identified, their structure determined, and cDNA-predicted amino acid sequences synthesized and characterized (4-7). There is remarkable homology throughout a large N-terminal region among cDNApredicted PTHRPs derived from these two species. The Rice H500 Leydig cell tumor in rats is a well characterized animal model of HHM (8, 9). While it seems likely that rat PTHRPs are responsible for this syndrome, methods for measuring circulating PTHRP in rats have not been available. We have taken advantage of the rathuman N-terminal PTHRP homology in adapting a senReceived March 5, 1990. Address all correspondence and requests for reprints to: Gregory Gaich, M.D., c/o William J. Burtis, M.D., Ph.D., Research/151, West Haven VA Medical Center, 950 Campbell Avenue, West Haven, Connecticut 06516. * This work was supported by the Department of Veterans Affairs, West Haven, CT.
calcium (r = 0.88) and tumor size (r = 0.80). Circulating rat PTHRP averaged 12.8 pM on day 9 and 27.5 pM on day 10 or 11. PTHRP was undetectable in the plasma of 19 control rats. In 3 rats, plasma calcium returned to normal, and PTHRP became undetectable within 24 h after tumor excision. Rat milk displayed a PTHRP concentration of 2000 pM, while acid-urea extract of the rat tumor contained 0.32 pmol/mg protein. Dilutions of rat plasma, milk, and tumor extract displayed response curves that were parallel to the human PTHRP-(l-74) standard in the assay. This two-site immunoradiometric assay is a sensitive and easily performed means of measuring rat PTHRP. It should be useful in studying this animal model of HHM and the function of PTHRP in normal tissues. {Endocrinology 127: 1444-1449, 1990)
sitive human PTHRP two-site immunoradiometric assay (IRMA) for use in rat plasma and other tissues.
Materials and Methods Animals Male Fisher 344 rats (Harlan Sprague-Dawley, Indianapolis, IN), ranging in age from 8-12 weeks, were used in all studies involving the H500 Leydig cell tumor. Two lactating female Wistar rats (Harlan Sprague-Dawley) were used for the experiments involving rat milk. All animals were fed standard rodent chow and allowed access to water ad libitum. The Rice H500 Leydig cell tumor was passaged by serial sc transplantation in the flanks of anesthetized animals, as previously described (8), and allowed to grow for 9-14 days. Animals were typically moribund by days 10-14. Since the tumor masses were ovoid in shape, we estimated tumor volume using the formula V = 1/6 7r D2L, where D is the diameter, and L is the length of the tumor. Tumor volume was calculated in a blinded fashion before knowing the results of the plasma calcium or PTHRP measurements. Three of 12 hypercalcemic animals survived complete surgical excision of their tumors and had plasma
1444
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 11:17 For personal use only. No other uses without permission. . All rights reserved.
RAT PTHRP IRMA calcium and PTHRP measurements performed before and 24 h after surgery. Control rats were matched with respect to age and bled simultaneously with the experimental rats. Blood and milk sampling
All plasma samples (1.5 ml) were obtained in iced heparinized tubes containing 50 pi of a protease inhibitor mixture, including aprotinin (500 klU/ml), leupeptin (5 /ig/ml), pepstatin (5 ng/ ml), and EDTA (1 mM). Samples were immediately placed on ice, and the plasma was separated by centrifugation at 4 C and frozen at — 70 C. Plasma calcium measurements were performed by atomic absorption. In the time-course studies, 1.5 ml blood were obtained every 3 days by tail vein bleeds in anesthetized animals. At the end of the experiment, the rats were anesthetized, and the tumors were excised and frozen at —70 C. The rats were then exsanguinated by open cardiac puncture. Rat milk was manually expressed from anesthetized lactating rats (after separation from their pups for 2-8 h) and promptly frozen at -70 C. Tumor and control tissue extraction All extraction procedures were performed on ice, as detailed previously (10). Frozen Leydig cell tumor or normal rat testis was homogenized in a Waring blender in 0.1 M Tris-HCl, pH 7.4 (3 ml/g tissue), then centrifuged at 27,000 x g for 15 min. The resultant pellet was extracted in 8 M urea, 0.2 N HC1, and 0.1 M cysteine for 1 h at 0 C and centrifuged at 27,000 x g for 15 min. The supernatant was dialyzed against deionized water and then lyophilized. Protein was measured using the method of Lowry et al. (11), with BSA as standard. PTHRP-C1-74) IRMA The IRMA has been previously described in detail as it pertains to human plasma (12). The only modification was the use of rat plasma for the standard curve and sample diluent, as described below. Briefly, the two-site IRMA uses affinity-purified rabbit polyclonal anti-PTHRP-(37-74) antibody bound to a solid phase as the "capture" antibody, and iodinated affinity-purified rabbit polyclonal anti-PTHRP-(l-36) antibody as the "signal" antibody. Wells of polyvinyl chloride microtiter plates (Dynatech Laboratories, Chantilly, VA) were filled with 100 n\ anti-PTHRP-(37-74) (1:100) in borate buffer (100 mM boric acid, 25 mM sodium tetraborate, and 75 mM sodium chloride), pH 8.4, and incubated overnight at 4 C. The wells were washed with 0.1% Tween-20 (Sigma Chemical Co., St. Louis, MO) in 10 mM phosphate-buffered 150 mM sodium chloride, pH 7.4 (PBS), followed by three washes with PBS alone, blocked with 300 n\ 1% BSA in borate buffer for 2 h at 25 C, and then washed again as described above. Two hundredmicroliter plasma samples or standards were added to duplicate wells and incubated overnight at 4 C. After washing, 100 n\ radiolabeled anti-PTHRP-(l-36) antibody (-50,000 cpm/well) in PBT buffer (PBS, 0.1% BSA, and 0.1% Triton X-100) with 1% normal rabbit serum were added to each well and incubated overnight at 4 C. After the final wash, the wells were cut from the plate and counted individually. Synthetic human PTHRP(1-74) was employed as the assay standard. In the human
1445
PTHRP IRMA, normal human plasma is the standard and sample diluent. To determine whether normal rat plasma contained measurable PTHRP immunoreactivity, in an initial experiment normal rat plasma was stripped of endogenous Nterminal PTHRP using an anti-PTHRP-(l-36) antibody column, and this PTHRP-stripped plasma was used as diluent for the standard curve and as the assay zero standard. When the assay was thus configured, unstripped normal rat plasma displayed no detectable immunoreactivity. Normal rat plasma was accordingly used as a diluent for the subsequent assays. The detection limit for the assay was 1.4 pM PTHRP-(1-74). The antibodies used in the assay have been previously shown to be highly specific for PTHRP-(1-74), demonstrating no significant cross-reactivity with PTHRP-(l-36), PTHRP-(37-74), PTHRP-(109-138), or human PTH-(l-84) (12). The intra- and interassay variations for the rat averaged 3.4% and 8.9%, respectively. Statistics Statistical analysis was performed using Student's two-tailed t test, and simple regression using the StatView program on an Apple Macintosh computer.
Results Results using the PTHRP-(1-74) IRMA on serial dilutions of rat milk, H500 rat tumor extract, normal rat testis extract, and two representative plasma samples from hypercalcemic rats bearing the H500 tumor are compared with those using the PTHRP-(1-74) standard diluted in normal rat plasma in Fig. 1. The standard curve was linear over the range of 1.4-200 pM and exhibited saturation at 1000 pM. Rat milk examined at 100-, 400-, and 1600-fold dilutions displayed a response curve parallel to that of the PTHRP-(1-74) standard and contained 2000 pMeq PTHRP-(1-74). Rat Leydig cell tumor and normal rat testis extract were matched for total protein content and assayed at protein concentration of 4830, 483, 48.3, and 4.83 Mg/ml. As with milk, the Leydig cell tumor extract displayed parallelism with the PTHRP-(1-74) standard. Leydig cell tumor extract contained 0.32 pmol/mg protein or 3.1 pmol/gm tumor. In contrast, normal rat testis extract displayed marginal binding only at the highest concentration examined, yielding PTHRP concentrations some 100-fold lower than those observed in the Leydig cell tumor extract. Figure 2a shows the serum calcium concentration as a function of days of tumor growth. The shaded area represents the normal range, as defined by mean ± 2 SD of 44 samples from control rats (10.4 ± 0.8 mg/dl). The tumor-bearing rats achieved statistically significant hypercalcemia on day 9. Figure 2b displays immunoreactive PTHRP-(1-74) concentrations in tumor-bearing rats as a function of days after transplantation. Immunoreactive rat PTHRP was undetectable (
