from Patrick Borgen, M.D., Memorial Sloan-Kettering Cancer. Center (New York, New York, U.S.A.). ..... Nigel J. Bundred, M.D.. University Department of Surgery, ...
World J. Surg. 19, 292-298, 1995
WORLD Journal of
SURGERY 9 1995 by the Soci~t~ Internationale de Chirurgie
Parathyroid Hormone-Related Protein: Primary Osteolytic Factor Produced by Breast Tumor Cells in Vitro? A r n o l d Tabuenca, M.D., 1 S u b b u r a m a n M o h a n , Ph.D., 2' 4, 6 Carlos A. Garberoglio, M.D., 1 Patrick I. Borgen, M.D., 5 T h o m a s Rosol, D.V.M., Ph.D., 7 T h o m a s A. Linkhart, Ph.D. 2' 3, 6 aDepartment of Surgery, Loma Linda University Medical Center, Loma Linda, California 92357, USA ZDepartment of Biochemistry, Loma Linda University Medical Center, Loma Linda, California 92357, USA 3Department of Pediatrics, Loma Linda University Medical Center, Loma Linda, California 92357, USA 4Department of Physiology, Loma Linda University Medical Center, Loma Linda, California 92357, USA 5Department of Surgery, Memorial Sloan-Kettering Cancer Center, 444 East 68 Street, New York, New York 10021, USA 6Research Service (151), Jerry L. Pettis Veterans Hospital, 11201 Benton Street, Loma Linda, California 92357, USA 7Department of Veterinary Pathobiology, Ohio State University, Columbus, Ohio 43210, USA Abstract. Tumor cells in bone metastases are thought to induce bone resorption primarily by releasing paracrine factors. Parathyroid hormone related protein (PTHrp) has been proposed to mediate osteolytic activity of many tumors. PTHrp is produced by 40% to 60% of breast tumors and is elevated in the serum of up to 50% of patients with breast cancer metastases to bone. Most biologic processes in humans are heterogeneous in nature, so the purpose of this study was to investigate the hypothesis that paracrine factors other than PTHrp could mediate bone resorption by breast tumor cells. Serum-free conditioned medium (CM) was collected from five human breast tumor cell lines and tested for bone resorption-stimulating activity (BRSA) in mouse calvaria organ cultures. CM from all tumor cells studied produced significant bone resorption, comparable to that produced by 10 nM PTH. Small amounts of immunoreactive PTHrp (1.4-12.5 pM) were produced by all breast tumor cell lines. When tested in vitro, equivalent amounts of human PTHrp[1-36] did not .produce significant bone resorption. Indomethacin (1 /~M) significantly blocked BRSA by CM from all cell lines but did not decrease BRSA by PTHrp. In contrast PTHrp antibody (130 t~g/ml) completely blocked BRSA by I nM PTHrp but did not modify BRSA by CM of breast tumor cells. The results of this study support the hypothesis that breast cancer cells release paracrine factors in vitro that stimulate bone resorption by a mechanism that is partially dependent on prostaglandin synthesis and at least in part different from that of PTHrp. These results suggest that breast tumor cells may produce osteolytic factors that are distinct from PTHrp and that may constitute a more significant portion of the osteolytic activity produced at sites of metastasis in bone, although additional in vivo studies are needed to test this hypothesis.
Bone metastases are a common cause of morbidity in cancer patients; pathologic fractures impair ambulation, and cord compression with severe neurologic impairment may follow spinal metastases. Debilitating pain seen in many patients with advanced malignancies is usually produced by bone metastases. Breast cancer along with prostate and lung cancer, are accountable for more than 80% of bone metastases. Bone is the first site of distant spread in 26% of patients treated by radical mastectomy [1]; in large series 70% of patients have obvious skeletal metasCorrespondence to: T.A. Linkhart, Ph.D.
tases by the time of death compared with 30% for metastases to either the lung or liver [2]. Although presently incurable, metastatic breast cancer can be controlled for up to 5 years or even 10 years in selected patients; median survival for all patients with breast cancer metastatic disease is approximately 2 to 3 years [3]. These figures emphasize the importance of a better understanding of the mechanisms by which breast tumor cells induce osteolysis. The precise mechanism by which breast tumor cells induce bone resorption is not clear [4]. Tumor cell secretion of parathyroid hormone-related protein (PTHrp) has been implicated in the pathogenesis of hypercalcemia associated with several types of cancer [5-9]. Elevated levels of PTHrp were found in serum of 23% to 50% of hypercalcemic patients with breast cancer [10-13]. These findings along with the immunohistochemical identification of PTHrp in 92% of breast cancer bone metastases [14] supported the hypothesis that PTHrp may stimulate bone resorption by both systemic and local paracrine mechanisms [15, 16]. However, because PTHrp production in breast cancer is inconsistent [17] and breast tumor cells produce several cytokines with osteolytic activity (interleukin-1, transforming growth factor-a, tumor necrosis factor) [18-20], it is possible that factors in addition to PTHrp could mediate bone resorption by breast tumor cells [4, 21, 22]. The purpose of the present study was to determine whether PTHrp is the predominant osteolytic factor produced by human breast tumor cells in vitro. Using a neonatal mouse calvaria organ culture assay, we examined serum-free conditioned media (CM) from a number of human breast tumor cell lines for the secretion of bone resorption-stimulating factors. We measured the amounts of PTHrp secreted by the tumor cells using a specific immunoradiometric assay. To assess whether cytokines other than PTHrp contributed to tumor cell-derived osteolytic activities, we used a specific neutralizing PTHrp antibody and indomethacin (a cyclooxygenase inhibitor) to determine how it would affect the
Tabuenca et al.: Osteolytic Factor in Breast Tumor Cells
induction of bone resorption by PTHrp and CM from the cultured tumor cells. Materials and Methods
Materials Culture plastic ware was purchased from Coming (Corning, New York, U.S.A.); culture media and bovine calf serum from Mediatech Inc. (Herndon, Virginia, U.S.A.) and Hyclone (Logan, Utah, U.S.A.), respectively; human PTHrp [1-36] and synthetic bovine PTH[1-34] from Bachem Corporation (Torrance, California, U.S.A.), 45CAC12(7.4 TBq/mol) from International Chemical and Nuclear (Irvine, California, U.S.A.); indomethacin and bovine serum albumin (BSA) from Fluka (Ronkonkoma, New York, U.S.A.); ultrafiltration (MW 5000 cutoff) membranes from Spectrum (Houston, Texas, U.S.A.); and MCF-7, ZR75-B, T47-D, MDA-MB-468, and MDA-MB-231 human breast tumor cell lines, from Patrick Borgen, M.D., Memorial Sloan-Kettering Cancer Center (New York, New York, U.S.A.). The PTHrp concentration in the CM samples was assessed by two assays. A two-site immunoradiometric assay (IRMA) from Diagnostic Systems (Webster, Texas, U.S.A.) uses PTHrp[1-86] standards. The IRMA is a noncompetitive assay in which the analyte to be measured is "sandwiched" between two antibodies. The first antibody is immobilized to the inside wall of the tubes, and the other antibody is radiolabeled for detection ([~25I]-labeled anti-PTHrp). A two site IRMA kit (Allegro PTHrp kit) was obtained from Nichols Institute Diagnostics (San Juan Capistrano, California, U.S.A.); this assay uses PTHrp[I-86] standards and has two polyclonal antibodies to synthetic human PTHrp: One antibody binds only the amino acid sequence 60-72 and is coupled to biotin; the other recognizes the N-terminal 1-40 and is radiolabeled for detection. Chicken polyclonal anti-human PTHrp antibody was prepared against a synthetic peptide corresponding to the first 36 Nterminal amino acids of PTHrp by immunizing laying hens; polyclonal IgG to PTHrp was purified from the egg yolks with high levels of PTHrp specific binding [23].
Collection and Preparation of Breast Tumor Cell CM Breast tumor cells were grown to near confluence in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum. At this point the medium was changed to serum-free DMEM with BSA 0.2 mg/ml. After 24 hours this medium was discarded and replaced with fresh DMEM + BSA. After 24 hours of incubation the CM was collected and acetic acid added to a concentration of 1 M. The CM was concentrated and dialyzed with 1 M acetic acid using an Amicon stirred cell with a 5000-dalton molecular weight cutoff low-protein-binding membrane; it was then lyophilized and reconstituted in 0.02 M acetic acid at 50• the original volume. CM batches were stored at -70~
Assays for Osteolytic Activity Release of 45Ca from prelabeled mouse calvaria was assessed as previously described [24] to determine bone resorption-stimulating activity (BRSA). Newborn mice were injected subcutaneously with 2/xCi 45Ca and euthanized after 3 days. Calvariae (frontal
293
and parietal bone) were dissected and precultured for 24 hours in DMEM with penicillin (100 units/ml), streptomycin (100 txg/ml), and BSA (1 mg/ml). Calvaria were then cut in half and randomly placed in 24 well plates with 1.5 ml of medium per well. The factors were then added, including CM from different breast tumor cell lines (six wells per group), at a final concentration of IX (0.03 ml of the 50• concentration). When testing the effect of PTHrp blocking antibody, CM samples and human PTHrp[I-40] were preincubated with the antibody for 1.5 hours at 37~ A solvent control group was treated with acetic acid at the same final concentration as in l x CM. Human PTHrp[l-36] at 1 to 10 nM or 10 nM synthetic bovine PTH[1-34] was used as a positive control in each experiment, Calvariae were incubated for 3 days, after which the medium was collected, and factors were added again. The experiment was stopped on day 6 and the amount of 45Ca remaining in each calvaria was determined after disolving the calvaria in formic acid. The 45Ca released was normalized to the initial 45Ca content of each calvaria (amount released + amount remaining) and, in some experiments, expressed as a percentage of 45Ca released from untreated control cultures during the same collection period. Treated groups were compared to the control by the Student t-test for unpaired variates and one-way A N O V A with Fisher LSD multiple comparison test. The PTHrp concentrations in the 50x CM samples were assessed by three assays and normalized to 1• concentration. A two-site IRMA from Diagnostic Systems Laboratory was used in which the lowest detectable level of PTHrp is 0.3 pM at the 95% confidence limit. A second two-site IRMA kit from Nichols Institute Diagnostics had a sensitivity of 0.3 pM at the 95% confidence limit. Additional standards were obtained, and various amounts were added to different CM samples to determine the recovery. PTHrp levels measured by an N-terminal PTHrp radioimmunoassay (RIA) from Incstar (Stillwater, Minnesota, U.S.A.) were compared to the other two assays; with this method, human PTHrp[1-40] was used to determine the recovery. Results
The CM was collected from five human breast tumor cell lines: three estrogen receptor-positive lines (MCF-7, ZR75-B, T47-D) and two estrogen receptor-negative lines (MDA-MB-231, MDAMB-468). In a previous study CM from all five cell lines studied induced significant BRSA in a dose-dependent manner [25]. Representative results from the highest concentration tested (equivalent to 1• are shown in Figure 1. Similar results were obtained when resorption was assessed by an increase in 4~ concentration in the medium. The PTHrp concentrations in CM from four cell lines were assessed by two assays. CM from each cell line was analyzed for PTHrp by two two-site IRMAs. Readings from all samples were in the linear region of the standard curve. Small amounts of PTHrp (1.4-12.5 pM) were present in CM from all cell lines (Table 1). Recovery of PTHrp standard when added to CM samples was 100% or more. The quantitative difference between the two kits appear to be due to the difference in the standards used. The correlation between the two assays was 0.98 with a two-tailed significance o f p = 0.003. To determine whether equivalent concentrations of PTHrp were effective in stimulating bone resorption, synthetic human
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World J. Surg. Vol. 19, No. 2, Mar./Apr. 1995
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