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Bone and Kidney Adenylate Cyclase-stimulating Activity Produced by a Hypercalcemic Canine Adenocarcinoma Line (CAC-8) Maintained in Nude Mice¹

Department of Veterinary Pathobiology, The Ohio State University, Columbus, Ohio 43210

The tumor line, CAC-8, is a serially transplantable adenocarcinoma maintained in nude mice which originated from a hypercalcemic dog. Nude mice with CAC-8 developed a syndrome of humoral hypercalcemia of malignancy. CAC-8 contained a protein factor which stimulated adenylate cyclase of bone and kidney cells in vitro. The adenylate cyclase (AC) of rat osteosarcoma cell lines, ROS 17/2.8 (ROS) and UMR-106, was stimulated by the tumor extract and potentiated by forskolin (0.1 µM). The ROS cells responded to the lowest concentration of CAC-8 extract, but UMR cells responded with a greater increase in AC activity compared to controls following exprosure to CAC-8 extract. Pretreatment of ROS 17/2.8 cells with dexamethasone enhanced the response to CAC-8 extract. The opossum kidney cell line (OK) was less sensitive to the AC-stimulating activity of CAC-8 extract, but AC stimulation was increased in the presence of forskolin. Bovine (1–34) parathyroid hormone (BPTH) (10 nM) stimulated AC equally in ROS, UMR, and OK cells. Isoproterenol (1.0 µM) stimulated AC activity in ROS and UMR cells but not in OK cells. The AC-stimulating activity of CAC-8 appeared to bind to the parathyroid hormone receptor of ROS, UMR, and OK cells since addition of the parathyroid hormone receptor antagonist, [^8,18norleucine, ³⁴tyrosine]BPTH (3–34) amide, inhibited CAC-8-mediated cyclic adenosine 5'-monophosphate production and alone did not stimulate AC activity. The AC-stimulating activity of CAC-8 was acid and heat stable. Trypsin digestion reduced BPTH and CAC-8 stimulation of AC to near basal levels and treatment of CAC-8 extract with dithiothreitol reduced AC stimulation in UMR cells by approximately 50%.

Extracts of the hypercalcemic tumor line (CAC-8) contained bone and kidney AC-stimulating activity which was enhanced by forskolin and dexamethasone, inhibited by [^8,18Nle, ³⁴Tyr]BPTH (3–34) amide, heat stable, trypsin sensitive, inactivated by reduction, and had a relative molecular weight of 34,000 by gel exclusion chromatography. Isolation and characterization of the factor(s) produced by CAC-8 that stimulate AC activity will be useful in further understanding the pathogenesis of humoral hypercalemia of malignancy in animal and human patients.

¹ This work was supported by grants from The Ohio Canine Research Fund (611216) and the Morris Animal Foundation (20322-55-00). Nude mouse facilities were provided by the Comprehensive Cancer Center of The Ohio State University (USPHS Grant CA16058-12). T. J. R. was supported by a Schering-Plough Corporation fellowship and a National Research Service Award (1 F32 CA07818-01A1) from the National Cancer Institute.

² To whom requests for reprints should be addressed, at The Ohio State University, Department of Veterinary Pathobiology, 1925 Coffey Road, Columbus, OH 43210.

Received 6/26/86. Revised 9/15/86. Accepted 10/21/86.