Separation of parathyroid hormone-like activity from transforming growth factor-alpha and -beta in the canine adenocarcinoma (CAC-8) model of humoral hypercalcemia of malignancy.
A canine adenocarcinoma model (CAC-8) of humoral hypercalcemia of malignancy was evaluated for transforming growth factors (TGF)-alpha and -beta, PTH-like activity [adenylate cyclase-stimulating activity (ACSA)], and in vitro bone-resorbing activity. Biological activities present in CAC-8 were separated by reverse phase or cation exchange HPLC. TGF alpha in tumor extract was separated from TGF beta and ACSA by reverse phase HPLC. TGF alpha eluted between 26-30% acetonitrile and was identified by RIA. After the initial reverse phase separation, TGF beta and ACSA in tumor extract coeluted between 36-38% acetonitrile. Sequential cation exchange followed by reverse phase HPLC separated TGF beta from ACSA. Evaluation of fractions containing ACSA using an in vitro bone-resorbing assay demonstrated copurification of ACSA and bone-resorbing activity. The PTH receptor antagonist [Nle8,18,Tyr34]bovine PTH-(3-34)-amide, but not [Nle8,18,Tyr34]bovine PTH-(7-34)-amide, completely inhibited ACSA in column eluates. Conditioned cell culture medium from CAC-8 primary cultures contained predominantly latent TGF beta that could be activated by acidification. These findings indicate that the CAC-8 model of cancer-associated hypercalcemia produces a PTH-like factor, TGF alpha, and TGF beta that were separable by reverse phase or cation exchange HPLC. This feature should be useful to investigate the role of TGFs and PTH-like proteins in the pathogenesis of humoral hypercalcemia of malignancy.
Merryman
J I
JI
Department of Veterinary Pathobiology, Ohio State University, Columbus 43210.
Rosol
T J
TJ
Brooks
C L
CL
Capen
C C
CC
D000230:Adenocarcinoma / Q000150:complications*; D000818:Animals; D001862:Bone Resorption; D003470:Culture Media; D004285:Dogs; D006934:Hypercalcemia / Q000209:etiology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism*; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000502:physiology; D018016:Receptors, Parathyroid Hormone; D015291:Transforming Growth Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D003470:Culture Media; D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; C053371:parathyroid hormone-like adenylate cyclase-stimulating protein, human; D015291:Transforming Growth Factors