ASBMR 97 Abstract:

19th Annual Meeting Sept 10th-14th

Cincinnati Convention Centre

Cincinnatti, Ohio

USA

Oral Presentation: Peter Rowe:

CANDIDATE GLYCOPROTEIN(S) FOR PHOSPHATURIA AND ALTERED VITAMIN-D METABOLISM IN TUMOUR OSTEOMALACIA.

Rowe, P.S.N. (Intr. by J.L.H. O'Riordan) University College London, Dept of Med, London W1N 8AA. UK.

Tumour osteomalacia (OHO), is an oncogenic bone disease that results from a renal phosphate leak, hypophosphataemia, and low levels of 1,25 dihydroxy vitamin D (1,25VitD3). Removal of the tumour corrects the defects in bone and renal metabolism. The aim of this study was to identify the tumour-derived circulating factor(s) responsible for mediating the clinical changes.

An intra-cranial tumor from a patient with OHO was obtained together with surrounding sub-dura and dura. The patients phosphate and Vitamin D metabolism became normal after tumour resection. Half of the tumour and tissue samples were frozen in N2(l), and the rest incubated at 37 oC overnight in DMEM - Eagles 10%-FCS / Glutamine / antibiotic - antimycotic, supplemented with collagenase-A 0.2mg/ml. Supernatants were then removed from the flasks, the cell/cellular debris spun down and resuspended in media without serum, and then incubated for a further 8h. After centrifugation, serum free conditioned-media from tumour (TCM), sub-dura (SD-CM), and dura (D-CM), were collected. The cell pellets were then resuspended in media with 10% FCS and the process repeated.

TCM inhibited Na+-dependent phosphate co-transport (NaPI), in a dose dependent manner in a human renal SV40-transformed cell line (CL8). Fractionation of conditioned media (CM), from TCM, SD-CM and D-CM, was carried out using affinity chromatography (concanavalin-A Sepharose), and cation exchange chromatography (SP-Sepharose). A weakly bound concanavalin-A peak was present in all serum free conditioned media. A second peak eluted with a-methyl D-glucopyranoside was present in TCM and to a lesser extent in SD-CM (proportionate with the ability of the CM to inhibit NaPI), and absent in D-CM. The high affinity concanavalin-A peak was further purified by SP-Sepharose cation exchange chromatography, and a single peak was obtained that eluted at 0.37M NaCl in both TCM and SD-CM. SDS polyacrylamide gel electrophoresis (SDS-PAGE), of the purified samples from TCM and SD-CM, revealed two closely running diffuse glycoproteins of approximately 50 to 58 kD.

The single purified peak from conditioned media (serum free), derived from TCM and SD-CM, but not D-CM, is evidence for a candidate tumour-derived phosphaturic factor. This peak resolves into two glycoprotein bands on SDS-PAGE of ~50-58 kD, and may well be a new hormonal agent involved in bone-mineral homeostasis.