Effects of cyclofenil diphenol, an agent which disrupts Golgi structure, on proteoglycan synthesis in chondrocytes.
1. Cyclofenil diphenol (F6060), a weak non-steroidal oestrogen, was shown previously to inhibit [35S]proteoglycan synthesis [Mason, Lineham, Phillipson & Black (1984) Biochem. J. 223, 401-412] and to induce fragmentation of the Golgi apparatus into small vesicles [Lancaster, Fryer, Griffiths & Mason (1989) J. Cell Sci. 92, 271-280] in cultures of Swarm chondrosarcoma chondrocytes. Two structurally related compounds, F6204 and F6091, show a similar concentration-related effect, with complete inhibition of [35S]proteoglycan synthesis at 90 micrograms/ml. The apparent [3H]protein synthesis is only approx. 40% inhibited with [3H]lysine as precursor. Stilboestrol, clomiphene and tamoxiphen are also potent inhibitors of [35S]proteoglycan synthesis. 2. Syntheses of chondroitin 4-[35S]sulphate and chondroitin 6-[35S]sulphate, which are Golgi-mediated events, are inhibited 40-68% and 3-48% respectively by concentrations of cyclofenil between 50 and 70 micrograms/ml. [3H]Hyaluronan synthesis, which occurs by a different mechanism at the plasma membrane, is inhibited by 47-66%. These results suggest that cyclofenil may act via more than one inhibitory mechanism. Cyclofenil diphenol inhibits polymerization of chondroitin sulphate on to p-nitrophenyl beta-xyloside even when the chondrocytes are loaded with the initiator prior to treatment. 3. Cyclofenil diphenol interferes with the cellular uptake of amino acids via the system A carrier, as shown by inhibition of uptake of methylaminoisobutyric acid, a specific substrate for this system. The drug had no effect on the uptake of 2-deoxyglucose by the cells. 4. Cyclofenil diphenol (90 micrograms/ml) caused a decrease in the pool size of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-hexoses, but this was insufficient to account for the accompanying profound inhibition of [35S]proteoglycan synthesis. Entry of [3H]glucosamine into the cell and into the UDP-N-acetylhexosamine pool did not appear to be affected. 5. Cyclofenil diphenol inhibited the substitution of 3H-labelled proteoglycan core protein with chondroitin sulphate chains. Core protein was identified in treated cultures on the basis of immunoprecipitation with an antiserum against the hyaluronate-binding region and distinguished from precipitated proteoglycan on SDS/PAGE.
Mason
R M
RM
Department of Biochemistry, Charing Cross and Westminster Medical School, London, U.K.
Lancaster
C A
CA
D000818:Animals; D002356:Cartilage / Q000187:drug effects* / Q000378:metabolism; D002809:Chondroitin Sulfates / Q000096:biosynthesis; D002813:Chondrosarcoma; D003506:Cyclofenil / Q000031:analogs & derivatives* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D006820:Hyaluronic Acid / Q000096:biosynthesis; D011233:Precipitin Tests; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D014407:Tumor Cells, Cultured
D011509:Proteoglycans; C004382:F 6060; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D003506:Cyclofenil