Rat Chondrosarcoma Cells Research Articles

Effect of tunicamycin on insulin binding and on proteoglycan synthesis and distribution in Swarm rat chondrosarcoma cell cultures.

Tunicamycin, an inhibitor of dolichol-diphospho-N-acetylglucosamine formation and hence an inhibitor of N-linked oligosaccharide biosynthesis, suppressed total proteoglycan synthesis by Swarm rat chondrosarcoma chondrocytes without affecting the size of the proteoglycan molecule, its secretion from the cell, or its ability to be retained in the extracellular matrix. In addition, tunicamycin did not substantially alter the ability of the chondrocytes to polymerize glycosaminoglycan onto an exogenous beta-D-xyloside acceptor. A secondary effect of tunicamycin suppression of proteoglycan synthesis was that a lesser amount of newly synthesized proteoglycan diffused from the extracellular matrix into the culture medium. The ability of exogenous hyaluronic acid and proteoglycan to increase the percentage of newly synthesized 35S-proteoglycan in the medium in tunicamycin-treated cultures indicates that matrix retention of 35S-proteoglycan is related to the total extracellular uronic acid content rather than to the presence or absence of mannose oligosaccharides bound to the proteoglycan molecule. These noncytotoxic concentrations of tunicamycin (33-333 ng/ml) decreased [3H]mannose incorporation to the same extent that they decreased total [35S]sulfate and [3H]serine incorporation, and caused the chondrocyte to synthesize and secrete a species of beta-hexosaminidase that was mannose-deficient as assessed by its failure to bind to concanavalin A. The additional finding of decreased insulin binding to tunicamycin-treated chondrosarcoma chondrocytes suggested that the inhibition of proteoglycan synthesis was due to diminution of receptors which respond to stimulatory hormones.

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.

Monensin inhibits synthesis of proteoglycan, but not of hyaluronate, in chondrocytes.

Monensin (10nm-1mum) inhibited the incorporation of [(35)S]sulphate and [(3)H]glucosamine into proteoglycans by rat chondrosarcoma cells, but the incorporation of [(3)H]glucosamine into hyaluronate was unaffected. The results suggest that hyaluronate synthesis occurs in a cell compartment separate from chondroitin sulphate synthesis.

Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes.

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.

The effects of cycloheximide on the biosynthesis and secretion of proteoglycans by chondrocytes in culture.

Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulphate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t 1/2 less than 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t 1/2 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM-cycloheximide, but the incorporation of [35S]sulphate into proteoglycans was only inhibited gradually(t 1/2 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulphated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulphate and chased for up to 6 h in the presence of 1 mM-cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t 1/2 120 min to t 1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion, as well as inhibiting further protein core synthesis. The results suggested that the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulphate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labelled with [35S]sulphate showed an increase with time of both the average proteoglycan size and the length of the constituent chondroitin sulphate chain. However, the proportion of synthesized proteoglycans able to form stable aggregates did not alter.

In Vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation

AbstractIntermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L‐cells ODC. The dose‐response pattern of the IC effect indicated an all‐or‐none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The km of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The Vmax, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non‐competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in 3H‐thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum‐deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G1 phase. Exposure to IC early in G1 (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on 3H‐thymidine incorporation in L‐cells.