| Abstract: | Earlier studies from this laboratory suggested that embryonic chick bones in organ culture released into the culture medium a specific inhibitor of bone cell proliferation as defined by inhibition of 3H]TdR incorporation into DNA. Dialysis and membrane ultrafiltration experiments suggested that the inhibitory substance (IS) had a molecular weight between 6000 and 14,000. However, subsequent studies on the purification of IS have revealed that the inhibitory activity in bone-conditioned medium is of lower molecular weight and has several properties in common with thymidine (TdR): (1) IS coeluted with 3H]TdR upon gel filtration chromatography on Sephadex G-10. (2) IS bound to charcoal but not to cation or anion exchange resins. (3) Bone-conditioned medium decreased incorporation of 3H]TdR into the free 3H]TdR pool of cells in monolayer culture. (4) Conditioned medium inhibited 3H]TdR incorporation into 3H]thymidine monophosphate in a reaction catalyzed by thymidine kinase. The equivalent concentration of TdR in conditioned medium as estimated by thymidine kinase assay was sufficient to account for the reduction in 3H]TdR incorporation into bone cell DNA. No evidence was found for a specific inhibitor of bone cell proliferation other than TdR. Hence we conclude that the inhibitory effect of IS is due to dilution of 3H]TdR by nonradioactive TdR. Furthermore, media conditioned by several tumor cell lines also contained a low-molecular-weight component which inhibited 3H]TdR incorporation. The results suggest that organ- and cell-conditioned media can contain significant concentrations of TdR which can artifactually inhibit 3H]TdR incorporation in cell proliferation assays. |
