INFLUENCE OF 1,25-DIHYDROXYVITAMIN-D3 ON CULTURED OSTEOGENIC-SARCOMA CELLS - CORRELATION WITH THE 1,25-DIHYDROXYVITAMIN-D3 RECEPTOR

Abstract

The effects of the steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] were characterized on a series of rat osteogenic sarcoma cell lines of increasing osteoblastic-like nature (ROS 24/1, ROS-2/3 and ROS-17/2.8). When these cells were grown in monolayer culture in the presence of 10 nM, 1,25-(OH)2D3, there was a dramatic and selective inhibition of proliferation in the ROS 17/2.8 line. Similar concentrations of other vitamin D metabolites did not elicit this effect. The aggregated cuboidal ROS 17/2.8 cells showed a marked change after 6 days of treatment with 10 nM 1,25-(OH)2D3 to an apparently less transformed spindle-like morphology. ROS-2/3 displayed only a slight morphological alteration, and ROS-24/1 was unchanged by treatment with 1,25-(OH)2D3. Anchorage-independent growth studies performed in soft agar indicated that 1,25-(OH)2D3 inhibited colony formation to the greatest degree in ROS-17/2.8, with a lesser effect in ROS-2/3. Based upon analyses by sucrose gradient centrifugation, DNA cellulose chromatography and saturation of specific binding, the level of the 1,25-(OH)2D3 receptor was quantitated in these cells. ROS-17/2.8 cells possess 18,000 copies of the receptor per cell, while ROS-2/3 contains only 5000 binding sites per cell, and no detectable high-affinity 1,25(OH)2D3 receptor is found in ROS-24/1. The receptor in ROS cells is indistinguishable from other mammalian 1,25-(OH)2D3 receptors in that it is a DNA-binding protein that sediments on sucrose gradients at 3.3S and specifically binds the hormone with high affinity (Kd = 2 to 3 .times. 10-11 M). Since the biological responses of these 3 cell lines to 1,25-(OH)2D3 exhibit a strong correaltion with the respective number of receptor molecules per cell, it was proposed that the actions of this hormone are mediated by the specific 1,25-(OH)2D3 receptor.