Influence of 1,25-Dihydroxyvitamin D3 on Cultured Osteogenic Sarcoma Cells: Correlation with the 1,25-Dihydroxyvitamin D3 Receptor

Abstract We have characterized the effects of the steroid hormone 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] 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) 2 D 3 , 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. Furthermore, the aggregated cuboidal ROS 17/2.8 cells showed a marked change after 6 days of treatment with 10 nm 1,25-(OH) 2 D 3 to an apparently less transformed spindle-like morphology. In contrast, ROS 2/3 displayed only a slight morphological alteration, and ROS 24/1 was unchanged by treatment with 1,25-(OH) 2 D 3 . Anchorage-independent growth studies performed in soft agar indicated that 1,25-(OH) 2 D 3 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) 2 D 3 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 500 binding sites per cell, and no detectable high-affinity 1,25-(OH) 2 D 3 receptor is found in ROS 24/1. The receptor in ROS cells is indistinguishable from other mammalian 1,25-(OH) 2 D 3 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 (K d = 2 to 3 × 10 -11 m). Since the biological responses of these three cell lines to 1,25-(OH) 2 D 3 exhibit a strong correlation with the respective number of receptor molecules per cell, we propose that the actions of this hormone are mediated by the specific 1,25-(OH) 2 D 3 receptor.