Takada J, Baylink D J, Lau K H
Department of Medicine, Loma Linda University, California, USA.
J Bone Miner Res. 1995 Oct;10(10):1512-22. doi: 10.1002/jbmr.5650101012.
We recently reported that picomolar doses of norethindrone (NET), a synthetic analog of 19-nortestosterone, significantly stimulated human TE85 osteosarcoma cell proliferation, differentiation, and activity in vitro. In the present study, we investigated the possibility that NET interacts with another osteogenic agent, i.e., fluoride, to stimulate human TE85 osteosarcoma cell proliferation, differentiation, and activities. Bone cell proliferation was measured by the stimulation in [3H]thymidine incorporation. Differentiation was monitored by the increase in alkaline phosphatase-specific activity. Osteoblastic activity was assessed by the stimulations in collagen synthesis and in osteocalcin secretion (in the presence of 1 nM 1,25-dihydroxyvitamin D3). When the human TE85 cells were incubated with mitogenic doses of NET and fluoride concurrently, the stimulatory effects of the two agents on these parameters exhibited no significant interaction. The enhancing effect of NET on the osteogenic effect of fluoride was not due to a shift of the fluoride dose response curve. Pretreatment with NET for 24 h followed by a treatment with a mitogenic dose (i.e., 100 microM) of fluoride for an additional 24 h significantly and synergistically potentiated the effects of fluoride on the [3H]thymidine incorporation, alkaline phosphatase-specific activity, collagen synthesis, and osteocalcin secretion, compared with those with the subsequent vehicle (0.05% ethanol) treatments. In contrast, pretreatment with fluoride for 24 h before the addition of NET for 24 h did not produce significant synergistic stimulations in the test parameters. Pretreatment of TE85 cells with the same doses of dihydrotestosterone or progesterone prior to treatment with fluoride under the same conditions did not induce synergistic potentiation of fluoride in [3H]thymidine incorporation, suggesting that the synergistic interaction with fluoride is probably not a common property of anabolic sex steroids. In summary, we found that: (1) the osteogenic effects of fluoride and NET were additive when cells were treated with both agents concurrently; (2) a 24-h pretreatment with picomolar doses of NET potentiated the osteogenic actions of fluoride in human TE85 osteosarcoma cells; and (3) pretreatment with NET produced a subsequent fluoride response that was synergistic. In conclusion, these findings led us to speculate that the osteogenic actions of NET and fluoride act through different mechanisms, and that NET at low doses has a permissive effect on the osteogenic effects of fluoride, and as such NET may be used in concert with fluoride to increase osteoblast proliferation, differentiation, and activity.
我们最近报道，皮摩尔剂量的炔诺酮（NET），一种19-去甲睾酮的合成类似物，在体外能显著刺激人TE85骨肉瘤细胞的增殖、分化及活性。在本研究中，我们探究了NET与另一种成骨剂即氟化物相互作用以刺激人TE85骨肉瘤细胞增殖、分化及活性的可能性。通过[3H]胸腺嘧啶核苷掺入的刺激来测量骨细胞增殖。通过碱性磷酸酶特异性活性的增加来监测分化。在1 nM 1,25-二羟基维生素D3存在的情况下，通过胶原蛋白合成和骨钙素分泌的刺激来评估成骨细胞活性。当人TE85细胞同时用促有丝分裂剂量的NET和氟化物孵育时，这两种试剂对这些参数的刺激作用未表现出显著的相互作用。NET对氟化物成骨作用的增强效应并非由于氟化物剂量反应曲线的偏移。先用NET预处理24小时，随后用促有丝分裂剂量（即100 microM）的氟化物再处理24小时，与随后用赋形剂（0.05%乙醇）处理相比，显著且协同地增强了氟化物对[3H]胸腺嘧啶核苷掺入、碱性磷酸酶特异性活性、胶原蛋白合成和骨钙素分泌的作用。相反，在添加NET之前先用氟化物预处理24小时，然后再用NET处理24小时，在测试参数中未产生显著的协同刺激作用。在相同条件下，在氟化物处理之前先用相同剂量的双氢睾酮或孕酮预处理TE85细胞，在[3H]胸腺嘧啶核苷掺入中未诱导氟化物的协同增强作用，这表明与氟化物的协同相互作用可能不是合成代谢性甾体激素的共同特性。总之，我们发现：（1）当细胞同时用这两种试剂处理时，氟化物和NET的成骨作用是相加的；（2）用皮摩尔剂量的NET预处理24小时可增强氟化物对人TE85骨肉瘤细胞的成骨作用；（3）用NET预处理产生了随后的氟化物反应，该反应是协同的。总之，这些发现使我们推测NET和氟化物的成骨作用通过不同机制起作用，并且低剂量的NET对氟化物的成骨作用具有允许作用，因此NET可与氟化物协同使用以增加成骨细胞的增殖、分化及活性。
