Findlay D M, Raggatt L J, Bouralexis S, Hay S, Atkins G J, Evdokiou A
Department of Orthopaedics and Trauma, University of Adelaide, The Royal Adelaide Hospital, Adelaide 5000, South Australia, Australia.
J Endocrinol. 2002 Dec;175(3):715-25. doi: 10.1677/joe.0.1750715.
We recently reported that calcitonin (CT) can profoundly inhibit the growth of HEK-293 cells transfected with the human calcitonin receptor (hCTR). We also obtained preliminary evidence that suggested a role for CT in cell survival, and in the present study we have investigated the pro-apoptotic action of CT, which we observe in conditions of low serum concentration. Under these conditions, we have found that CT treatment of HEK-293 cells stably transfected with the insert-negative form of the human CTR (HR12 cells) caused a time-dependent decrease in cell number associated with loss of cellular attachment. Loss of cellular adherence in CT-treated cultures caused programmed cell death, as shown by Annexin V staining of cells, failure of cells to exclude Trypan Blue dye, condensation and cleavage of nuclear DNA, and appearance of hypodiploid cells in fluorescence-activated cell sorting (FACS) analysis. The accumulation of non-adherent cells and cell death was concomitant with increased intracellular activity of caspase-3. However, inhibition of caspase activation in HR12 cells did not prevent CT-mediated loss of attachment and did not maintain the viability of non-adherent cells, indicating that caspase activation accompanied, but was probably not the cause of, the loss of cell viability. Neither the effects of CT on cell survival nor the activation of caspase-3 were observed in serum-replete conditions, suggesting that serum-derived factors provide protection of cells from CT-induced apoptosis. The inhibitory effects of CT on cell growth were found previously to be related to activation of Erk1/2 MAP kinase. In the present experiments, it was found that the Erk1/2 inhibitor, PD 98059, inhibited the CT-induced loss of cellular adherence and the consequent reduction in cell numbers. These results demonstrate that CT can negatively affect cell survival and they identify roles for cell adherence and MAP kinase activation in this process.
我们最近报道，降钙素（CT）可显著抑制转染了人降钙素受体（hCTR）的HEK - 293细胞的生长。我们还获得了初步证据，提示CT在细胞存活中发挥作用，在本研究中，我们研究了CT在低血清浓度条件下所观察到的促凋亡作用。在这些条件下，我们发现用CT处理稳定转染人CTR插入阴性形式的HEK - 293细胞（HR12细胞）会导致细胞数量随时间减少，并伴有细胞贴壁丧失。CT处理的培养物中细胞贴壁丧失导致程序性细胞死亡，这表现为细胞的膜联蛋白V染色、细胞不能排斥台盼蓝染料、核DNA凝聚和裂解以及荧光激活细胞分选（FACS）分析中出现亚二倍体细胞。非贴壁细胞的积累和细胞死亡与细胞内caspase - 3活性增加同时发生。然而，抑制HR12细胞中的caspase激活并不能阻止CT介导的贴壁丧失，也不能维持非贴壁细胞的活力，这表明caspase激活伴随但可能不是细胞活力丧失的原因。在血清充足的条件下未观察到CT对细胞存活的影响以及caspase - 3的激活，这表明血清来源的因子可保护细胞免受CT诱导的凋亡。先前发现CT对细胞生长的抑制作用与Erk1/2丝裂原活化蛋白激酶的激活有关。在本实验中，发现Erk1/2抑制剂PD 98059可抑制CT诱导的细胞贴壁丧失以及随之而来的细胞数量减少。这些结果表明CT可对细胞存活产生负面影响，并确定了细胞贴壁和丝裂原活化蛋白激酶激活在此过程中的作用。
