Medh R D, Saeed M F, Johnson B H, Thompson E B
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch at Galveston, 77555-0645, USA.
Cancer Res. 1998 Aug 15;58(16):3684-93.
Glucocorticoids (GCs) induce apoptosis in lymphoid cells that contain functional GC receptors (GRs). However, GC resistance often is seen in cells with demonstrable GRs; one such line is CEM-C1. We have tested the hypothesis that positive interactions between GC and cyclic AMP (cAMP) regulate GC actions in CEM clones. Treatment of both GC-resistant CEM-C1 [resistant to 1 microM dexamethasone (Dex)] and the sensitive sister clone, CEM-C7 (approximately 65% cell death with 20 nM Dex, approximately 99% death with 1 microM Dex), with a < or = 20 microM concentration of the protein kinase A activator, forskolin, had no significant effect on cell viability. Cotreatment with Dex and forskolin resulted in a strong synergistic death response, with only approximately 10% CEM-C1 cells surviving treatment with 1 microM Dex and 20 microM forskolin. This death was blocked by the GR antagonist RU 38486. However, the extent of apoptosis did not correlate with the amount of GR protein or binding activity in either C7 or C1 cells. As reported previously, Dex-evoked cell death was associated with suppression of c-Myc in C7 cells. In CEM-C1 cells, Dex alone did not affect c-Myc; however, Dex plus forskolin suppressed c-Myc levels. To evaluate mechanisms of Dex-forskolin synergism, fresh subclones of CEM-C7 (clone 14) and CEM-C1 (clone 15) were isolated, to ensure purity of phenotype. In these, forskolin (with or without Dex) caused a similar increase in cAMP (approximately 300-fold) and phospho-cAMP-responsive element binding protein (approximately 4-5-fold) levels, whereas total cAMP-responsive element binding protein expression was not affected. GR transcription function, as tested from a GR-responsive 330-bp mouse mammary tumor virus promoter-luciferase reporter construct, was induced 8- and 4-fold by 1 microM Dex treatment of CEM-C7-14 and CEM-C1-15 cells, respectively. Forskolin (10 microM) significantly potentiated Dex response in CEM-C1-15 cells (13.5-fold) but had only a modest effect (1.5-fold) in CEM-C7-14 cells. These studies suggest that sensitization of CEM-C1 cells by cross-talk between GR and protein kinase A pathways may occur via cooperative effects on GR-mediated gene transcription.
糖皮质激素（GCs）可诱导含有功能性糖皮质激素受体（GRs）的淋巴细胞凋亡。然而，在具有可证实的GRs的细胞中常常可见GC抵抗现象；CEM-C1细胞系就是这样一种细胞系。我们检验了如下假说：GC与环磷酸腺苷（cAMP）之间的正向相互作用调节CEM克隆中的GC作用。用浓度≤20 μM的蛋白激酶A激活剂福斯可林处理GC抵抗的CEM-C1细胞系（对1 μM地塞米松有抵抗）及其敏感的姐妹克隆CEM-C7细胞系（20 nM地塞米松处理时约65%的细胞死亡，1 μM地塞米松处理时约99%的细胞死亡），对细胞活力均无显著影响。地塞米松与福斯可林联合处理导致强烈的协同死亡反应，用1 μM地塞米松和20 μM福斯可林处理后，只有约10%的CEM-C1细胞存活。这种死亡可被GR拮抗剂RU 38486阻断。然而，凋亡程度与C7或C1细胞中GR蛋白的量或结合活性均不相关。如先前报道，地塞米松诱发的细胞死亡与C7细胞中c-Myc的抑制有关。在CEM-C1细胞中，单独使用地塞米松不影响c-Myc；然而，地塞米松加福斯可林可抑制c-Myc水平。为评估地塞米松-福斯可林协同作用的机制，分离了CEM-C7（克隆14）和CEM-C1（克隆15）的新鲜亚克隆，以确保表型的纯度。在这些细胞中，福斯可林（无论有无地塞米松）使cAMP水平升高程度相似（约300倍），使磷酸化的cAMP反应元件结合蛋白水平升高程度相似（约4至5倍），而总的cAMP反应元件结合蛋白表达不受影响。用GR反应性的330 bp小鼠乳腺肿瘤病毒启动子-荧光素酶报告构建体检测GR转录功能，1 μM地塞米松处理CEM-C7-14和CEM-C1-15细胞后，GR转录功能分别诱导增强8倍和4倍。福斯可林（10 μM）显著增强CEM-C1-15细胞中地塞米松的反应（13.5倍），但对CEM-C7-14细胞只有适度影响（1.5倍）。这些研究提示，GR与蛋白激酶A途径之间的相互作用可能通过对GR介导的基因转录的协同作用使CEM-C1细胞致敏。
