Geley S, Hartmann B L, Hala M, Strasser-Wozak E M, Kapelari K, Kofler R
Institute for General and Experimental Pathology, Division of Molecular Pathophysiology, University of Innsbruck, Medical School, Austria.
Cancer Res. 1996 Nov 1;56(21):5033-8.
The ability of glucocorticoids (GCs) to induce death in lymphoid-origin cells is the basis for their frequent use in the therapy of various human hematological malignancies. However, the occurrence of primary or secondary GC resistance limits their clinical usefulness. Prior investigations into the mechanism of GC resistance in established human leukemic cell lines revealed loss-of-function mutations in the GC receptor (GR) gene. In this study, we analyzed the GC-resistant human acute T-cell leukemia line CEM-C1, which has been reported to express biochemically functional GR and, thus, was thought to owe its GC resistance to signal transduction changes distal from the GR. Radioligand binding assays revealed a 2-3-fold lower expression of GR in CEM-C1 than in the GC-sensitive sister cell line CEM-C7H2. Analysis of transcriptional activity using mouse mammary tumor virus-long terminal repeat-controlled chloramphenicol acetyltransferase expression in transient transfection assays confirmed the expression of functional GR in CEM-C1 but at levels lower than those in CEM-C7H2 cells. Upon molecular analyses of the GR gene and its transcripts, we found that CEM-C1 cells were heterozygous for the ligand binding domain L753F point mutation in exon 9, which is also present in GC-sensitive CEM-C7H2. No mutations, however, were found on the second GR allele of CEM-C1. To test the possibility that resistance in CEM-C1 cells might be caused by insufficient expression of GR, we established several cell lines stably transfected with rat GR expression vectors. These cell lines differed in exogenous GR expression as determined by Northern blotting and radioligand binding assays. The GR expression level in individual lines correlated well with their sensitivity to GC-induced apoptosis. Thus, GC resistance of CEM-C1 cells might be due to subthreshold expression of functional GR rather than defects in signal transduction pathways distal from the GR. Since several clinical investigations showed a correlation between reduced GR expression and poor response to GC-containing treatment, the CEM-C1 line may represent a valid model for GC resistance in human acute T-cell leukemia.
糖皮质激素（GCs）诱导淋巴起源细胞死亡的能力是其在各种人类血液系统恶性肿瘤治疗中频繁使用的基础。然而，原发性或继发性GC抵抗的发生限制了它们的临床应用。先前对已建立的人类白血病细胞系中GC抵抗机制的研究揭示了GC受体（GR）基因的功能丧失突变。在本研究中，我们分析了GC抵抗的人类急性T细胞白血病细胞系CEM-C1，该细胞系已被报道表达具有生化功能的GR，因此被认为其GC抵抗归因于GR远端的信号转导变化。放射性配体结合试验显示，CEM-C1中GR的表达比GC敏感的姐妹细胞系CEM-C7H2低2-3倍。在瞬时转染试验中，使用小鼠乳腺肿瘤病毒-长末端重复序列控制的氯霉素乙酰转移酶表达分析转录活性，证实了CEM-C1中功能性GR的表达，但水平低于CEM-C7H2细胞。对GR基因及其转录本进行分子分析后，我们发现CEM-C1细胞在外显子9的配体结合域L753F点突变上是杂合的，该突变在GC敏感的CEM-C7H2中也存在。然而，在CEM-C1的第二个GR等位基因上未发现突变。为了测试CEM-C1细胞中的抵抗可能是由GR表达不足引起的可能性，我们建立了几个稳定转染大鼠GR表达载体的细胞系。通过Northern印迹和放射性配体结合试验确定，这些细胞系在外源GR表达上存在差异。各个细胞系中的GR表达水平与其对GC诱导凋亡的敏感性密切相关。因此，CEM-C1细胞的GC抵抗可能是由于功能性GR的表达低于阈值，而不是GR远端信号转导途径的缺陷。由于几项临床研究表明GR表达降低与对含GC治疗的反应不佳之间存在相关性，CEM-C1细胞系可能代表人类急性T细胞白血病中GC抵抗的有效模型。
