Rishi A K, Yu M, Tsai-Wu J J, Belani C P, Fontana J A, Baker D L, Periasamy M, Hussain A
Division of Oncology, Department of Medicine, Greenebaum Cancer Center, University of Maryland and Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA. Cincinnati, O.
Nucleic Acids Res. 1998 Oct 1;26(19):4529-37. doi: 10.1093/nar/26.19.4529.
We have selected a series of cell lines from the parental Syrian hamster smooth muscle cell line DDT1-MF2that are resistant to thapsigargin (TG), a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+transport ATPases (SERCAs). Cells were selected for resistance to TG in the presence or absence of cyclosporin (CSA), which is a competitive inhibitor of the multidrug transporter p-glycoprotein (pgp). Since TG is a known substrate for pgp, selection for TG resistance was carried out in the presence of CSA in an attempt to minimize the contribution of pgp, and to identify the potential range of adaptive responses of the SERCA pump itself, during the development of the TG-resistant phenotype. Irrespective of whether the selection is carried out in the presence or absence of CSA, pgp is overexpressed in the TG-resistant DDT1-MF2cells. SERCA protein is also overproduced in the TG-resistant cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcription of the gene. Enhanced transcription is observed only upon long-term selection and occurs through the SERCA gene proximal promoter elements. Although SERCA transcription in wild-type cells is dependent upon the -284 to -72 bp region of the SERCA promoter, the TG-resistant cells utilize both the -284 to -72 bp and the -72 to +80 bp promoter regions for enhanced SERCA transcription. That is, additional elements within the -72 to +80 bp region are recruited in the TG-resistant cells to allow for increased SERCA expression. A post-transcriptional step may also be recruited by the TG-resistant cells in their overall strategy to produce increased amounts of the SERCA protein. These studies demonstrate that the DDT1-MF2cells can utilize different mechanisms which lead to increased levels of SERCA protein as the cells adapt to inhibition of the ATPase by TG.
我们从叙利亚仓鼠平滑肌细胞系DDT1-MF2亲本中筛选出了一系列对毒胡萝卜素（TG）具有抗性的细胞系，毒胡萝卜素是肌浆网/内质网Ca2+转运ATP酶（SERCAs）的特异性抑制剂。在存在或不存在环孢菌素（CSA）的情况下筛选细胞对TG的抗性，环孢菌素是多药转运蛋白p-糖蛋白（pgp）的竞争性抑制剂。由于TG是已知的pgp底物，因此在CSA存在的情况下进行TG抗性筛选，以尽量减少pgp的作用，并确定在TG抗性表型形成过程中SERCA泵自身潜在的适应性反应范围。无论筛选是在CSA存在还是不存在的情况下进行，pgp在对TG具有抗性的DDT1-MF2细胞中均过度表达。SERCA蛋白在TG抗性细胞系中也过量产生，这是通过几种机制之一发生的。其中包括SERCA基因的扩增和基因转录增强。仅在长期筛选后才观察到转录增强，并且是通过SERCA基因近端启动子元件发生的。虽然野生型细胞中的SERCA转录依赖于SERCA启动子的-284至-72 bp区域，但TG抗性细胞利用-284至-
