Hussain A, Garnett C, Klein M G, Tsai-Wu J J, Schneider M F, Inesi G
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, USA.
J Biol Chem. 1995 May 19;270(20):12140-6. doi: 10.1074/jbc.270.20.12140.
Thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG.
毒胡萝卜素（TG）是细胞内Ca2+转运ATP酶（SERCA）的特异性抑制剂，当以纳摩尔浓度范围添加到培养基中时，它会抑制细胞增殖。然而，长期暴露于逐渐增加浓度的TG会使亲本中国仓鼠肺成纤维细胞DC - 3F及其通过转染全长编码禽SERCA1 ATP酶的cDNA并稳定表达而衍生的亚系（DC - 3F/Ca细胞）对TG抑制产生抗性。TG抗性的产生与选择表达更高水平内源性SERCA2以及外源性转染的SERCA1 ATP酶的细胞同时发生，其Ca2+转运功能可通过成像技术在原位进行研究，并在微粒体组分分离后进行跟踪。从抗性细胞中分离出的微粒体含有两种功能不同的ATP酶群体：一种群体可被与TG进行化学计量滴定所抑制，另一种群体即使TG超过酶化学计量也表现出对抑制的抗性。显然，对TG的抗性与（a）选择表达高水平SERCA ATP酶的细胞以及（b）选择对TG具有抗性的ATP酶同时发展。
