Tracking cellular stress with labeled FMAU reflects changes in mitochondrial TK2.
Tracking cellular stress with labeled FMAU reflects changes in mitochondrial TK2.
复制标题
使用标记的 FMAU 跟踪细胞应激反映了线粒体 TK2 的变化。
DOI：
10.1007/s00259-008-0738-9
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发表时间：
2008
影响因子：
9.1
通讯作者：
Shields,AnthonyF
中科院分区：
文献类型：
--
作者：
Tehrani,OmidS;Douglas,KirkA;Lawhorn-Crews,JawanaM;Shields,AnthonyF
目的氟嘧啶类化合物1-（2′-脱氧-2 ′-氟-β-d-阿拉伯呋喃糖基）-胸腺嘧啶（FMAU）和3′-脱氧-3 ′-氟胸苷（FLT）在肿瘤中蓄积，被用作正电子发射断层扫描（PET）肿瘤显像的示踪剂。具有高胸苷激酶1（TK 1）活性的再生组织保留FLT;方法在前列腺癌细胞系PC 3、LNCaP、DU 145和乳腺癌细胞系MD-MBA-231中测量[3 H]FLT和[3 H]FMAU的保留，并通过高效液相色谱法（HPLC）分析示踪代谢物。测定细胞中FMAU保留、胸苷激酶2（TK 2）活性和线粒体质量，这些细胞通过耗尽细胞培养基或通过用氧化、还原和能量应激或特异性腺苷一磷酸活化蛋白激酶激活剂或eIF 2抑制剂处理进行应激。TK 1和TK 2的活动和线粒体质量测定FLT磷酸化，1-β-D-阿拉伯呋喃糖基胸腺嘧啶（Ara-T）磷酸化，和流式cytometer.ResultsFMAU保留在快速增殖的癌细胞系是5至10倍低于FLT孵育10分钟后。细胞提取物的HPLC分析表明，磷酸化示踪剂是主要保留的代谢产物。营养胁迫降低TK 1活性和FLT保留，但增加保留FMAU。TK 2抑制降低FMAU保留和磷酸化，对FLT的影响可忽略不计。氧化、还原或能量应激增加FMAU保留并与线粒体质量相关（r2= 0.88，p = 0.006）。FMAU磷酸化与TK 2活性增加相关（r2= 0.87，p = 0.0002）。FMAU可提供治疗效果的早期标志物。
PurposeFluoropyrimidines like 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-thymine (FMAU) and 3′-deoxy-3′-fluorothymidine (FLT) accumulate in tumors and are being used as positron emission tomography tumor-imaging tracers. Proliferating tissues with high thymidine kinase 1 (TK1) activity retain FLT; however, the mechanism of selective accumulation of FMAU in tumors and certain other tissues requires further study.MethodsRetention of [3H]FLT and [3H]FMAU was measured in prostate cancer cell lines PC3, LNCaP, DU145, and the breast cancer cell line MD-MBA-231, and the tracer metabolites were analyzed by high-performance liquid chromatography (HPLC). FMAU retention, thymidine kinase 2 (TK2) activity, and mitochondrial mass were determined in cells stressed by depleted cell culture medium or by treating with oxidative, reductive, and energy stress, or specific adenosine monophosphate-activated protein kinase activator, or eIF2 inhibitor. TK1 and TK2 activities and mitochondrial mass were determined by FLT phosphorylation, 1-β-d-arabinofuranosylthymine (Ara-T) phosphorylation, and flow cytometry, respectively.ResultsFMAU retention in rapidly proliferating cancer cell lines was five to ten times lower than FLT after 10 min incubation. HPLC analysis of the cellular extracts showed that phosphorylated tracers are the main retained metabolites. Nutritional stress decreased TK1 activity and FLT retention but increased retained FMAU. TK2 inhibition decreased FMAU retention and phosphorylation with negligible effects on FLT. Oxidative, reductive, or energy stress increased FMAU retention and correlated with mitochondrial mass (r2= 0.88,p= 0.006). FMAU phosphorylation correlated with increased TK2 activity (r2= 0.87,p= 0.0002).ConclusionFMAU is preferably phosphorylated by TK2 and can track TK2 activity and mitochondrial mass in cellular stress. FMAU may provide an early marker of treatment effects.