Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan™ assays
Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan™ assays
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DOI：
10.1016/j.ejcb.2009.06.005
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发表时间：
2009-12-01
影响因子：
6.6
通讯作者：
Eriksson, Staffan
中科院分区：
文献类型：
--
作者：
Gasparri, Fabio;Wang, Naining;Eriksson, Staffan
胸苷激酶1（TK 1）是一种DNA补救酶，参与合成S期所需的三磷酸胸苷。尽管TK 1已被用作细胞增殖标记物多年，但没有得到充分表征的抗体。描述了两种类型的多聚和单克隆抗TK 1肽抗体的制备和性质，它们用于测定完整细胞中TK 1的水平。用高容量ArrayScan（TM）荧光显微镜研究了TK 1、c-fos、细胞周期蛋白B1、Ki 67、磷酸化组蛋白H3、磷酸化核糖体蛋白S6的表达以及人正常真皮成纤维细胞培养物中溴脱氧尿苷（BrdU）掺入。血清再加入饥饿细胞后6- 7 h，TK 1蛋白表达水平升高，并随着细胞进入G1期、S期和G2/M期而升高，这早于Ki 67蛋白表达水平的升高，也早于BrdU掺入的检测。因此，可以鉴定具有高TK 1和低Ki 67表达的活化G1细胞群体，并且现在可以阐明它们在细胞增殖中的作用。(C)2009年Elsevier GmbH。All rights reserved.
Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan (TM) fluorescence microscopy. The levels of TK1 increased 6-7h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified. (C) 2009 Elsevier GmbH. All rights reserved.