Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.
PLoS One. 2013;8(2):e55989. doi: 10.1371/journal.pone.0055989. Epub 2013 Feb 6.
Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1) phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1)/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1) exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.
基因组不稳定性源于细胞周期检查点和 DNA 损伤反应 (DDR) 的失调，是许多癌症的共同特征。成人 T 细胞白血病 (ATL) 可发生在感染人 T 细胞白血病病毒 1 型 (HTLV-1) 的个体中，ATL 细胞含有广泛的染色体异常，表明它们在识别或修复 DNA 损伤方面存在缺陷。由于 Tax 是 HTLV-1 编码的转化蛋白，我们想知道 Tax 是否可以影响细胞周期检查点和 DDR。我们使用流式细胞术和 DNA 修复测定的组合表明，Tax 表达的细胞在损伤后过早地从 G1 期退出并开始 DNA 复制。在 Tax 表达的细胞中，还观察到 H2AX（γH2AX）和 RPA2 的磷酸化减少，这两种磷酸化蛋白对正确启动 DDR 是必不可少的。为了确定 Tax 表达细胞中 DDR 蛋白磷酸化减少的原因，我们检查了已知可以去磷酸化 γH2AX 的细胞磷酸酶 WIP1。我们发现 Tax 可以在体内和体外与 Wip1 相互作用，并且 Tax 表达的细胞显示出升高的 Wip1 mRNA 水平。体外磷酸酶测定表明，Tax 可以将 Wip1 在 γH2AX 肽靶标上的活性增强 2 倍。因此，体内 γH2AX 的丢失部分可能是由于 Tax 存在时 WIP1 的表达和活性增加。在 Tax 表达的细胞中用 siRNA 敲低 WIP1 可挽救损伤后 γH2AX 的恢复，证实了 WIP1 在 DDR 中的作用。这些研究表明，Tax 可以通过增强 WIP1 的活性来解除 G1/S 检查点，从而导致 DDR 减少。在存在 DNA 损伤的情况下，Tax 表达细胞过早退出 G1 期会产生一种环境，从而容忍将随机突变掺入宿主基因组中。
