Division of Oncology, Stanford University School of Medicine, Stanford, CA 94305-5151, United States.
Department of Medicine/Division of Medical Oncology, University of Miami Miller School of Medicine, Miami, FL 33136, United States; Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, FL 33136, United States.
DNA Repair (Amst). 2019 Nov;83:102644. doi: 10.1016/j.dnarep.2019.102644. Epub 2019 Jul 5.
Cellular homeostasis is dependent on a balance between DNA damage and DNA repair mechanisms. Cells are constantly assaulted by both exogenous and endogenous stimuli leading to high levels of reactive oxygen species (ROS) that cause oxidation of the nucleotide dGTP to 8-oxodGTP. If this base is incorporated into DNA and goes unrepaired, it can result in G > T transversions, leading to genomic DNA damage. MutT Homolog 1 (MTH1) is a nucleoside diphosphate X (Nudix) pyrophosphatase that can remove 8-oxodGTP from the nucleotide pool before it is incorporated into DNA by hydrolyzing it into 8-oxodGMP. MTH1 expression has been shown to be elevated in many cancer cells and is thought to be a survival mechanism by which a cancer cell can stave off the effects of high ROS that can result in cell senescence or death. It has recently become a target of interest in cancer because it is thought that inhibiting MTH1 can increase genotoxic damage and cytotoxicity. Determining the role of MTH1 in normal and cancer cells is confounded by an inability to reliably and directly measure its native enzymatic activity. We have used the chimeric ATP-releasing guanine-oxidized (ARGO) probe that combines 8-oxodGTP and ATP to measure MTH1 enzymatic activity in colorectal cancer (CRC), non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) along with patient-matched normal tissue. MTH1 8-oxodGTPase activity is significantly increased in tumors across all three tissue types, indicating that MTH1 is a marker of cancer. MTH1 activity measured by ARGO assay was compared to mRNA and protein expression measured by RT-qPCR and Western blot in the CRC tissue pairs, revealing a positive correlation between ARGO assay and Western blot, but little correlation with RT-qPCR in these samples. The adoption of the ARGO assay will help in establishing the level of MTH1 activity in model systems and in assessing the effects of MTH1 modulation in the treatment of cancer.
细胞内环境稳定依赖于 DNA 损伤与 DNA 修复机制之间的平衡。细胞不断受到内外源刺激的攻击，导致活性氧（ROS）水平升高，从而使核苷酸 dGTP 氧化为 8-氧代-dGTP。如果该碱基被掺入 DNA 且未被修复，它可能导致 G>T 颠换，从而导致基因组 DNA 损伤。MutT 同源物 1（MTH1）是一种核苷二磷酸 X（Nudix）焦磷酸酶，可通过将 8-氧代-dGTP 水解为 8-氧代-dGMP，从而在其掺入 DNA 之前从核苷酸池中去除。MTH1 的表达在许多癌细胞中升高，被认为是癌细胞的一种生存机制，可以避免高 ROS 引起的细胞衰老或死亡的影响。最近，它已成为癌症研究的一个目标，因为据认为抑制 MTH1 可以增加遗传毒性损伤和细胞毒性。由于无法可靠且直接地测量其天然酶活性，因此确定 MTH1 在正常和癌细胞中的作用受到阻碍。我们使用了结合 8-氧代-dGTP 和 ATP 的嵌合 ATP 释放鸟嘌呤氧化（ARGO）探针来测量结直肠癌（CRC）、非小细胞肺癌（NSCLC）和胰腺导管腺癌（PDAC）中的 MTH1 酶活性以及患者匹配的正常组织。在所有三种组织类型的肿瘤中，MTH1 8-氧代-dGTPase 活性均显著升高，表明 MTH1 是癌症的标志物。在 CRC 组织对中，通过 ARGO 测定法测量的 MTH1 活性与通过 RT-qPCR 和 Western blot 测量的 mRNA 和蛋白质表达进行了比较，揭示了 ARGO 测定法与 Western blot 之间存在正相关，但在这些样品中与 RT-qPCR 相关性不大。采用 ARGO 测定法将有助于确定模型系统中 MTH1 活性的水平，并评估 MTH1 调节在癌症治疗中的作用。
