Cowell Ian G, Durkacz Barbara W, Tilby Michael J
Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
Biochem Pharmacol. 2005 Dec 19;71(1-2):13-20. doi: 10.1016/j.bcp.2005.09.029. Epub 2005 Nov 15.
DNA-PK and ATM are members of the phosphatidylinositol 3'-kinase like kinase (PIKK) family of serine/threonine protein kinases and have critical roles in the cellular response to DNA double-strand breaks. Genetic loss of either activity leads to pronounced sensitivity to ionizing radiation (IR). Hence, these enzymes are potential targets to confer enhanced radiosensitivity on tumour cells. We show that novel inhibitors of either DNA-PK or ATM sensitize breast carcinoma cells to IR. Radiosensitization was accompanied by an apparent DNA repair deficit as measured by the persistence of IR-induced foci of phosphorylated histone H2AX (gammaH2AX foci). These specific inhibitors also allowed us to probe the biochemistry and kinetics of histone H2AX phosphorylation following gamma-irradiation in breast cancer cells with the aim of validating H2AX as a biomarker for DNA-PK or ATM inhibition in vivo. ATM inhibition reduced the initial average intensity of gammaH2AX foci while inhibition of DNA-PK had only a small effect on the initial phosphorylation of H2AX. However, simultaneous treatment with both compounds dramatically reduced gammaH2AX focus intensity, consistent with the reported role of ATM and DNA-PK in IR induced phosphorylation of H2AX.
DNA依赖蛋白激酶（DNA-PK）和共济失调毛细血管扩张突变蛋白（ATM）是丝氨酸/苏氨酸蛋白激酶的磷脂酰肌醇3'-激酶样激酶（PIKK）家族成员，在细胞对DNA双链断裂的反应中起关键作用。这两种活性的基因缺失都会导致对电离辐射（IR）的显著敏感性。因此，这些酶是使肿瘤细胞放射敏感性增强的潜在靶点。我们发现，DNA-PK或ATM的新型抑制剂可使乳腺癌细胞对IR敏感。放射增敏伴随着明显的DNA修复缺陷，这可通过磷酸化组蛋白H2AX（γH2AX灶）的IR诱导灶的持续存在来衡量。这些特异性抑制剂还使我们能够探究γ射线照射后乳腺癌细胞中组蛋白H2AX磷酸化的生物化学和动力学，目的是验证H2AX作为体内DNA-PK或ATM抑制的生物标志物。ATM抑制降低了γH2AX灶的初始平均强度，而DNA-PK抑制对H2AX的初始磷酸化只有很小的影响。然而，两种化合物同时处理可显著降低γH2AX灶强度，这与ATM和DNA-PK在IR诱导的H2AX磷酸化中的作用报道一致。
