Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX, 77054, USA.
School of Health Professions, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Cancer Chemother Pharmacol. 2020 Apr;85(4):661-672. doi: 10.1007/s00280-020-04035-x. Epub 2020 Feb 18.
We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG.
Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines.
The 6-NH derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH, compared to the wild type. Furthermore, CNDAG 6-NH induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase.
CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.
我们假设脱氧鸟苷类似物 CNDAG [9-(2-C-氰基-2-去氧-β-D-阿拉伯呋喃戊糖基)鸟嘌呤] 在掺入 DNA 后可能会导致单链断裂，类似于其胞嘧啶类似物 CNDAC 的作用，并且随后在未修复的缺口处进行 DNA 复制会产生双链断裂。本研究旨在鉴定 CNDAG 前药 2-氨基-9-(2-C-氰基-2-去氧-β-D-阿拉伯呋喃戊糖基)-6-甲氧基嘌呤（6-OME）和 9-(2-C-氰基-2-去氧-β-D-阿拉伯呋喃戊糖基)-2,6-二氨基嘌呤（6-NH）的细胞反应和修复机制。每种化合物都是腺苷脱氨酶的底物，该酶的作用会产生 CNDAG。
使用生长抑制测定、集落形成存活测定、免疫印迹和细胞遗传学分析（染色体畸变和姐妹染色单体交换）来研究 CNDAG 对细胞系的影响。
6-NH 衍生物在 T 细胞恶性细胞系中具有选择性效力。两种前药均导致 ATM 及其下游底物 Chk1、Chk2、SMC1、NBS1 和 H2AX 的磷酸化增加，表明激活了 ATM 依赖性 DNA 损伤反应途径。相比之下，DNA-PKcs 的磷酸化没有增加，后者参与非同源末端连接修复双链断裂。ATM、RAD51D、XRCC3、BRCA2 和 XPF 的缺陷，但不是 DNA-PK 或 p53，使 CNDAG 或前药对细胞集落形成具有显著的敏感性。此外，与野生型相比，缺乏 XPF 的仓鼠细胞在用 CNDAG 6-NH 孵育两个细胞周期后获得了明显更多的染色体畸变。此外，与一个细胞周期相比，在暴露于两个细胞周期后，野生型细胞中 CNDAG 6-NH 诱导更高水平的姐妹染色单体交换，这与第二个 S 期后双链断裂增加一致。
CNDAG 诱导的双链断裂主要通过同源重组修复。
