Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI 48109-5633, USA.
Cancer Gene Ther. 2011 Oct;18(10):751-9. doi: 10.1038/cgt.2011.51. Epub 2011 Aug 26.
The herpes simplex virus thymidine kinase (HSV-TK) is the most widely used suicide gene in cancer gene therapy due to its superior anticancer activity with ganciclovir (GCV) compared with other HSV-TK substrates, such as 1-β-D-arabinofuranosyl thymine (araT). We have evaluated the role of DNA damage as a mechanism for the superiority of GCV. Using γ-H2AX foci as an indicator of DNA damage, GCV induced ≥ sevenfold more foci than araT at similar cytotoxic concentrations. The number of foci decreased after removal of either drug, followed by an increase in Rad51 foci indicating that homologous recombination repair (HRR) was used to repair this damage. Notably, only GCV produced a late and persistent increase in γ-H2AX foci demonstrating the induction of unrepairable DNA damage. Both drugs induced the ATR damage response pathway, as evidenced by Chk1 activation. However, GCV resulted in greater activation of ATM, which coincided with the late induction of γ-H2AX foci, demonstrating the presence of DNA double-strand breaks (DSBs). The increase in DSBs after Rad51 induction suggested that they occurred as a result of a failed attempt at HRR. These data demonstrate that the late and unrepairable DSBs observed uniquely with GCV account for its superior cytotoxicity and further suggest that inhibition of HRR will enhance cytotoxicity with HSV-TK/GCV.
单纯疱疹病毒胸苷激酶（HSV-TK）是癌症基因治疗中最广泛使用的自杀基因，因为与其他 HSV-TK 底物（如 1-β-D-阿拉伯呋喃糖胸腺嘧啶（araT））相比，它与更昔洛韦（GCV）具有优越的抗癌活性。我们已经评估了 DNA 损伤作为 GCV 优越性的机制。使用 γ-H2AX 焦点作为 DNA 损伤的指标，与类似细胞毒性浓度的 araT 相比，GCV 诱导的焦点数量增加了≥七倍。在去除任一药物后，焦点数量减少，随后 Rad51 焦点数量增加，表明同源重组修复（HRR）被用来修复这种损伤。值得注意的是，只有 GCV 产生了晚期和持续增加的 γ-H2AX 焦点，表明诱导了不可修复的 DNA 损伤。两种药物都诱导了 ATR 损伤反应途径，这可以通过 Chk1 激活来证明。然而，GCV 导致 ATM 的更大激活，这与晚期诱导的 γ-H2AX 焦点相吻合，表明存在 DNA 双链断裂（DSB）。Rad51 诱导后 DSB 的增加表明它们是 HRR 失败的结果。这些数据表明，仅与 GCV 观察到的晚期和不可修复的 DSB 解释了其优越的细胞毒性，并进一步表明抑制 HRR 将增强 HSV-TK/GCV 的细胞毒性。
