Sun Yi, Huang Yue-Chen, Xu Qin-Zhi, Wang Hui-Ping, Bai Bei, Sui Jian-Li, Zhou Ping-Kun
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing, People's Republic of China.
Int J Radiat Oncol Biol Phys. 2006 Jul 1;65(3):842-50. doi: 10.1016/j.ijrobp.2006.02.040.
There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation.
Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and gamma-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry.
The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and gammaH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy gamma-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G(2)/M arrest as compared with parental cells. In addition, the G(2)/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein.
HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS cancer patients to radiotherapy.
越来越多的证据表明，患有人类免疫缺陷病毒1型/获得性免疫缺陷综合征（HIV-1/AIDS）的癌症患者在接受放射治疗时会出现更严重的组织反应，且常发生皮肤毒性作用。在此，我们探讨了HIV-1 Tat蛋白对细胞对电离辐射反应的影响。
通过用HIV-1 tat基因转染，从人横纹肌肉瘤细胞中获得了两种表达Tat的细胞系，TT2和TE671-Tat。使用集落形成能力测定放射敏感性。通过cDNA微阵列和免疫杂交评估基因表达。采用彗星试验和γ-H2AX焦点检测来检测DNA双链断裂（DSB）及其修复情况。通过流式细胞术检测辐射诱导的细胞周期变化。
与亲本TE671细胞或对照TE671-pCI细胞相比，TT2和TE671-Tat细胞的放射敏感性显著增加。Tat还增加了增殖活性。彗星试验和γH2AX焦点检测显示，表达Tat的细胞修复辐射诱导的DNA DSB的能力下降。微阵列分析表明，DNA修复基因DNA-PKcs以及细胞周期相关基因Cdc20、Cdc25C、KIF2C和CTS1在表达Tat的细胞中下调。RT-PCR和免疫杂交分析进一步证实了表达Tat细胞中DNA-PKcs的抑制。与亲本细胞相比，表达Tat的细胞在4 Gy γ射线照射后表现出延长的S期停滞，并且在辐射诱导的G(2)/M停滞的起始和消除方面有明显延迟。此外，TT2细胞中的G(2)/M停滞不完全。而且，HIV-1 Tat导致细胞周期蛋白B1蛋白的组成性过表达。
HIV-1 Tat蛋白通过抑制DNA修复和失调细胞周期检查点使细胞对电离辐射敏感。这些观察结果为AIDS癌症患者对放射治疗的组织反应增加提供了新的见解。
