HeLa细胞中hR24L基因的表达、DNA损伤修复、G2／M阻滞与电离辐射之间的关系（简报）

The hR24L gene ORF was cloned from the total RNA of HeLa cells by RT-PCR method. There is no mutation of the hR24L gene in HeLa cells. Ion radiation significantly increased the expression of hR24L gene, and the sense hR24L enhanced and accelerated the cell cycle arrest at G2/M phase in HeLa cells. Besides, it should seem that the overexpression of hR24L gene could enhance the repair ability of DNA damage induced by ion radiation, and vice versa.     

TAB182对电离辐射诱发细胞周期G2/M阻滞的调节作用

TAB182是一个端锚聚合酶1（tankyrase 1）结合蛋白,它在体外能够被tankyrase 1发生二磷酸腺苷核糖基化(PAR)修饰,其生物学功能目前尚不明确.本研究发现,TAB182蛋白水平受电离辐射诱导表达,HeLa细胞经过4 Gy照射处理时,TAB182在2 h表达含量最高; 经过不同剂量照射处理,2 h后2 Gy、4 Gy照射剂量组HeLa细胞中TAB182的表达有明显增加. 通过shRNA沉默HeLa细胞中TAB182基因表达,导致其对4 Gy及以下剂量 辐射的敏感性增加,但对8 Gy大剂量照射的敏感性没有明显变化. 与对照组相比,4 Gy照射诱发TAB182基因沉默细胞的G2/M期阻滞时间显著延长.抑制TAB182表达导致细胞中DNA损伤反应蛋白DNA PKcs、ATM、Chk2的表达水平显著降低. 实验结果提示,TAB182蛋白参与放射DNA损伤信号反应和调控细胞周期G₂/M进程.     

塞来昔布诱导HCT-116结肠癌细胞G2/M阻滞

目的:研究选择性COX-2抑制剂塞来昔布诱导结肠癌细胞株HCT-116细胞周期阻滞的作用及其可能的机制。方法:应用流式细胞仪检测塞来昔布对HCT-116细胞周期的影响,定量PCR检测细胞周期素cyclinB1及COX-2 mRNA表达水平,Western-Blot检测细胞周期素cyclinB1的蛋白水平。结果:塞来昔布诱导HCT-116细胞G2/M阻滞的作用呈剂量依赖性,塞来昔布在mRNA及蛋白水平下调HCT-116细胞的cyclinB1。结论:塞来昔布能在体外抑制HCT-116细胞的增殖,诱导G2/M的阻滞,其作用与下调细胞周期素cyclinB1有关。     

与细胞周期G2/M期进程相关的H2AX磷酸化

γH2AX焦点(foci)被普遍当做DNA双链断裂（DSB）损伤的分子标志物.为探 讨细胞周期进程相关的H2AX磷酸化规律特征,采用胸腺嘧啶双阻滞结合噻氨酯哒唑(nocodazole)的后续处理,将HeLa细胞同步于有丝分裂的前中期．然后,用流式细胞仪检测细胞周期、Western印迹和免疫荧光法,观察γH2AX表达和γH2AX焦点的形成．结果显示,细胞进入G2/M期和有丝分裂过程中,γH2AX水平显著增加 ;在无DNA DSB发生的情况下,部分M期细胞中也存在大量的γH2AX焦点．随着细 胞完成有丝分裂从M期退出再进入G1期,γH2AX的表达水平逐渐降低．这种 γH2AX表达变化特征与G2/M期密切关联的PLK1和Cyclin B1的表达规律相类似． 在4 Gy大剂量照射下,HeLa细胞于照后8 到12 h出现明显的G2/M期阻滞．γH2AX 焦点数在照后1 h达高峰,随后降低,照后8 h又上升,出现了第2个峰值．与之不同的是,在1 Gy低剂量照射下,细胞的G2/M期阻滞微弱,γH2AX焦点数在照后 0.5 h最高,随后下降,且无反弹,符合DNA DSB的修复动力学特征．因此,将γ H2AX当做DNA DSB分子标志物时,还需要考虑细胞周期变化的影响．γH2AX适合 作为1 Gy以下照射的DNA双链断裂损伤的分子标志．     

Gö6976转变8-氯-腺苷引起的G2/M期阻滞为S期阻滞并促进K562细胞凋亡

8-氯-腺苷可抑制多种人类肿瘤细胞生长．8-氯-腺苷可引起细胞有丝分裂异常、G2/M 期阻滞和晚期凋亡．为探索增强8-氯-腺苷的抗肿瘤作用,本研究以人慢性髓性白血病细胞株K562为靶细胞,联合使用Chk1抑制剂Gö6976与8-氯-腺苷,观察Gö6976处理后肿瘤细胞对8-氯-腺苷的增敏效果,探索其作用机制．流式细胞分析发现,Gö6976 可消除8-氯-腺苷引起的K562细胞G₂/M期阻滞,使转换为S期阻滞．蛋白质印迹及免疫共沉淀实验显示,Gö6976可灭活Chk1,激活Chk2,使Chk1-Cdc25C-CDK1级联反应转换为Chk2-Cdc25A-CDK2级联反应,从而引起细胞周期阻滞发生改变．蛋白质印迹实验证明,Gö6976 可明显增强8-氯-腺苷作用引起的凋亡相关分子procasepase-3和PARP的激活;流式细胞分析显示,Gö6976促进8-氯-腺苷引起的细胞凋亡．研究结果提示,Gö6976增强了靶细胞对8-氯-腺苷的敏感性,通过转换8-氯-腺苷引起的G₂/M期阻滞为S期阻滞,促进细胞凋亡．     

DADS诱导HL-60 G2/M期阻滞消减文库的构建与初步筛选

目的:构建DADS诱导HL60-细胞G2/M期阻滞的差异表达文库,初步筛选相关基因.方法:分别提取无DADS和有DADS处理HL-60细胞的总RNA和mRNA,构建消减cDNA文库.随即挑选正向SSH的阳性克隆,PCR检测插入片段,将含插入片段的克隆测序.Blastn分析差异cDNA片段的同源性.结果:构建了DADS诱导人白血病HL-60细胞G2/M期阻滞差异表达文库,其中包含120个正向SSH的克隆和100个反向SSH的克隆.50个随机正向SSH的克隆测序、比较同源性,发现5个新EST片段,已经在GenBank中登录.结论:所构建的DADS诱导人白血病HL-60细胞G2/M期阻滞消减文库为进一步筛选白血病HL-60细胞G2/M期阻滞相关基因奠定了基础.     

八氯腺苷诱导SH-SY5Y和SK-N-SH细胞G2/M期阻滞的分子机制不同

为探索八氯腺苷的抗肿瘤作用机制,以神经母细胞瘤SH-SY5Y和SK-N-SH细胞为对象,采用四唑盐比色实验（MTT法）证明,八氯腺苷具有明显的抑制肿瘤细胞增殖的作用,这种抑制作用呈剂量-时间依赖性.流式细胞分析显示,10 μmol/L八氯腺苷作用48 h后可导致靶细胞生长停滞于G ^₂/M期;SH-SY5Y细胞发生明显细胞凋亡,但SK-N-SH细胞却未见凋亡.Hoechst 33342染色显示,SK-N-SH细胞发生了核分裂异常.蛋白质免疫印迹分析证明,10 μmol/L 八氯腺苷处理SH SY5Y 48～72 h后,G^₂检验点调节蛋白ATM、Chk1、Cdc25C和Cdc2磷酸化形式明显上调,同时伴有caspase-3的激活,提示SH-SY5Y细胞发生了G^₂检验点通路和细胞凋亡途径的激活.与SH-SY5Y细胞不同,在SK-N-SH细胞中,八氯腺苷处理24～96 h时,磷酸化ATM、磷酸化Chk1/Chk2、磷酸化Cdc25C以及磷酸化Cdc2的水平呈现逐渐降低的趋势.结果提示,SK-N-SH细胞在八氯腺苷处理后发生了G^₂检验点失败.蛋白质免疫印迹分析还显示,八氯腺苷可诱导p53在SH-SY5Y细胞的表达,但却不能影响SK—N-SH细胞的p53组成性表达水平.p21在SK-N-SH的组成性表达随八氯腺苷处理时间延长而逐渐减少,但在处理前后的SH-SY5Y细胞均未检测到p21蛋白的表达.上述实验结果提示,八氯腺苷抑制两种细胞增殖的机制不同：在SH-SY5Y细胞,八氯腺苷可激活ATM-Chk-Cdc25C-Cdc2/cyclin途径和凋亡通路,使细胞发生G_²/M期阻滞和细胞凋亡;在SK-N-SH细胞,八氯腺苷诱导G^₂检验点失败,导致细胞阻滞在有丝分裂期,并发生有丝分裂异常.2种不同的细胞命运可能还与p53和p21表达不同有关.     

癌基因D52家族新基因PC-1在前列腺癌细胞系LNCaP和C4-2B的G2／M期高表达

目的：检测癌基因D52家族新基因船一,在细胞周期各时相的表达变化。方法：用1mmol／L羟基脲处理前列腺癌细胞系LNCaP和C4-2B 40h,使细胞同步化于G1／S期,在药物撤除后0-16h不同时间点收获细胞,分别进行流式分析和Westernblot检测。结果：流式分析和Westernblot检测在LNCaP和C4-2B细胞系中得到了趋势一致的结果,印PC-1基因在GVM期高表达。结论：PC-1基因的表达与前列腺癌细胞的细胞周期有关,表明PC-1蛋白可能在G2／M期发挥作用。     

HBXIP,a binding protein of HBx,regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity

To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Fas-mediated apoptosis in Jurkat cells is suppressed in the pre-G2/M phase

The relationship between the cell cycle and Fas-mediated apoptosis was investigated using Jurkat cells. Analysis of the inducibility of apoptosis by anti-Fas antibody during the cell cycle synchronized by the thymidine double-block method, showed that apoptosis was induced in only 50% of the G2/M phase cells, while most of cells in the other phases underwent apoptosis. These observations indicate that G2/M phase cells are more resistant to Fas-mediated apoptosis than cells in other phases. Furthermore, a detailed analysis of G2/M phase found that only 20–30% of the cells underwent apoptosis 12 h after the removal of the second thymidine block (pre-G2/M phase). This suggests that Fas-mediated apoptosis is potently suppressed during the pre-G2/M phase. A possible explanation for the observation that cells in the pre-G2/M phase are less sensitive to anti-Fas antibody is lower expression level of Fas. To test this possibility, Fas expression levels on the cell surface during the cell cycle were examined. The content of Fas on the cell surface, however, did not change appreciably during the cell cycle. Thus, the suppression of apoptosis in the pre-G2/M phase is determined downstream after the receipt of the apoptotic signal through Fas.     

Persistence of RNA transcription during DNA replication delays duplication of transcription start sites until G2/M

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G2/M cell cycle arrest and induction of apoptosis by a stilbenoid, 3,4,5-trimethoxy-4'-bromo-cis-stilbene, in human lung cancer cells

Stilbenoids, including resveratrol (3,5,4'-trihydroxy-trans-stilbene) which is a naturally occurring phytoalexin abundant in grapes and several plants, have been shown to be active in inhibiting proliferation and inducing apoptosis in human cancer cell lines. Using resveratrol as the prototype, we have synthesized various analogs and evaluated their growth inhibitory effects in cultured human cancer cells. In the present study, we show that one of the stilbenoids, 3,4,5-trimethoxy-4'-bromo-cis-stilbene (BCS), was more effective than its corresponding trans-isomer and resveratrol on the inhibition of cancer cell growth. Prompted by the strong growth inhibitory activity of BCS (IC50; 0.03 microM) compared to its trans-isomer (IC50; 6.36 microM) and resveratrol (IC50; 33.0 microM) in cultured human lung cancer cells (A549), we investigated its mechanism of action. BCS induced arrest at the G2/M phase cell cycle in the early time and subsequently increased in the sub-G1 phase DNA contents in a time-dependent manner, indicating induction of apoptosis. Morphological observation with round-up shape and DNA fragmentation was also revealed the apoptotic phenomena. BCS treatment elevated the expression levels of the pro-apoptotic protein p53, the cyclin-dependent kinase inhibitor p21, and the release of cytochrome c in the cytosol. The down-regulation of checkpoint protein cyclin B1 by BCS was well correlated with the cell cycle arrest at G2/M. These data suggest the potential of BCS to serve as a cancer chemotherapeutic or chemopreventive agent by virtue of arresting the cell cycle and induction of apoptosis of human lung cancer cells.