参与细胞衰老的蛋白质结构域

  大多数正常体细胞有有限的复制周期,并最终进入生长停滞状态被称为复制性衰老.迄今比较公认的3条细胞衰老信号转导途径是：p16INK4a/Rb、p19ARF/p53/p21Waf1以及PTEN/p27.目前发现,在基因转录水平上,有些转录因子的结构域对调节p16INK4a、p53/p21Waf1以及p27等与细胞衰老相关基因的表达有重要作用,如E2DBD、环指域(RING finger)等;其次,各条通路要发挥作用,必然要借助其上下游蛋白质的相互作用,其中结构域发挥了纽带作用.本文对其中某些蛋白质相互作用的结构域进行了描述.最后,还总结了其他一些参与细胞衰老的结构域.     

三氟拉嗪通过诱导p16INK4a抑制BGC-823细胞增殖

为探索三氟拉嗪（trifluoperazine, TFP）抗肿瘤作用机制,对胃癌BGC-823细胞进 行TFP（5、10 μmol/L）处理后,利用计数法、BrdU脉冲标记法、Western印迹等方法从细胞形态、细胞增殖、S期细胞百分比以及相关因子表达水平等方面进行分析. 结果显示,TFP处理后,细胞形态发生明显改变,细胞增殖受到明显抑制且呈时间计量 效应关系;S期细胞比例下降;p16INK4a表达水平升高.为进一步研究TFP诱导 p16INK4a表达的分子机制,本实验采用插入p16INK4a启动子片段及荧光素酶报告系统 的载体pGL3-Basic-p16INK4a（-967~-165 bp）,研究了TFP在转录水平对p16INK4a启 动子活性的影响.结果表明, TFP能够提高p16INK4a的启动子活性.上述结果提示,TFP 通过诱导p16INK4a表达抑制BGC-823细胞增殖.     

5-氮胞苷对转化后间充质干细胞增殖、衰老及p16~(INK4a)表达的影响

目的观察5-氮胞苷对体外自发转化的间充质干细胞增殖、衰老及p16INK4a基因表达的影响。方法采用细胞计数、SA-β-gal染色、Western Blot和重硫酸盐修饰后测序(BSP)检测体外自发转化的大鼠间充质干细胞在不同浓度5-氮胞苷处理后增殖、衰老、p16INK4a表达及p16INK4a基因启动子区CpG岛甲基化水平的变化。结果细胞计数显示5-氮胞苷可剂量依赖性地抑制转化后间充质干细胞的增殖,但增殖受抑的细胞SA-β-gal染色仍呈阴性。BSP及Western Blot分析显示:转化后间充质干细胞p16INK4a基因启动子区CpG岛呈现高水平甲基化(87.30±2.39%)。5-氮胞苷可在一定程度上降低该基因的甲基化,使p16INK4a表达得以部分恢复;但即使是高浓度高达100μmol/L,5-氮胞苷也只能使p16INK4a基因甲基化降低到46.20±1.65%。结论 5-氮胞苷可显著抑制转化后间充质干细胞的增殖,但并不能促使这些细胞重新恢复衰老状态;其原因是5-氮胞苷单独作用不足以完全解除p16INK4a基因启动子区DNA的高甲基化。     

候选抑癌基因 ING1的研究进展

人候选抑癌基因ING1定位于13q33-34,有3个外显子和2个内含子。ING1mRNA至少有3种变位剪接形式,分别编码3种不同的蛋白质：p47ING1a,p33ING1b和p24ING1。ING1与p53相互作用能够抑制细胞的生长,促进细胞凋亡。在一些肿瘤中还发现ING1表达下调或ING1突变。P33ING1b参与mSin3复合体的形成而介导转录抑制。     

低剂量顺铂通过p38MAPK介导的DNMT1下调降低p21与p16启动子甲基化上调p21与p16表达

低剂量顺铂可通过诱导p21与p16表达而诱导肿瘤细胞早衰,但其机制不明。本研究探讨了低剂量顺铂诱导的HeLa细胞衰老过程中p21与p16的上调机制。低剂量顺铂（4 μmol/L）处理HeLa细胞后,DNA甲基转移酶DNMT1蛋白水平降低;p21与p16启动子甲基化水平降低,二者mRNA及蛋白质水平升高;顺铂对DNMT1蛋白水平的降低作用与其激活p38MAPK有关,用SB203580抑制p38MAPK可部分逆转顺铂对DNMT1蛋白水平以及p21与p16启动子甲基化的降低作用,从而部分逆转顺铂对p21与p16表达的诱导;抑制p38MAPK 也可部分逆转低剂量顺铂诱导的HeLa细胞早衰。上述结果表明,低剂量顺铂可通过p38MAPK信号通路下调p21与p16启动子甲基化水平,进而上调二者的表达。这些结果为解析低剂量顺铂诱导肿瘤细胞早衰的信号转导机制提供了实验依据。     

小鼠胚胎干细胞p16INK4a基因外显子1α打靶研究

在活体水平上 ,小鼠p16 INK4a基因是否具有抑制肿瘤发生和发展的功能是一个悬而未决的问题。利用筛选基因组文库得到的小鼠p16 INK4a基因组DNA片段 ,构建了针对小鼠p16 INK4a 基因外显子 1α的基因打靶载体 ,其短臂为1.5kbEco81Ⅰ AccⅡ片段 ,长臂为 5 .9kbXbaⅠ XhoⅠ片段。打靶载体经线性化和纯化后通过电穿孔转导小鼠R1ES细胞 ,获得 37个G418和Gancyclovir双药抗性克隆。用Southern杂交法对双药抗性克隆进行鉴定 ,获得一个敲除了p16 INK4a基因外显子 1α的阳性ES细胞克隆。     

p16INK4a基因的功能及其调控

p16INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．     

p16INK4a基因的功能及其调控

p16^INK4a蛋白能抑制CDK4和CDK6的活性,使pRb处于非磷酸化或低磷酸化状态而能与转录因子E2Fs结合,从而抑制DNA 的合成,阻止细胞由G1期进入S期.p16^INK4a的表达受Ets1和Ets2的正调控,受Bmi-1的负调控.p16^INK4a基因缺失、突变、甲基化、RNA剪接加工错误可导致细胞周期失控和癌变.应用p16^INK4a对某些肿瘤进行基因治疗的研究正在进行中.     

A new chronological survival assay in mammalian cell culture

c-Abl is activated by oxidative stress but its precise function in cell response to this stress is elusive. Studies of c-Abl-/- osteoblasts revealed that c-Abl played a negative role in the induction of peroxiredoxin I (Prx I, Prdx I), an anti-oxidant protein with tumor suppression activity, but it provided a protective role against oxidative stress. In contrast, Atm, a signaling molecule that interacts with c-Abl and is required for c-Abl activation, served a totally different function. The significance of these findings is discussed here in the context of aging and tumorigenesis and their links to reactive oxygen species.     

核糖体S6蛋白激酶p90rsk与卵母细胞减数分裂

丝裂原活化蛋白激酶（MAPK）信号途径对减数分裂有重要调节作用,p90^rsk是迄今研究最清楚的MAPK下游靶分子,介导MAPK途径在卵母细胞减数分裂中的多种功能,包括卵母细胞减数分裂的启动、MⅠ/MⅡ期转化和MⅡ期阻滞的维持等.p90^rsk的磷酸化是MAPK激活的结果,而细胞退出减数分裂时,p90^rsk的去磷酸化也发生在MAPK失活以后.介绍了在卵母细胞中p90^rsk的研究进展.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.     

p57KIP2 control of actin cytoskeleton dynamics is responsible for its mitochondrial pro-apoptotic effect

p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57^KIP2 has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57^KIP2 is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57^KIP2 is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57^KIP2 is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57^KIP2''s pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57^KIP2''s pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57^KIP2''s effect on the actin cytoskeleton, was required for p57^KIP2''s death promoting effect. Finally, p57^KIP2-mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57^KIP2, by showing that the promotion of cell death by p57^KIP2 requires its actin cytoskeleton stabilization function.     

Controversial aspects of oncogene-induced senescence

Oncogene-induced senescence (OIS) is a fail-safe mechanism that is developed to suppress cell proliferation caused by aberrant activation of oncoproteins in normal cells. Most of the available literature considers senescence to be caused by activated RAS or RAF proteins. In the current review, we will discuss some of the controversial aspects of RAS- or RAF-induced senescence in different types of normal cells: are tumor suppressors important for OIS? What is the role of DNA damage in OIS? Are there different types of OIS?     

The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27^Kip1 and p21^Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.     

The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs

During the meiotic cell cycle in Xenopus oocytes, p90(rsk), the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1. However, p90(rsk) is inactivated after fertilization due to the degradation of Mos. Here we show that the Polo-like kinase Plx1, instead of p90(rsk), interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90(rsk)-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus.