核基质蛋白Ciz1与肿瘤北大核心CSCD

核基质蛋白Ciz1(Cdkn1A-interacting zinc finger protein 1)是在酵母双杂交系统中寻找能与p21Cip1/Waf1结合并调节其细胞核定位时发现的锌指蛋白。当分别过表达Ciz1和p21Cip1/Waf1时,它们均主要定位于细胞核,而当共转染时,则均从细胞核转位到细胞质。在小鼠3T3细胞中,Ciz1可以协同CDK2、细胞周期蛋白E和细胞周期蛋白A启动DNA的复制,并促进细胞由G1期进入S期。此外,Ciz1还具有结合DNA的能力并参与对转录因子的活性调控,同时,Ciz1还可能作为蛋白激酶ATM的底物参与DNA的损伤修复。近年来研究发现,Ciz1除与阿尔茨海默病和肌张力失常等疾病相关以外,还在肺癌、结肠癌和乳腺癌等多种肿瘤组织中呈现高表达,参与肿瘤的发生和发展过程。本文主要就Ciz1的结构功能及与肿瘤的关系作一综述。     

p21亚细胞定位与肿瘤

p21是一种重要的周期蛋白依赖性激酶抑制因子(cyclin-dependent-kinase inhibitor, CKI),主要通过调控细胞周期维持细胞的生长和增殖。此外, p21还参与调控细胞凋亡、细胞衰老以及细胞运动等过程。近年来越来越多的研究表明, p21的功能具有双重性。当p21定位在细胞核时,其主要通过抑制周期蛋白依赖性激酶(cyclin-dependent kinases, CDKs)的活性介导细胞周期停滞,抑制细胞增殖;当定位在细胞质时, p21能够促进细胞增殖。本文主要对p21的生物学功能、亚细胞定位调控机制及其在肿瘤研究中的最新进展予以综述。     

时钟节律与细胞周期

昼夜节律和细胞周期是生命有机体中两种主要的节律性、周期性的活动,参与机体代谢与生理节律．在分子水平上,它们的周期性活动是由一种周期性振荡的网络构成的,这种网络由一系列节律性表达的蛋白所形成．研究发现,多种节律因子通过调节周期蛋白的表达影响细胞周期进程,如G ₁-S期,REV-ERBa抑制p21促进细胞进程,RORα激活p21抑制细胞进程,DEC1抑制cyclinD1,CLOCK/BMAL1负调控c-Myc;G ₂-M期,BMAL1/CLOCK、BMAL1/NPAS2或Cry1作用于Wee1抑制或激活G_2-M期进程．此外,昼夜节律钟蛋白也参与了DNA损伤修复及细胞死亡的过程：Per1、Tim分别作用于ATM、ATR,因而促进细胞周期停滞,p53缺失的细胞中敲除Cry促进细胞凋亡过程,抑制了肿瘤的形成,DEC1以p53依赖的方式促细胞衰老等．同时,节律因子的紊乱引起多种疾病的产生．因此,阐明昼夜节律对细胞周期及死亡的影响,将为肿瘤的治疗提供分子理论基础．     

曲古抑菌素A对结肠癌细胞株SW480细胞周期影响的机制研究

为了研究组蛋白去乙酰化酶(HDACs)抑制剂曲古抑菌素A(TSA)对结肠癌细胞周期和凋亡的影响,初步探讨TSA作用细胞周期的可能机制,将人结肠癌细胞系SW480经TSA处理后,运用流式细胞术检测细胞周期、凋亡以及细胞周期素的变化,最后采用western-blot对细胞周期相关的基因进行检测.结果表明,TSA处理细胞后,TSA能够延缓细胞周期G1-S进程,阻滞细胞于G1期,并且影响细胞周期素cyclinE、cyclinA聚集,而对凋亡无明显的影响.Western-blot显示,TSA能够上调p21Waf1/Cip1、p27Kip1的表达,下调CDK2、cyclinE以及cycli-nA的表达.以上结果说明在结肠癌细胞中,TSA能够通过上调p21Waf1/Cip1、p27Kip1的表达以及下调CDK2、cy-clinE、cyclinA的表达,从而阻滞细胞周期于G1期,最终影响肿瘤细胞的生长,以上研究为HDAC抑制剂应用于结肠癌治疗提供了理论依据.     

过表达UHRF2通过上调p53及p21表达引起HEK293细胞G1期阻滞

UHRF2（ubiquitin like with PHD and ring finger domains 2）是新近发现的具有多个结构域的核蛋白,在细胞周期调控和表观遗传学中发挥重要作用．近期研究提示,UHRF2是肿瘤抑制蛋白p53的1个E3连接酶,在体内外能与p53相互结合并促进其泛素化,过表达UHRF2能使细胞周期停滞于G1期．然而,UHRF2介导的G1期阻滞及其与p53联系尚不清楚．通过共转染UHRF2质粒及p53特异性小干扰RNA(siRNAs)到HEK293细胞构建细胞模型,探索UHRF2引起细胞周期停滞与p53之间的关系．结果显示,UHRF2能促进HEK293细胞中p53的稳定,从而引起p21 (CIP1/WAF1)基因表达,并使细胞周期停滞于G1期;但在siRNA抑制p53的表达后p21(CIP1/WAF1)表达降低,UHRF2引起的细胞周期阻滞消除．研究结果提示,UHRF2可通过稳定p53,上调p21的表达,从而介导细胞周期阻滞于G1期;同时UHRF2可能参与细胞周期调控及DNA损伤反应（DNA damage response, DDR）．UHRF2稳定p53的具体分子机制及其在DDR中的作用有待进一步研究证明．     

丙戊酸钠抑制A549细胞生长

目的研究丙戊酸钠对肺癌A549细胞增殖和细胞周期的影响。方法MTT检测生长抑制,流式细胞仪检测细胞周期和凋亡,Western blot检测p21WAF1/CIP1蛋白表达。结果丙戊酸钠以剂量依赖性方式抑制A549细胞生长;丙戊酸钠上调G0/G1期比例,下调S期和G2/M期,不影响细胞凋亡;丙戊酸钠上调p21WAF1/CIP1蛋白表达。结论丙戊酸钠上调p21WAF1/CIP1表达,使细胞阻滞于G0/G1期,抑制A549细胞生长。     

p53基因与DNA修复

DNA的损伤修复是一个多因子参与的、多环节的复杂修复系统。p53基因以多条信号通路,多种调控方式参与DNA修复。它可以通过其下游一系列靶基因p21、gadd45等调控细胞周期,使细胞停滞于G1期、G2期等检测点,从而使受损DNA有足够的时间进行多因子参与的修复过程;也可以与DNA修复因子PRSA、PCNA、XPp48基因等相互作用,直接参与DNA修复;还可以蛋白－蛋白相互作用参与DNA修复。     

值得注意的细胞周期调节蛋白

细胞周期调节蛋白在细胞周期的G_1期和S期交界处开始合成,S期结束后消失。细胞内定位在核内,又称分裂细胞核抗原。细胞周期调节蛋白含量与细胞分裂状况直接有关。细胞周期调节蛋白为细胞DNA复制所必须,是DNA聚合酶δ的活化蛋白。因此细胞周期调节蛋白对于真核生物DNA复制的调控及细胞分裂具有重要意义,值得重视。     

乙肝病毒x基因致肝细胞癌机理探讨

构建HBx基因真核表达载体, 导入HepG2细胞中, 无血清培养同步化后, Western 杂交检测HepG2-X细胞IGF-ⅠR, PCNA和VEGF表达增强, 但无p21CIP1/WAF1表达. 流式细胞检测细胞周期, HepG-X0细胞70%进入G0～G1期, 而HepG2-X细胞仅56%进入G0～G1期, 与血清培养亲本HepG2细胞几乎相同. HBx基因增强IGF-ⅠR表达, 通过信号通路到细胞核, 同时使PCNA表达增强, p21CIP1/WAF1表达抑制, 推进细胞周期, 使G0～G1细胞明显减少. HBx基因增强VEGF表达, 通过旁分泌作用使血管内皮增生; 以上可能是HBx蛋白致HCC 的机理之一.     

rBTI通过上调PTEN的表达抑制Hep G2细胞增殖

磷酸酶及张力蛋白的同源基因(PTEN) 是一种抑癌基因,可以调控细胞的增殖,与癌症的发生和发展息息相关。本研究采用MTT法和流式细胞术分别检测了重组荞麦胰蛋白酶抑制剂(rBTI)对人肝癌细胞株Hep G2细胞的增殖以及周期的影响。免疫荧光及Western印迹法检测了PTEN和p PTEN的亚细胞定位及蛋白表达的变化。采用qRT-PCR及Western印迹法检测了周期相关蛋白的表达。旨在探究PTEN和p PTEN在rBTI抑制Hep G2细胞增殖和周期阻滞中的作用。结果表明,rBTI能显著抑制Hep G2细胞增殖,将细胞周期阻滞在G0/G1期,并呈时间和剂量依赖性;rBTI作用于Hep G2后,可显著上调PTEN和p-PTEN的表达。同时发现,p-PTEN主要分布于细胞核中,能与核仁发生共定位;周期相关蛋白检测表明,细胞内p53、p21转录水平和蛋白水平均增加。综上所述,rBTI通过上调PTEN的表达,使得细胞周期阻滞于G0/G1期,进而抑制Hep G2细胞的增殖。     

Cloning and characterization of a novel p21(Cip1/Waf1)-interacting zinc finger protein, ciz1.

p21(Cip1/Waf1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. Here, we report a novel p21(Cip1/Waf1)-interacting protein, Ciz1 (for Cip1 interacting zinc finger protein), which contains polyglutamine repeats and glutamine-rich region in the N-terminus as well as three zinc-finger motifs and one MH3 (matrin 3-homologous domain 3) in the C-terminal region. Ciz1 bound to the N-terminal, the CDK2-interacting part of p21(Cip1/Waf1), and the interaction was disrupted by the overexpression of CDK2. A region of about 150 amino acids containing the first zinc-finger motif in Ciz1 was the binding site for p21(Cip1/Waf1). When Ciz1 and p21(Cip1/Waf1) were individually overexpressed in U2-OS cells, they mostly localized in the nucleus. However, coexpression of Ciz1 induced cytoplasmic distribution of p21(Cip1/Waf1). These data indicate that Ciz1 is a unique nuclear protein that regulates the cellular localization of p21(Cip1/Waf1).     

Ciz1, Cip1 interacting zinc finger protein 1 binds the consensus DNA sequence ARYSR(0–2)YYAC

The matrin 3 family of nuclear proteins consists of members with potentially diverse activities. Matrin 3 and NP220 share RNA-binding domains, and NP220 has been shown to recognize and bind to the DNA sequence, CCCCC (G/C). We have isolated and characterized another member of the matrin 3 family, designated NP94, from a medulloblastoma. This protein, also named Ciz1, has previously been characterized for its ability to interact with p21(Cip1/Waf1) and contains 3 zinc finger domains and a matrin 3-homologous domain 3. Our immunofluorescence and Northern blot analysis data indicate that Ciz1 is localized in the nucleus and is expressed in a wide range of tissues, especially the pancreas and the brain; within the brain, the highest message levels are found in the cerebellum. A modified selected and amplified binding (SAAB) sequence method was used to identify DNA sequences recognized by Ciz1. From the analysis of the retrieved SAAB sequences and verification using electrophoretic mobility shift assays, we formulated a consensus DNA sequence, ARYSR(0-2)YYAC, recognized by Ciz1. The potential activities of Ciz1, including those involved in brain tumorigenesis, are discussed.     

Multifaceted p21 in carcinogenesis,stemness of tumor and tumor therapy

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21^Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.     

Sodium butyrate induces G2 arrest in the human breast cancer cells MDA-MB-231 and renders them competent for DNA rereplication

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21(Waf1) and of p21(Waf1) associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21(Waf1) accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21(Waf1) level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21(Waf1) by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.     

Functions of p21Waf1 in norm and in stress

Cyclin-dependent kinase inhibitor p2(Waf1/Cip1/Sdi1/CAP20) plays the key part in cell cycle arrest at the G1/S checkpoint in response to DNA damage, and is involved in the assembly of active cyclin-kinase complexes, in particular, cyclin D-Cdk4/6. Recent studies extended the range of known p21Waf1 functions. In addition to the cell-cycle control, p21Waf1 participates in important cell processes such as differentiation, senescence, and apoptosis. A balance of p21Waf1 functional activity seems to shift depending on the cell state (senescence, exposure to stress, expression of viral oncogenes). This is due to direct or indirect interaction with various modulators or to modification (phosphorylation, partial proteolysis) of p21Waf1. The review considers the structure of p21Waf1, its posttranslational modification, interactions with various cell or viral proteins, and their effects on the p21Waf1 function and the cell.     

Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells

The progression of cells from G₂ into mitosis is mainly controlled by formation of the cyclin B1/p34^cdc2 complex. The behaviour of this complex in the irradiation-induced G₂ cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G₂ block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin-dependent kinase p34^cdc2 in HeLa cells. Irradiation induces p34^cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G₂ maximum reverses these trends. This is also evident from the cyclin B1/p34^cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin B1 and p34^cdc2 protein expression are important events and act in concert to control the irradiation induced G₂ block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G₂ block is abrogated by pentoxifylline.     

Functions of p21Waf1 in Norm and in Stress

Cyclin-dependent kinase inhibitor p21^Waf1/Cip1 plays the key part in cell cycle arrest at the G1/S checkpoint in response to DNA damage, and is involved in the assembly of active cyclin–kinase complexes, in particular, cyclin D–Cdk4/6. Recent studies extended the range of known p21^Waf1/Cip1 functions. In addition to the cell-cycle control, p21^Waf1/Cip1 participates in important cell processes such as differentiation, senescence, and apoptosis. The balance of p21^Waf1/Cip1 functional activity appears to shift depending on the cell state (senescence, exposure to stress, expression of viral oncogenes). This is due to direct or indirect interaction with various modulators or to modification (phosphorylation, partial proteolysis) of p21^Waf1/Cip1. The review considers the structure of p21^Waf1/Cip1, its posttranslational modification, interactions with various cell or viral proteins, and their effects on the p21^Waf1/Cip1 function and on the cell.