新型抗肿瘤药物作用靶点Plk1

Polo样激酶1（Plk1）是一种参与有丝分裂调控的重要激酶,在大多数恶性肿瘤中呈现过表达,并与某些肿瘤的生物学行为及预后密切相关。综述Plk1在细胞周期调控方面的作用及Plk1作为抗肿瘤新靶点的研究进展。     

Polo样激酶1在细胞有丝分裂期作用的研究进展

Polo样激酶1（Plk1）是一类广泛存在于真核细胞中的丝氨酸/苏氨酸激酶,是细胞有丝分裂期的关键调控因子.在有丝分裂的不同时期,Plk1在细胞中的定位不同,并与不同的底物相互作用,从而具有多种生物学功能.文章对Plk1在细胞有丝分裂各个时期的功能作一综述.     

Chk1/2和Plk1蛋白在子宫内膜癌中的表达

目的 Chk1/2（checkpoint kinase 1、2）和Plk1（polo like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶,本研究检测3种激酶在子宫内膜癌及正常子宫内膜组织中的表达,探讨3种蛋白在两者之间的表达差异、与子宫内膜癌临床病理特征的关系及3种蛋白表达的相关性。方法 应用免疫组化SP法检测44例子宫内膜癌组织和21例正常子宫内膜组织中Chk1、Chk2和Plk1蛋白的表达情况。结果 Chk1、Chk2和Plk1蛋白在子宫内膜癌患者中的阳性率分别为47.7％、75.0％和31.8％,在正常子宫内膜中的阳性率分别为61.9％、61.9％和4.8％;Plk1蛋白在子宫内膜癌组织中的表达显著高于正常子宫内膜组织（P＜0.01）,而Chk1、Chk2的表达差异无统计学意义（P>0.05）。Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的子宫内膜癌患者中差异无统计学意义（P>0.05）;但Chk1的表达在不同分化程度的子宫内膜癌患者中差异有统计学意义（P<0.01）。Spearman等级相关分析,在44例子宫内膜癌患者中,Chk2与Plk1间的表达呈正相关（r＝0.482,P＝0.001）。结论 Plk1可能成为子宫内膜癌比较理想的治疗靶点,而CHK1/2在子宫内膜癌中表达及意义还有待进一步研究。     

Plk1与人类肿瘤生成

人类肿瘤生成过程主要表现为细胞生长的失调控,而Plk1是细胞周期进行有丝分裂、胞质分裂及DNA损伤反应的关键调节因子。通过对Plk1在中心体周期中作用的认识,推测在多种癌症中可检测到Plk1表达的失控,且与人类肿瘤生成有关。研究发现,Plk1在人类多种肿瘤中存在高表达,且其高表达与肿瘤患者的细胞增殖及预后相关。应用不同方法下调Plk1的表达水平在体外细胞培养及活体动物实验中均被证实有抑制肿瘤细胞增殖及促进其凋亡的作用,但对正常细胞却无明显杀伤。因此,靶向作用于Plk1的治疗可能成为人类肿瘤治疗的新的很有潜力的方法。     

Plk1与人类肿瘤生成

人类肿瘤生成过程主要表现为细胞生长的失调控,而Plk1是细胞周期进行有丝分裂、胞质分裂及DNA损伤反应的关键调节因子。通过对Plk1在中心体周期中作用的认识,推测在多种癌症中可检测到Plk1表达的失控,且与人类肿瘤生成有关。研究发现,Plk1在人类多种肿瘤中存在高表达,且其高表达与肿瘤患者的细胞增殖及预后相关。应用不同方法下调Plk1的表达水平在体外细胞培养及活体动物实验中均被证实有抑制肿瘤细胞增殖及促进其凋亡的作用,但对正常细胞却无明显杀伤。因此,靶向作用于Plk1的治疗可能成为人类肿瘤治疗的新的很有潜力的方法。     

Polo-like激酶1反义RNA对肺癌细胞A549细胞周期的影响

背景与目的Polo-Like激酶1(Polo-likekinase 1,Plk1)是参与有丝分裂调控的重要分子,已在肺腺癌细胞株A549中检测到Plk1的高表达,并认为Plk1高表达与肺癌患者的放化疗耐受和预后相关。本研究利用反义RNA技术,探讨Plk1基因表达下调对肺癌细胞细胞周期的影响。方法培养肺腺癌细胞株A549,构建表达Plk1反义RNA的质粒pcDNA3.0-Plk1(pc3.0P),通过脂质体介导转入A549细胞,Westernblot、RT-PCR检测Plk1的表达,BrdU脉冲标记和流式细胞术分析细胞周期变化;免疫荧光染色检测α微管蛋白的表达。结果A549细胞转染pc3.0P后,Plk1mRNA的表达较对照组显著下降(P<0.05),转染24h后Plk1mRNA的表达下降46.75%,转染48h后下降61.84%;蛋白表达亦有下降;S期细胞百分数(BrdU标记指数)较对照组明显下降(P<0.05),转染后48h仅有25.59%;转染后72h出现G2/M期阻滞(P<0.05),并出现细胞凋亡;微管染色显示细胞周边缺乏微管的聚集,单极纺锤体形成。结论Plk1影响纺锤体微管的形成,使A549细胞增殖速度减慢,细胞周期阻滞并发生凋亡。     

Chk1/2和Plk1蛋白在宫颈良恶性病变组织中的表达及其意义

背景与目的：Chk1/2（checkpoint kinase 1/2）和Plk1（polo-like kinase 1）是各细胞周期检测点启动DNA损伤修复的主要激酶.本研究主要探讨3种激酶蛋白在宫颈良恶性病变中的表达差异、与宫颈癌临床病理特征的关系及3种激酶在宫颈癌组织中表达之间的相互关系.方法：应用免疫组化SP法检测43例宫颈癌组织和20例慢性宫颈炎性组织中Chk1、Chk2和Plk1蛋白的表达情况.结果：Chk1、Chk2和Plk1蛋白在宫颈癌患者中的阳性率分别为76.7%、60.5%和32.6%,在慢性宫颈炎患者中的阳性率分别为30.0%、35.0%和0;Chkl和Plk1蛋白在宫颈癌组织中的表达显著高于慢性宫颈炎组织（P＜0.01）,而Chk2的表达差异无显著性（P＞0.05）.Chk1、Chk2和Plk1蛋白的表达在不同年龄、病理类型和临床分期的宫颈癌患者中差异无显著性（P＞0.05）;但Chk1和Plkl的表达在不同分化程度的宫颈癌患者中差异有显著性（P＜0.05）.Spearman等级相关分析,在43例宫颈癌患者中,Chk1与Chk2的表达呈正相关（r=0.492,P=0.001）.结论：Chk1和Plk1可能成为比较理想的宫颈癌治疗靶点.     

敲低Plk1对人肝癌血管内皮细胞迁移影响研究

目的研究人Polo样激酶1(polo-like kinase 1,Plk1)在人肝癌血管内皮细胞(human hepatocellular carcinoma tumor-derived endothelial cell,TEC)迁移过程中的作用。方法用含有20%胎牛血清的RPMI1640培养基培养TEC细胞,合成Plk1和阴性对照小干扰片段。实验设置Plk1siRNA处理组和siRNA阴性组(对照组)。分别用Plk1siRNA和阴性对照siRNA转染TEC,采用蛋白质印迹法验证Plk1siRNA干扰能否有效降低TEC细胞的Plk1蛋白表达水平;采用Transwell细胞迁移实验和细胞划痕实验评估Plk1siRNA转染后TEC迁移能力的变化。结果蛋白质印迹法检测结果显示,Plk1siRNA转染后TEC细胞中Plk1蛋白表达降低。Transwell细胞迁移实验发现,Plk1siRNA处理组12h穿膜细胞数为130.9±23.0,低于siRNA阴性组的184.9±26.6,t=-5.943,P<0.001。细胞划痕实验显示,Plk1siRNA处理组细胞12h迁移距离为(17.5±7.0)像素,低于siRNA阴性组的(72.6±12.4)像素,t=-6.698,P=0.006;Plk1siRNA处理组细胞24h迁移距离为(70.9±6.9)像素,低于siRNA阴性组的(137.9±18.9)像素,t=-5.761,P=0.016。结论敲低Plk1能显著抑制人肝癌血管内皮细胞的迁移能力,提示Plk1可能在人肝癌血管内皮细胞转移中具有一定作用。     

Polo样激酶3针对P73蛋白的磷酸化位点分析

目的：探讨P73蛋白上存在的能够被polo样激酶3（pololike kinases 3,Plk3）磷酸化的结构域或位点,并分析Plk3对P73介导肿瘤细胞凋亡的影响。方法：免疫共沉淀法检测COS7细胞中Plk3与P73蛋白之间的相互作用,荧光免疫染色法检测Plk3与P73蛋白在细胞中的定位。制备不同的P73缺失突变体GST融合蛋白,体外点突变法构建GSTP73(1~130)点突变的P73(T86A) （第86位苏氨酸点突变为丙氨酸）质粒,体外磷酸化实验分析P73中被Plk3磷酸化的结构域或位点。通过检测PARP蛋白的裂解分析Plk3对P73介导人宫颈癌HeLa细胞凋亡的影响。结果：Plk3与P73蛋白之间存在相互作用,Plk3与P73蛋白共定位于COS7细胞核中。制备获得不同的P73缺失突变体GST融合蛋白,Plk3在P73蛋白N端第63~113位氨基酸残基之间磷酸化P73蛋白。GSTP73(1~130)融合蛋白第86位苏氨酸点突变为丙氨酸(T86A)之后,不影响GSTP73(1~130)蛋白的磷酸化状态。Plk3可抑制P73介导的HeLa细胞凋亡。结论：Plk3通过与P73蛋白结合,诱导P73蛋白N端第63~113位氨基酸磷酸化,但第86位苏氨酸并非Plk3的特异作用位点;此外Plk3抑制P73介导的HeLa细胞凋亡。     

Plk1和p27在甲状腺乳头状癌中的表达及预后意义

目的:探讨Plk1和p27在甲状腺乳头状癌中的表达及与甲状腺乳头状癌预后的关系。方法:采用免疫组织化学技术检测Plk1和p27在甲状腺乳头状癌中的表达情况。结果:甲状腺乳头状癌中Plk1的阳性表达率明显高于正常甲状腺组织（P<0.05）。Plk1在微灶癌（直径≤1cm）中的阳性表达率明显高于在癌灶直径>1cm的甲状腺乳头状癌(P<0.05)。甲状腺乳头状癌中p27阳性表达率（44.12%）明显低于正常甲状腺组织（80.00%,P<0.05）。Plk1和 p27表达与包膜浸润、淋巴结转移情况相关。结论:Plk1在早期甲状腺乳头状癌的发生、发展中起重要作用,联合检测Plk1和p27 有助于甲状腺乳头状癌的诊断及预后评估。     

细胞周期与肿瘤转化医学

恶性肿瘤最基本的生物学特征之一是细胞周期调控紊乱导致的细胞恶性转化和肿瘤细胞失控性增殖。了解细胞周期的调控机制能够揭示肿瘤发生发展的本质，阐释癌症的发生机制，从而为肿瘤的早期诊断和临床治疗提供分子标志物及药物靶点。本综述通过分析参与细胞周期运转的重要蛋白分子以及这些分子在细胞癌变和临床肿瘤防治过程中的作用，探讨基于这些分子的药物研发，从而系统地阐述细胞周期紊乱对肿瘤发生发展的生物学影响及其对肿瘤诊断与治疗的理论和实践意义。      

The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death.

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16ink4a, PSM-RB is also a potent inhibitor of cell cycle progression in RB-deficient tumor cells. Surprisingly, we identified a tumor cell line that is resistant to the cell cycle inhibitory effects of PSM-RB. This finding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this 'resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the first to show that RB inhibits cellular proliferation through at least two distinct mechanisms - inhibition of cell cycle progression and induction of cell death.     

Development of cell cycle active drugs for the treatment of gastrointestinal cancers: a new approach to cancer therapy.

The cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate. An essential part of the cell cycle machinery is the cyclin-dependent kinases (CDKs). When activated, the CDKs provide a means for the cell to move from one phase of the cell cycle to the next (G1 to S or G2 to M). The cell cycle serves to protect the cell from genotoxic stress. In the setting of DNA damage, the CDKs are inhibited and the cell undergoes cell-cycle arrest. This provides the cell the opportunity to repair its own damaged DNA before it resumes cell proliferation. If a cell continues to cycle with its damaged DNA intact, the apoptotic machinery is triggered and the cell will undergo apoptosis. In essence, cell cycle arrest at these critical checkpoints represents a survival mechanism, which provides the tumor cell the opportunity to escape the effects of lethal DNA damage induced by chemotherapy. Over the past several years, a series of new targeted agents has been developed that promote apoptosis of DNA damaged tumor cells either during cell cycle arrest or following premature cell cycle checkpoint exit, such that tumor cells re-enter the cell cycle before DNA repair is complete. An understanding of the cell cycle and its relationship to p53 are critical for the successful clinical development of these agents for the treatment of patients with gastrointestinal cancers.     

细胞周期对肿瘤坏死因子诱导Hela 细胞凋亡的影响

 目的　研究细胞周期对肿瘤坏死因子 ( TNF)诱导 Hela细胞凋亡的影响。方法　通过胸腺嘧啶核苷酸 ( Td R)阻断法阻滞 Hela细胞的细胞周期 ,以 MTT法、流式细胞术和荧光染色分析 Td R阻滞细胞和周期化的 Hela细胞对 TNF诱导凋亡的敏感性。结果　Td R阻滞细胞周期较周期化的 Hela细胞对 TNF诱导的凋亡的敏感性降低。结论　揭示 TNF诱导 Hela细胞凋亡与细胞周期有关.     

蛋白酶体抑制剂在恶性血液病靶向治疗中的研究进展

 蛋白酶体是一种多酶复合物,能够降解细胞周期调节蛋白,而这些被降解的蛋白控制着细胞周期的进程和细胞凋亡。蛋白酶体抑制剂可通过抑制泛素-蛋白酶体通路（UPP）调节细胞周期,促进细胞凋亡,体内外实验均发现蛋白酶体抑制剂对多种血液系统恶性肿瘤细胞具促凋亡的生物活性,可通过不同的机制引起细胞凋亡。因此蛋白酶体抑制剂是一种新型的肿瘤靶向治疗药物。     

热休克蛋白70参与癌变及肿瘤耐药的分子机制

热休克蛋白70(HSP70)在许多肿瘤组织和细胞中过表达,与肿瘤的恶性程度、治疗预后密切相关.HSP70作为分子伴侣参与细胞周期调控、细胞凋亡和细胞增殖与分化,在肿瘤治疗中,抑制抗癌药物、放疗射线照射等因素介导的凋亡.HSP70不仅参与了肿瘤的发生、发展,还在肿瘤耐药中起重要作用.     

Mathematic models for cancer chemotherapy: pharmacokinetic and cell kinetic considerations.

This paper presents a theoretic study of pharmacokinetic and cell kinetic models for cancer chemotherapeutic systems. The mathematic analysis is based on a modified procedure deduced from DeVita's scheme of the relationship between the cellular kinetics of normal and tumor tissues and the pharmacokinetics of antitumor agents for designing an optimal dose and schedule for cancer treatment. In this scheme pharmacokinetic models and cell-drug interactions at the tumor site are incorporated into the cell cycle kinetic models to form the cancer chemotherapeutic model systems. Three cell cycle kinetic models are presented under alternative hypotheses concerning the mechanism of the resting cells, while each tumor mass is comprised of cells in proliferating (consisting of the four cycle phases G1, S, G2, and M), resting (Go), and non dividing (D, dead) states. An algorithm and a computer program for simulating the tumor populations during scheduled treatments have been prepared. By a suitable selection of expressions for cell-drug interactions, the program is able to simulate tumor behavior during scheduled treatments with different classes of anticancer agent such as cell cycle phase-specific, cell cycle-specific, or cell cycle-specific, or cell cycle-nonspecific drugs. A preliminary study of the L1210-ara-C therapeutic system is included to demonstrate the computer simulation procedures.     

Non-metabolic function of MTHFD2 activates CDK2 in bladder cancer

Bladder cancer is a common tumor with a high recurrence rate and high fatality rate, and its mechanism of occurrence and development remains unclear. Many proteins and metabolites reprogram at different stages of tumor development to support tumor cell growth. The moonlighting effect happens when a protein performs multiple functions simultaneously in a cell. In this study, we identified a metabolic protein, MTHFD2, which participates in the cell cycle by binding to CDK2 in bladder cancer. MTHFD2 has been shown to affect bladder cancer cell growth, which is independent of its metabolic function. We found that MTHFD2 was involved in cell cycle regulation and could encourage cell cycle progression by activating CDK2 and sequentially affecting E2F1 activation. In addition, moonlighting MTHFD2 might be regulated by the dynamics of the mitochondria. In conclusion, MTHFD2 localizes in the nucleus to perform a distinct function of catalyzing metabolic reactions. Moreover, the nuclear MTHFD2 activates CDK2 and promotes bladder cancer cell growth by modulating the cell cycle.     

蟾蜍活性多肽对不同肿瘤细胞的抗癌功能及机制研究

目的:研究来源于中华大蟾蜍卵子内一种活性多肽对人不同组织类型肿瘤细胞株的抗癌作用及可能机制。方法:采用MTT法检测活性多肽对肿瘤细胞增殖能力的影响。构建肿瘤移植瘤模型,检测该活性多肽对肿瘤细胞成瘤能力的影响。流式细胞仪检测细胞周期及凋亡情况。采用real-time PCR检测活性多肽对细胞周期和凋亡相关基因表达的影响。结果:蟾蜍活性多肽对不同组织类型的肿瘤细胞具有增殖抑制和/或诱导凋亡的作用,并呈时间依赖性和剂量依赖性。蟾蜍活性多肽可能通过下调周期相关基因cyclinB、CDK1的表达阻滞肿瘤细胞周期于G2/M期检查点。对凋亡诱导的作用可能与凋亡相关基因Survivin、Caspase-3表达和bcl/bak表达的比值上调有关。结论:蟾蜍活性多肽能够通过阻滞细胞周期进展和诱导凋亡抑制肿瘤细胞的体内外增殖能力。