Polo样激酶1功能及其在肿瘤靶向治疗中的应用

Polo样激酶1(polo-like kinase1,PLK1)是一种广泛存在于真核细胞中的丝/苏氨酸激酶,在细胞周期调控中发挥关键的作用。其主要功能包括参与激活cyclin B/CDK1复合体,协助中心体的功能成熟,活化细胞分裂后期促进复合物(anaphase promoting complex,APC),促进染色体正常分离、分配和调控胞质分裂等。现已发现PLK1在多种肿瘤中表达增高并与某些肿瘤的预后密切相关。利用反义寡核苷酸、RNA干扰技术和化学合成PLK1小分子抑制剂等方法阻断PLK1的表达或降低其激酶活性,能够有效抑制肿瘤细胞的增殖并介导肿瘤细胞的凋亡,但对正常细胞没有明显影响,因此PLK1在肿瘤靶向治疗中具有重要的应用前景。     

Bora在细胞功能调控中的作用和机制研究进展

Aurora蛋白激酶A及Polo样蛋白激酶1（PLK在）作为重要的细胞周期调节蛋白可参与调控纺锤体组装、有丝分裂等细胞进程,但其激活机制及在有丝分裂中的作用机制仍然不是很清楚。Bora作为Aurora蛋白激酶A的结合蛋白,在果蝇和脊椎动物中功能高度保守,其主要通过结合Aurora蛋白激酶A从而调节Aurora蛋白激酶A的活性、促进PLK1的磷酸化、调节纺锤体的组装以及调控细胞周期进程等。随着对Bora研究的深入,人们对AuroraA和PLK1的激活机制以及Bora、Aurora蛋白激酶A、PLK1三者对细胞的调控也有了进一步的认识。主要综述Bora在细胞功能调控中的作用和研究机制。     

抑癌基因P16在肺癌中的研究进展

抑癌基因P16与细胞周期调控密切相关,其主要作用是参与细胞周期过程,它通过细胞周期与其它癌基因及肿瘤抑制基因相互作用成为正常细胞增殖的调控因子。P16基因主要通过基因缺失、点突变及甲基化而失活,已证实该基因的失活与多种肿瘤的形成与转移密切相关。通过各种分子生物学技术检测出它在肿瘤中的表达,将有助于判断肿瘤的恶性程度、浸润深度及预后,为制定合理的治疗方案提供依据。P16是已知的抑癌基因中唯一通过直接抑制细胞周期而抑制细胞生长的基因,在肿瘤治疗方面有很大的应用前景。P16基因突变在人类恶性肿瘤中普遍存在,因此,有关p16基因的研究已经成为当前分子生物学和分子遗传学研究的重要课题。本文就p16基因的分子生物学特性及它与肺癌的关系作一综述。     

细胞周期蛋白依赖的蛋白激酶抑制剂与肿瘤

细胞周期蛋白依赖的蛋白激酶抑制剂与肿瘤李月彬查锡良（上海医科大学生化教研室卫生部糖复合物重点实验室,上海２０００３２）关键词细胞周期蛋白依赖的蛋白激酶抑制剂肿瘤多细胞生物的正常发育需要精确的增殖和分化调节。一系列调节基因组成了一个复杂的网络调控着细胞...     

细胞周期抑制蛋白p27的研究进展

p27基因位于人类染色体12p13,其编码的蛋白对cyclinsCDKs具有广泛的抑制活性,是细胞周期调控的抑制蛋白。它以化学剂量的方式调节细胞周期的进程,参与细胞的生长、分化等过程。对p27基因的发现、基因结构、对细胞周期和细胞分化的调控机制以及与肿瘤的关系作一介绍。     

抗凋亡蛋白survivin研究与癌治疗新策略的进展

survivin是凋亡抑制蛋白家族中的一个新成员,在近乎所有的人类肿瘤中都显示高表达,而在终末分化的正常组织中未能检测出表达。survivin表达的另一个独特性质是,其表达受细胞周期调控,在G2／M期有异常高的特异性表达。survivin的功能意义在于参与细胞凋亡调控和细胞分裂调控。引人兴趣的是,新近癌生物学基础与应用研究则更关注它是一个枢纽癌基因。本文将重点讨论与survivin在细胞死亡和细胞分裂功能相联系的、当前一些新的癌治疗策略的进展。     

抑癌蛋白p33(ING1)对肿瘤作用的研究进展

p33(ING1)是生长抑制基因(ING1)编码的重要抑癌蛋白,具有抑制细胞生长﹑促进细胞老化﹑维持基因组稳定性、作用于细胞周期调控点等作用,其失活与肿瘤的发生、发展密切相关。本文就近年来有关p33(ING1)的结构、功能及其在肿瘤中的失活机制、临床应用前景等方面的研究进展进行了概述。     

CDK12主要生理功能与其在肿瘤发生中作用的研究进展

周期蛋白依赖性激酶(cyclin-dependent kinase, CDK)是细胞周期和基因转录的关键调节因子,其调控异常是促进肿瘤发生的重要因素。CDK12是一种与转录相关的周期蛋白依赖性激酶,可使RNA聚合酶Ⅱ碳端氨基酸(carboxy terminal domain of RNA polymeraseⅡ, RNA pol II CTD)中的丝氨酸磷酸化,并参与多种细胞生理过程,如DNA损伤反应、细胞增殖和分化以及m RNA剪接和转录前m RNA加工等。此外, CDK12编码基因的突变将导致多种细胞过程调控异常,基因不稳定性增加,这都可能促进肿瘤的发生发展。本文将重点讨论细胞中CDK12调节转录调控、RNA剪接、细胞成熟和分化、DNA损伤修复(DNA damage repair, DDR)的机制以及其基因突变对于正常细胞的影响,旨在阐明CDK12的主要生理功能及其在肿瘤发生发展中的作用,为临床各类肿瘤的靶向药物研究提供帮助。     

肿瘤抑制基因ARHI的研究进展

ARHI是Ras超家族中第一个被报道的肿瘤抑制基因,定位于人染色体lp31,属小GTP结合蛋白,与Ras拥有相似的GTP／GDP结构域,却具有抑癌作用。ARHI是母源性印迹、父源性表达,可参与细胞周期调控和信号通路转导,从而负向调节细胞生长。在正常人类多种组织中都存在ARHI基因的表达,但在肿瘤组织中其表达却下调。ARHI的表达异常可能与印迹基因的杂合性丢失,DNA甲基化和染色体乙酰化修饰等转录水平的调节失常有关。     

RNA干扰在肿瘤基因治疗中的应用策略

运用RNA干扰（RNAi）技术可以通过以下策略进行肿瘤的靶向治疗：抑制癌基因、生长因子及其受体的过表达,从而抑制细胞生长;干扰细胞周期蛋白及其相关基因的表达,从而抑制细胞增殖;抵抗致癌病毒的入侵;抑制抗凋亡基因的表达;上调和恢复抑癌基因的功能;抑制肿瘤发生过程中的关键酶;抑制与肿瘤转移有关的血管生成;靶向端粒酶;靶向耐药基因。尽管目前RNAi已经较为广泛地应用于基因功能研究和肿瘤疾病的基因治疗研究中,但其在应用过程中还有许多亟待解决的问题。我们就RNAi及其在肿瘤疾病基因治疗中的应用策略和存在的问题做一综述。     

Tazarotene-induced gene 1 interacts with Polo-like kinase 2 and inhibits cell proliferation in HCT116 colorectal cancer cells

Tazarotene-induced gene 1 (TIG1) is considered to be a tumor suppressor gene that is highly expressed in normal or well-differentiated colon tissues, while downregulation of TIG1 expression occurs in poorly differentiated colorectal cancer (CRC) tissues. However, it is still unclear how TIG1 regulates the tumorigenesis of CRC. Polo-like kinases (Plks) are believed to play an important role in regulating the cell cycle. The performance of PLK2 in CRC is negatively correlated with the differentiation status of CRC tissues. Here, we found that PLK2 can induce the growth of CRC cells and that TIG1 can prevent PLK2 from promoting the proliferation of CRC cells. We also found that the expression of PLK2 in CRC cells was associated with low levels of Fbxw7 protein and increased expression of cyclin E1. When TIG1 was coexpressed with PLK2, the changes in Fbxw7/cyclin E1 levels induced by PLK2 were reversed. In contrast, silencing TIG1 promoted the proliferation of CRC, and when PLK2 was also silenced, the proliferation of CRC cells induced by TIG1 silencing was significantly inhibited. The above research results suggest that TIG1 can regulate the tumorigenesis of CRC by regulating the activity of PLK2.     

特异小干扰RNA敲除PLK1基因的表达

为研究特异小干扰RNA（siRNA）作用于大肠癌细胞株SW480中PLK1 (Polo-like kinase 1)基因表达的mRNA对该细胞分裂生长的影响,设计了对应于PLK1基因表达mRNA不同位点的10种特异siRNA,经化学合成后,用脂质体转染SW480细胞,实时定量PCR检测PLK1基因的表达,观察不同的siRNA作用强度,并计数细胞了解相应细胞的生长情况,western-blot观察PLK1表达蛋白的变化和流式细胞计数分析细胞周期改变。发现10种siRNA均可敲除PLK1基因表达的20 %以上,其中P1、P4和P9 3组敲除mRNA达80 ％以上,这3种siRNA及其混合物对PLK1基因mRNA的作用具有相应浓度效应,在25 nmol/L时达到最佳作用效果,而且相同浓度的混合物作用效果更好（超过95%）,PLK1表达蛋白质明显降低,细胞周期在G2期受到阻碍。72 h后的各种siRNA浓度下细胞生长变化与PLK1基因的mRNA水平变化相一致。结果表明化学合成的特异siRNA对SW480细胞中PLK1基因表达具有消除作用,混合物作用更强,在细胞水平上抑制了SW480细胞的分裂生长。     

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.     

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.     

Non-mitotic functions of polo-like kinases in cancer cells

Inhibitors of mitotic protein kinases are currently being developed as non-neurotoxic alternatives of microtubule-targeting agents (taxanes, vinca alkaloids) which provide a substantial survival benefit for patients afflicted with different types of solid tumors. Among the mitotic kinases, the cyclin-dependent kinases, the Aurora kinases, the kinesin spindle protein and Polo-like kinases (PLKs) have emerged as attractive targets of cancer therapeutics.The functions of mammalian PLK1-5 are traditionally linked to the regulation of the cell cycle and to the stress response. Especially the key role of PLK1 and PLK4 in cellular growth and proliferation, their overexpression in multiple types of human cancer and their druggability, make them appealing targets for cancer therapy. Inhibitors for PLK1 and PLK4 are currently being tested in multiple cancer trials. The clinical success of microtubule-targeting agents is attributed not solely to the induction of a mitotic arrest in cancer cells, but also to non-mitotic effects like targeting intracellular trafficking on microtubules. This raises the question whether new cancer targets like PLK1 and PLK4 regulate critical non-mitotic functions in tumor cells. In this article we summarize the important roles of PLK1-5 for the regulation of non-mitotic signaling. Due to these functions it is conceivable that inhibitors for PLK1 or PLK4 can target interphase cells, which underscores their attractive potential as cancer drug targets. Moreover, we also describe the contribution of the tumor-suppressors PLK2, PLK3 and PLK5 to cancer cell signaling outside of mitosis. These observations highlight the urgent need to develop highly specific ATP-competitive inhibitors for PLK4 and for PLK1 like the 3^rd generation PLK-inhibitor Onvansertib to prevent the inhibition of tumor-suppressor PLKs in- and outside of mitosis. The remarkable feature of PLKs to encompass a unique druggable domain, the polo-box-domain (PBD) that can be found only in PLKs offers the opportunity for the development of inhibitors that target PLKs exclusively. Beyond the development of mono-specific ATP-competitive PLK inhibitors, the PBD as drug target will support the design of new drugs that eradicate cancer cells based on the mitotic and non-mitotic function of PLK1 and PLK4.     

Abortive Cell Cycle Events in the Brains of Scrapie-Infected Hamsters with Remarkable Decreases of PLK3/Cdc25C and Increases of PLK1/Cyclin B1

Polo-like kinases (PLKs) consist of a family of kinases which play critical roles during multiple stages of cell cycle progression. Increase of PLK1 and decrease of PLK3 are associated with the developments and metastases of many types of human malignant tumors; however, the situations of PLKs in prion diseases are less understood. Using Western blots and immunohistochemical and immunofluorescent assays, marked increase of PLK1 and decrease of PLK3 were observed in the brains of scrapie strain 263K-infected hamsters, presenting obviously a time-dependent phenomenon along with disease progression. Similar alterations of PLKs were also detected in a scrapie infectious cell line SMB-S15. Both PLK1 and PLK3 were observed in neurons by confocal microscopy. Accompanying with the changes of PLKs in the brains of 263K-infected hamsters, Cdc25C and its phosphorylated forms (p-Cdc25C-Ser198 and p-Cdc25C-Ser216) were significantly down-regulated, whereas Cyclin B1 and PCNA were obviously up-regulated, while phospho-histone H3 remained almost unchanged. Moreover, exposure of the cytotoxic peptide PrP106-126 on the primary cultured cortical neuron cells induced similar changes of cellular PLKs and some cell cycle-related proteins, such as Cdc25C and its phosphorylated forms, phospho-histone H3. Those results illustrate obviously aberrant expressions of cell cycle regulatory proteins in the prion-infected neurons, which may lead to the cell cycle arrest at M phase. Possibly due to the ill-regulation of some key cell cycle events during prion infection, together with the fact that neurons are unable to complete mitosis, the cell cycle reentry in prion-infected neurons is definitely abortive, which may lead to neuron apoptosis and neuron degeneration.     

Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377-385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.     

CCNA1基因与肿瘤的相关性

细胞周期调节蛋白A1即细胞周期素A1（cyclinA1,CCNA1）在细胞周期调节中起关键作用,而细胞周期与肿瘤发生的关系是近年来肿瘤研究的热门课题之一。研究发现,CCNA1与恶性肿瘤的发生、生长、侵袭和转移等有着密切的关系,许多肿瘤的发生与CCNA1基因的启动子甲基化异常有直接或间接的关系,但在不同肿瘤中其发生机制并非相同。本文就CCNA1基因与肿瘤发生的相关性进行综述。     

Plk3 functions as an essential component of the hypoxia regulatory pathway by direct phosphorylation of HIF-1alpha

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.