过表达CDK11~(p58)促进人胰腺导管腺癌MIAPaCa-2细胞增殖

CDK11p58属于CDK11/PITSLRE蛋白激酶家族成员,由Cdc2L2编码,是一种重要的细胞周期调控蛋白.为了研究CDK11p58与胰腺癌细胞增殖的关系,我们通过采用脂质体转染真核表达载体及G418筛选的方式,获得了稳定过表达CDK11p58的MIAPaCa-2(人胰腺导管腺癌细胞)单克隆细胞,并通过流式细胞分析、MTT检测及real-time PCR的方法检测了细胞周期、细胞增殖能力及G1/S期相关调控基因的转录水平.结果显示,该单克隆细胞(实验组)与空载体组细胞和空白对照组细胞相比G1期细胞比例明显下降(P0.01),S期细胞比例明显上升(P0.01);细胞增殖能力明显提高(P0.01);cyclin D1、cyclin D3、p21基因mRNA水平较两组对照细胞明显升高(P0.01).提示过表达的CDK11p58通过上调cyclin D1、cyclin D3和p21基因的mRNA水平促进MIAPaCa-2细胞通过细胞增殖的关键限速点G1/S期,加快细胞增殖.     

植物来源的蛋白磷酸酶Cdc25C的新抑制剂

蛋白磷酸酶Cdc25C能够使有丝分裂激酶CDK1/cyclin B去磷酸化,从而促进细胞周期的进程.已经在一些肿瘤细胞中检测到Cdc25C的过量表达,这使得Cde25C成为肿瘤治疗中的潜在靶标.通过随机筛选,发现了八个CAe25C的天然新抑制剂(1-8),其IC50值在1.66～75.07umol/L之间.肿瘤细胞毒试验结果表明,其中四个化合物(化合物3,4,5,7)对十种肿瘤细胞株显示一定的细胞毒活性,其IC50值皆小于10ug/ml.     

多态性蛋白Mad2与其配体Cdc20121-138的相互作用研究

多态性蛋白Mad2是有丝分裂纺锤体检测点(SAC)的关键蛋白,也是多态性蛋白质家族中研究最广泛的成员之一.Mad2有两种不同的天然构象:O-Mad2和C-Mad2.Mad2构象间的转变及其与配体Cdc20间的相互作用对SAC发挥其生物学功能至关重要.本文利用荧光各向异性技术对O-Mad2和C-Mad2与配体TAMRA-Cdc20~(121-138)间相互作用的热力学及动力学过程进行了系统表征.结果表明:在无盐和低盐溶液(100 mmol/L NaCl)中,Mad2两种构象与Cdc20~(121-138)的平衡解离常数(K_D)均在10~(-6) mol/L,但C-Mad2与Cdc20~(121-138)结合的K_D值约为O-Mad2的1/5;在高盐(300 mmol/L NaCl)溶液中,Mad2两种构象与TAMRA-Cdc20~(121-138)结合的K_D值无明显差别.动力学实验结果显示,在同一种缓冲液中Mad2两种构象与Cdc20~(121-138)相互作用的解离速率常数k_d没有显著差别,而C-Mad2与Cdc20~(121-138)的结合速率常数k_a却比O-Mad2高一个数量级,这表明C-Mad2与Cdc20~(121-138)不仅结合力更强,且结合速率要快很多.Mad2与Cdc20~(121-138)突变体间的相互作用以及离子强度对二者相互作用的影响结果提示,Mad2和Cdc20间的相互作用不是通过静电相互作用,而可能是通过疏水相互作用来实现的.本研究为揭示多态性蛋白Mad2的构象转变机理及其在有丝分裂过程中的作用机制提供了重要的实验基础.     

酵母细胞周期调控的研究进展

酵母是一种研究细胞周期调控的好材料,在细胞周期的调控研究中具有重要作用。现在通常以芽殖酵母和裂殖酵母为代表进行研究。这两种酵母的细胞周期进程与高等真核生物相比各有其特点。 酵母细胞周期运行中存在有三个不同的控制点,即start点、S期启动点、G2/M转换处。在这三个不同的控制点起作用的CDK的组成是不同的。芽殖酵母分别是Cdc28-Clns;Cdc28-Clb5和Cdc28-Clb6;Cdc28-Clb1、Cdc28-Clb2、Cdc28-Clb3和Cdc28-Clb4。裂殖酵母中分别是Cdc2-Cig2;Cdc2-Cig2;Cdc2-Cdc13和Cdc2-Cig1,其中芽殖酵母中的Cdc28和裂殖酵母中的cdc2是等效基因。不同的控制点存在着不同 的调控机制,它们涉及到大量的基因,其中以G2/M转换处的调控机制研究得最早也最透彻。另外,APC途径在M中期/后期转化中起着重要作用。     

CyclinD1、CDK4、P16和结核菌L型感染在前列腺癌中的表达及临床意义

目的探讨CyclinD1、CDK4和P16在前列腺癌中的表达以及结核菌L型感染率及临床意义。方法应用免疫组化和抗酸染色等方法检测了65例前列腺癌（carcinoma of prostate,PCa）和30例良性前列腺增生（benignprostatic hyperplasia,BPH）中的CyclinD1、CDK4和P16的表达,以及结核菌L型的检出率。并对前列腺肿瘤主要临床资料和病理分级参数进行比较,用χ^2检验进行统计学处理。结果 CyclinD1、CDK4阳性表达前列腺癌明显高于前列腺增生（P〈0.01）;并与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈正相关。P16阳性表达前列腺增生明显高于前列腺癌（P〈0.01）;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.01-0.05）呈负正相关。结核菌L型检出率前列腺癌明显高于前列腺增生;与前列腺癌的临床分期、病理分级及淋巴结转移差异有统计学意义（P〈0.05）。结论 CyclinD1、CDK4和P16在前列腺肿瘤中不同程度异常表达以及结核菌L型检出率与肿瘤的临床分期、病理分级和转移相关,因此研究CyclinD1、CDK4和P16的阳性表达和结核菌L型感染与前列腺癌发生发展中可能有协同作用,具有重要的临床应用价值。     

dbcAMP通过Cdc25B蛋白调控小鼠1-细胞期受精卵发育

为了研究PKA激活剂dbcAMP通过调控小鼠Cdc25B蛋白S149和S321位点磷酸化状态影响 小鼠1-细胞期受精卵的发育,将质粒pBSK-Cdc25B-WT、pBSK-Cdc25B-S149A、pBSK- Cdc25B-S321A和pBSK-Cdc25B-S149A/S321A体外转录成mRNA;显微注射入S期受精卵中 ,在2 mmol/L dbcAMP的M16培养基中培养,观察其对受精卵发育、MPF活性及CDC2- pTyr15磷酸化状态的影响. 结果显示,在有dbcAMP存在时,各组受精卵卵裂时间延迟 ,但Cdc25B-S/A mRNAs注射组受精卵卵裂率明显高于Cdc25B-WT mRNA注射组,MPF 活性提前达到高峰;CDC2-pTyr15磷酸化状态和MPF活性变化相一致. 因此,在小鼠1- 细胞期受精卵有丝分裂过程中,PKA对小鼠Cdc25B蛋白S149位点与S321位点的磷酸化 修饰是控制受精卵G2/M转换的重要方式.     

生理条件下的S期检查点

细胞周期检查点在细胞遭遇DNA损伤因子的攻击或遇到营养缺乏等不利因素作用时,能够暂时阻止或减慢细胞周期的进程,是细胞在长期进化中发展起来的抵御DNA损伤的重要机制.不仅如此,最近的研究表明,在正常生理条件下,存在一种S期检查点,对DNA复制的速度进行调控.从分子水平而言,这种调控作用可能是通过一系列细胞周期调控蛋白如ATR、9-1-1复合体、Chk1、Cdc25A和CDK2等的作用来实现的.这种调节作用对细胞至关重要,它使DNA复制速度不致于过快,从而减少复制过程中发生错误的几率,维护基因组的稳定性.     

胃癌中CDK1和CDK2的表达及预后意义

目的探讨胃癌中CDK1和CDK2的表达情况及预后意义。方法应用免疫组化S-P法对48例各期胃癌和癌旁正常组织中CDK1和CDK2的表达情况进行检测。结果早期胃癌中CDK1和CDK2中表达较低(P0.05),进展期胃癌中表达更高(P0.05)。结论CDK1和CDK2可望成为胃癌早期预后指标和分子治疗的靶标。     

金葡菌L型感染致小鼠肿瘤中CyclinD1、CDK4和P16的表达及意义

目的探讨金黄色葡萄球菌(金葡菌)L型感染C57小鼠致瘤后,CyclinD1、CDK4和P16在小鼠肿瘤及癌前病变中的表达及相关性研究。方法动物实验观察发现金葡菌L型感染90只C57小鼠后11.1%(10/90)发生肿瘤;14.4%(13/90)引起癌前病变。革兰染色、免疫组化染色,检测小鼠肿瘤和癌前病变中金葡菌L型感染率和CyclinD1、CDK4和P16蛋白的阳性表达。结果 10只小鼠肿瘤及13只小鼠癌前病变中金葡菌L型检出率与正常对照组比较差异有明显统计学意义(P0.01)。CyclinD1、CDK4和P16蛋白的阳性表达与正常对照组比较差异有统计学意义(P0.01～0.05),呈正相关。结论金葡菌L型感染可能与CyclinD1、CDK4和P16蛋白在小鼠肿瘤发生和发展中有协同作用。     

Rac1和Cdc42在乳腺癌中的表达和临床意义

目的:研究Rac1和Cdc42在人乳腺癌中的表达及临床意义。方法:收集339例人乳腺癌组织样本,通过免疫组化的方法检测Rac1和Cdc42的表达情况,并分析其与乳腺癌临床病理学特征间的相关性。结果:Rac1和Cdc42在正常乳腺组织中几乎不表达,而在肿瘤组织的阳性表达率分别为35.9%和38.5%,均较正常乳腺组织显著升高,差异均具有统计学意义(P0.001和P0.05)。卡方检验分析表明,二者的表达与患者的年龄、肿瘤大小、组织分化程度、HER2状态无关(P0.05),而与TNM分期、淋巴结转移、肿瘤侵袭、ER状态和Ki-67表达有相(P0.05)。相关性分析表明,Rac1和Cdc42的表达与高TNM分期(r分别为0.443和0.295;P均0.001)、淋巴结转移阳性(r均为0.480和0.562;P均0.001)、肿瘤侵袭(r分别为0.412和0.440;P均0.001)、ER阴性表达(r分别为-0.517和-0.342;P均0.001)以及Ki-67高表达(r分别为0.338和0.454;P均0.001)呈正相关。结论:在乳腺癌组织中,Rac1和Cdc42作为癌基因表达增加,可能在乳腺癌恶性进程中发挥重要作用。     

3,3��-Diindolylmethane inhibits breast cancer cell growth via miR-21-mediated Cdc25A degradation

3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4, and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast cancer cell proliferation. These observations collectively suggest that by differentially modulating cellular signaling pathways DIM is able to arrest the cell-cycle progression of human breast cancer cells.     

Thr-370 is responsible for CDK11(p58) autophosphorylation, dimerization, and kinase activity

CDK11(p58), a member of the p34(cdc2)-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11(p58) interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11(p58) was autophosphorylated, and the main functions of CDK11(p58), such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11(p58) was autophosphorylated at Thr-370. By mutagenesis, we created CDK11(p58) T370A and CDK11(p58) T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11(p58), respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11(p58) and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivation of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11(p58). Moreover, Thr-370 mutants might affect CDK11(p58)-mediated signaling pathways.     

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.     

Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation

CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.     

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.