蛋白激酶C抑制剂Staurosporine对HeLa细胞周期的影响

用蛋白激酶C的抑制剂Staurosporine(10nmol/L)处理HeLa细胞,明显抑制HeLa细胞的增殖。这种抑制作用不是由于引起细胞死亡,而是因为细胞被阻断在G2期。这种阻断作用伴随着HeLa细胞多倍体的形成,提示Staurosporine抑制了HeLa细胞蛋白激酶C活性后引起的细胞阻滞,对细胞核的周期运转没有影响。进一步的探讨发现这种抑制作用可能是通过干扰细胞骨架的正确分布形成的,表明蛋白激酶C对于HeLa细胞由G2到M期正确过渡起重要作用。     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

蛋白激酶C与细胞周期

近年的研究表明,PKC涉及到细胞的周期调节。在酵母细胞和哺乳动物细胞均发现PKC参与细胞周期调控,从而提示PKC可能在进化上是一种保守的细胞周期调节子。一般认为PKC在两个点上对细胞周期起作用,即G1期和G2期到M期的过渡期（G2／M）。在G1期,PKC分别在早G1期和晚G1期作用有所不同,主要作用表现在使细胞停留在G1期的中末阶段,这一过程,主要涉及到抑制肿瘤抑制因子－成视网膜细胞瘤（Rb)蛋白的磷酸化。PKC的主要作用是降低周期素依赖激酶CDK2的活性、降低周期素E和A的表达和增加周期素依赖的周期抑制蛋白p21^WAF1和p27^KIP1的表达;在G2／M期,PKC对细胞周期的调节主要与Cdc2(CDK1)的活性抑制有关。     

The Replication Initiation Protein Sld3/Treslin Orchestrates the Assembly of the Replication Fork Helicase during S Phase

The initiation of DNA replication is a highly regulated process in eukaryotic cells, and central to the process of initiation is the assembly and activation of the replication fork helicase. The replication fork helicase is comprised of CMG (Cdc45, Mcm2–7, and GINS) in eukaryotic cells, and the mechanism underlying assembly of the CMG during S phase was studied in this article. We identified a point mutation of Sld3 that is specifically defective for Mcm3 and Mcm5 interaction (sld3-m10), and also identified a point mutation of Sld3 that is specifically defective for single-stranded DNA (ssDNA) interaction (sld3-m9). Expression of wild-type levels of sld3-m9 resulted in a severe DNA replication defect with no recruitment of GINS to Mcm2–7, whereas expression of wild-type levels of sld3-m10 resulted in a severe replication defect with no Cdc45 recruitment to Mcm2–7. We propose a model for Sld3-mediated control of replication initiation, wherein Sld3 manages the proper assembly of the CMG during S phase. We also find that the biochemical functions identified for Sld3 are conserved in human Treslin, suggesting that Treslin orchestrates assembly of the CMG in human cells.     

JNK相互作用蛋白通过JNK途径影响鼻咽癌的增殖和凋亡

EB病毒编码的瘤蛋白潜伏膜蛋白（LMP1）所介导的活化蛋白（AP-1）信号转导途径在细胞增殖、分化、转化与凋亡方面发挥着重要作用.越来越多的证据表明,AP-1信号转导通路中上游激酶JNK在鼻咽癌的发生发展过程中起着重要作用.最近克隆出来的JNK相互作用蛋白(JIP-1)是一种能抑制JNK核移位的胞浆锚蛋白.为探讨JIP在LMP1调控AP-1信号通路中的作用机制,采用间接免疫荧光法和报告基因法,发现JIP通过有效地抑制磷酸化的JNK从胞浆移位入核,从而抑制LMP1上调的AP-1活性.同时,JIP导入鼻咽癌细胞中,MTT法发现JIP能够明显抑制鼻咽癌细胞的生长.进一步发现转染JIP后细胞的集落形成率与对照组相比大约降低了53.6%,也抑制了细胞. 提示JIP可明显抑制细胞的增殖作用.进一步采用流式细胞术分析,结果发现JIP引起细胞G1/S期细胞阻滞,说明JIP是抑制细胞增殖的重要调节子.进一步采用流式细胞术定量发现,转染JIP后细胞的24 h凋亡百分率由1.25%上升到8.25%,上升约6.6倍,48 h由1.04%上升到31.45%,上升约30倍. 采用激光共聚焦显微镜发现,转染JIP后细胞核发生显著变化,核质由均匀状态固缩成高凝集状态,形成了典型的胞膜体.提示JIP可有效地促进细胞凋亡.结果表明,JIP可通过抑制活化的JNK核移位,降低LMP1所介导的AP-1信号通路.并进一步发现JIP可有效地抑制细胞增殖和细胞凋亡,从而提示JIP可作为新的治疗肿瘤潜在靶分子.     

Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes

PKD is a family of three serine/threonine kinases (PKD-1, -2, and -3) involved in the regulation of diverse biological processes including proliferation, migration, secretion, and cell survival. We have previously shown that despite expression of all three isoforms in mouse epidermis, PKD1 plays a unique and critical role in wound healing, phorbol ester-induced hyperplasia, and tumor development. In translating our findings to the human, we discovered that PKD1 is not expressed in human keratinocytes (KCs) and there is a divergence in the expression and function of other PKD isoforms. Contrary to mouse KCs, treatment of cultured human KCs with pharmacological inhibitors of PKDs resulted in growth arrest. We found that PKD2 and PKD3 are expressed differentially in proliferating and differentiating human KCs, with the former uniformly present in both compartments whereas the latter is predominantly expressed in the proliferating compartment. Knockdown of individual PKD isoforms in human KCs revealed contrasting growth regulatory roles for PKD2 and PKD3. Loss of PKD2 enhanced KC proliferative potential while loss of PKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability. This proliferation defect was correlated with up-regulation of CDK4/6 inhibitor p15^INK4B and induction of a p53-independent G1 cell cycle arrest. Simultaneous silencing of PKD isoforms resulted in a more pronounced proliferation defect consistent with a predominant role for PKD3 in proliferating KCs. These data underline the importance and complexity of PKD signaling in human epidermis and suggest a central role for PKD3 signaling in maintaining human epidermal homeostasis.     

Polo样激酶1在细胞周期及细胞周期监测点中的功能

Plk1（Polo-like kinase 1）是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶,是真核细胞有丝分裂的重要调控因子.Plk1随有丝分裂进程定位于不同位点,调节分裂期进入、纺锤体形成和胞质分裂等过程.Plk1能够与磷酸化的停靠蛋白结合,从而在不同空间被激活以满足其在细胞周期中的不同功能.Plk1还参与G2和M期DNA损伤监测点的调节,对于DNA损伤恢复后重新进入有丝分裂期是必须的.目前,Plk1的重要功能尤其是在DNA损伤监测点中发挥的重要功能正在被广泛研究.Plk1在多种恶性肿瘤中存在过表达且与肿瘤发生密切相关,对于Plk1功能的深入研究为以Plk1为靶的肿瘤治疗提供理论依据     

转录因子FOXM1在上皮性卵巢癌中的表达及临床意义

FOXM1(Forkhead box protein M1)是调控细胞增殖的重要转录因子,近年来研究表明与肿瘤发生密切相关,但与卵巢癌的关系尚不明确.通过检测68例卵巢癌标本、21例卵巢良性肿瘤标本、24例正常卵巢标本以及3株卵巢癌细胞系(A2780细胞、OVCAR3细胞、SKOV3细胞)中FOXM1的表达情况,分析其与临床参数之间的相关性及临床意义.结果显示卵巢癌中FOXM1表达明显高于卵巢良性肿瘤及正常卵巢组织,差异极为显著,其中低分化细胞中表达强于中高分化细胞(P=0.013),Ⅲ～Ⅳ期表达强于Ⅰ～Ⅱ期(P=0.011),但与病理类型无关;FOXM1在3株卵巢癌细胞系中均有较强表达.FOXM1在卵巢癌组织及3种卵巢癌细胞系中存在高表达,且与卵巢癌分化程度及临床分期有关.     

Protein tyrosine kinase pathway-derived ROS/NO productions contribute to G2/M cell cycle arrest in evodiamine-treated human cervix carcinoma HeLa cells

Abstract

A previous study indicated that reactive oxygen species (ROS) and nitric oxide (NO) played pivotal roles in mediating cytotoxicity of evodiamine in human cervix carcinoma HeLa cells. This study suggested that G2/M cell cycle arrest was triggered by ROS/NO productions with regulations of p53, p21, cell division cycle 25C (Cdc25C), Cdc2 and cyclin B1, which were able to be prevented by protein tyrosine kinase (PTK) activity inhibitor genistein or JNK inhibitor SP600125. The decreased JNK phosphorylation by addition of Ras or Raf inhibitor, as well as the increased cell viability by addition of insulin-like growth factor-1 receptor (IGF-1R), Ras, Raf or c-Jun N-terminal kinase (JNK) inhibitor, further demonstrated that the Ras-Raf-JNK pathway was responsible for this PTK-mediated signalling. These observations provide a distinct look at PTK pathway for its suppressive effect on G2/M transition by inductions of ROS/NO generations.     

Live-cell imaging of HP1α throughout the cell cycle of mouse C3H10T1/2 cells and rhythmical flickering of heterochromatin dots in interphase

Heterochromatin protein 1 alpha (HP1α) localizes to heterochromatin in interphase and shows dynamic molecular behavior in living cells. We previously reported that during mitosis, the majority of HP1α diffused into the cytoplasm but some remained in centromere heterochromatin. Here, we further characterize the molecular behavior of HP1α throughout the cell cycle. Time-lapse imaging of DsRed-HP1α through two successive cell divisions indicated that interphase can be divided into four phases. HP1α forms heterochromatin dots in early G1, which are maintained without any apparent changes (Phase 1). However, the HP1α dots begin to diffuse into the nucleoplasm and start flickering with a rhythmical cycle (Phase 2). Then, the HP1α dots diffuse further towards the periphery of the nucleus (Phase 3), and uniformly diffuse throughout the entire nucleus (Phase 4). Rhythmical flickering of HP1α dots in the middle of interphase may be useful for following cell cycle progression in mouse living cells.     

周期蛋白和周期蛋白依赖性激酶及相关激酶抑制剂在细胞周期进程中的调控机制研究进展

在细胞发育过程中,细胞周期起着至关重要的作用。细胞周期进程主要受细胞周期蛋白依赖性激酶(cyclin dependent kinase, CDK)、周期蛋白和内源性CDK抑制剂(cyclin-dependent kinase inhibitors,CKI)调控。其中,CDK是主要的细胞周期调节因子,可与周期蛋白结合形成周期蛋白-CDK复合物,从而使数百种底物磷酸化,调控分裂间期和有丝分裂进程。各类细胞周期蛋白的活性异常,可引起不受控制的癌细胞增殖,导致癌症的发生与发展。因此,了解CDK的活性变化情况、周期蛋白-CDK的组装以及CKI的作用,将有助于了解细胞周期进程中潜在的调控过程,为癌症与疾病的治疗和CKI治疗药物的研发提供基础。本文关注了CDK激活和灭活的关键事件,并总结了周期蛋白-CDK在特定时期及位置的调控过程,以及相关CKI治疗药物在癌症及疾病中的研究进展,最后简单阐述了细胞周期进程研究面临的问题和存在的挑战,以期为后续细胞周期进程的深入研究提供参考和思路。     

Polo-like kinase 1 (PLK1)-dependent phosphorylation of methylenetetrahydrofolate reductase (MTHFR) regulates replication via histone methylation

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the folate cycle and its genetic variations have been associated with various human diseases. Previously we identified that MTHFR is phosphorylated by cyclin-dependent kinase 1 (CDK1) at T34 and MTHFR underlies heterochromatin maintenance marked by H3K9me3 levels. Herein we demonstrate that pT34 creates a binding motif that docks MTHFR to the polo-binding domain (PBD) of polo-like kinase 1 (PLK1), a fundamental kinase that orchestrates many cell cycle events. We show that PLK1 phosphorylates MTHFR at T549 in vitro and in vivo. Further, we uncovered a role of MTHFR in replication. First, MTHFR depletion increased the fraction of cells in S phase. This defect could not be rescued by siRNA resistant plasmids harboring T549A, but could be restored by overproduction of Suv4–20H2, the H4K20 methyltransferase. Moreover, siMTHFR attenuated H4K20me3 levels, which could be rescued by Suv4–20H2 overproduction. More importantly, we also investigated MTHFR-E429A, the protein product of an MTHFR single nucleotide variant. MTHFR-E429A overexpression also increased S phase cells and decreased H4K20me3 levels, and it is linked to a poor glioma prognosis in the Chinese population. Collectively, we have unveiled a vital role of PLK1-dependent phosphorylation of MTHFR in replication via histone methylation, and implicate folate metabolism with glioma.     

Functional analysis of an auxin-inducible DNA-binding protein gene

Over the past decades, several studies indicate a correlation between the phytohormone auxin and cell division. The molecular players of this signaling pathway are now being uncovered. DNA Binding Protein1 from Arabidopsis (AtDBP1) is an auxin-inducible gene able to bind DNA non-specifically. In this work the tissue-expression pattern of this gene was investigated. Promoter-GUS analysis demonstrated that the AtDBP1 promoter is active in regions exhibiting intense cell division such as meristems and nematode feeding sites. Also, the promoter expression was modulated upon incubation with cell cycle blockers, indicating a potential role in cell division for this gene. Lastly, AtDBP1 antisense plants presented a higher insensitivity to auxin, and interfered negatively with auxin–induced callus formation and reduced apical dominance.     

Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.     

阳性表达表皮钙粘蛋白增加G0/G1期人乳腺癌细胞比例及其分子机制

表皮钙粘蛋白（E-cadherin）阴性的乳腺癌细胞株MDA-MB-231和MDA-MB-435转染野生型表皮钙粘蛋白基因,通过流式细胞仪测量细胞周期发现表皮钙粘蛋白阳性细胞生长变慢,更多细胞停滞在G0/G1期,蛋白质印迹证实由G0/G1期进入S期的重要调控分子细胞周期蛋白-D1（cyclin D1）下降了,并发现表皮钙粘蛋白还能降低直接激活细胞周期蛋白-D1基因转录的β-连环蛋白的蛋白质浓度.蛋白激酶B（PKB）能通过抑制糖原合成激酶-3β(GSK-3β)的活性来抑制β-连环蛋白降解,并在乳腺癌高转移细胞株中普遍过表达,其表达同样受到了表皮钙粘蛋白的抑制.并且在表皮钙粘蛋白阳性细胞中,作为PKB上游信号分子并能激活PKB的粘着斑激酶 (FAK) 和整联蛋白相关激酶(ILK)蛋白量也发生下降,能抑制PKB激活的PTEN蛋白量却增加了.结果显示,表皮钙粘蛋白能通过降低乳腺癌细胞中的PKB蛋白浓度,并通过上游信号分子抑制PKB的激活,进而降低PKB对β-连环蛋白降解的抑制作用,导致β-连环蛋白直接调控的靶基因细胞周期蛋白D1的表达量下降,引起更多的细胞停止在G0/G1期.