Cell cycle-related transformation of the E2F4-p130 repressor complex

During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.     

Cell membrane polarity in rat ascites hepatoma cells

Summary As previously described, a cell surface-associated adhesive factor (AF) was separated from differentiated rat ascites hepatoma AH136B cells (forming cell islands in vivo) and highly purified by chromatography. AF induces not only aggregation of dissociated AH136B cells or undifferentiated rat ascites hepatoma AH109A cells (present as free cells in vivo), but also adhesiveness characterized by the development of junctional complexes. The localization of AF on the surfaces of AH136B cell islands was investigated using anti-AF IgG (Fab fragment) coupled to peroxidase. AF was detected in the contact region of the lateral surfaces of the AH136B cells and in the intercellular spaces. In contrast, no AF was detectable on the apical non-contacted free cell surfaces of AH136B cells. Fluorescence studies revealed that biotin-labeled AF did not bind to the apical surface of AH136B cell islands. These results indicate a distinct difference between apical and lateral surfaces of AH136B cells; neither AF nor binding site for AF were localized on the apical surface of AH136B cells, whereas both were localized on the lateral surface. On the other hand, AH136B cells detached from the cell islands, or during the process of partial dissociation from them, showed the loss of the AF localization and binding site of AF on the surfaces. The results suggest that AH136B cell surfaces may be polarized in terms of the AF localization, and this polarization may be lost after cell dissociation.     

Effect of methylglyoxal on tumour microtubular protein

Methylglyoxal inhibits cell division, exerting an antiproliferative action on tumour cells. Supernatants from ascites hepatoma cell homogenate, previously incubated with the aldehyde, showed a decrease in colchicine binding activity dependent on methylglyoxal concentration. In contrast, in vivo treatment of tumour-bearing rats apparently did not cause a significant impairment of microtubular protein, suggesting that the aldehyde interaction with microtubules cannot be considered responsible for its carcinostatic action.     

姜黄素对两株不同转移力小鼠腹水型肝癌细胞生物学特性的影响

目的 研究姜黄素（Curcumin,Cur）对来源于同一亲本的小鼠腹水型肝癌高、低淋巴道转移株（HCa-F和HCa-P）增殖、细胞周期分布及皮下移植HCa-F和HCa-P的615小鼠生存期的影响,并探讨2株细胞对Cur的敏感性差异是否存在显著性.方法 用15～250 μmol/L Cur分别处理HCa-F和HCa-P 48 h,以MTT法检测细胞的生长活性,以生长曲线评估药物对细胞生长的作用,以流式细胞仪检测药物对细胞周期分布的影响,观察Cur对细胞形态的影响.给615小鼠右腋中线皮下接种HCa-F和HCa-P细胞,通过ig给药的方法,观察Cur对荷瘤鼠生存期的影响.结果 Cur可抑制HCa-F和HCa-P的生长,抑制率分别为7.23%～82.23%和6.04%～61.31%,且呈剂量依赖性;IG0在HCa-F为51.48μmol/L,在HCa-P为90.87μmol/L;在非细胞毒性最大剂量15μmol/L Cur作用下,2株细胞的生长速度均受到抑制,群体倍增时间延长;HCa-F细胞形态受到影响;细胞周期被重新分布,HCa-F被阻滞在S期,HCa-P被阻滞于S期和G2/M期.经ig给予150 mg/kg的Cur可使皮下接种HCa-F和HCa-P的615小鼠生存率提高分别达45.06%和49.68%.结论 Cur可抑制HCa-F和HCa-P的增殖,使细胞周期重新分布,且高转移细胞株HCa-F较低转移细胞株HCa-P对Cur更为敏感Cur可延长荷瘤鼠的生存期.     

人类主要Cyclins在MOLT-4细胞阻断动力学下的表达规律

细胞周期素与相应的细胞周期素依赖性蛋白激酶相结合,驱动着细胞通过细胞周期各时相,而细胞周期素时相性规律大多来自酵母研究或同步化细胞的分析。本研究着重于人类非同步化细胞的细胞周期素时相性规律的揭示。采用人类白血病细胞株MOLT-4,使其处于对数生长期,加以有丝分裂中期阻滞法,应用多参数流式细胞术分析人类主要细胞周期素B1、A和E。分析发现,细胞周期素B1峰值在M期,降解于M期后,细胞周期素A峰值在G2期,降解于M期,细胞周期素E峰值在G1晚期,降解于S期。以上结果,使我们首次在人类非同步化培养细胞展现了主要细胞周期素的时相性表达规律。     

Inactivation of TGF-beta signaling in lung cancer results in increased CDK4 activity that can be rescued by ELF

Escape from TGF-beta inhibition of proliferation is a hallmark of multiple cancers including lung cancer. We explored the role of ELF, crucial TGF-beta adaptor protein identified from endodermal progenitor cells, in lung carcinogenesis and cell-cycle regulation. Interestingly, elf-/- mice develop multiple defects that include lung, liver, and cardiac abnormalities. Four out of 6 lung cancer and mesothelioma cell lines displayed deficiency of ELF expression with increased CDK4 expression. Immunohistochemistry and Western blot analysis of primary human lung cancers also showed decreased ELF expression and overexpression of CDK4. Moreover, rescue of ELF in ELF-deficient cell lines decreased the expression of CDK4 and resulted in accumulation of G1/S checkpoint arrested cells. These results suggest that disruption in TGF-beta signaling mediated by loss of ELF in lung cancer leads to cell-cycle deregulation by modulating CDK4 and ELF highlights a key role of TGF-beta adaptor protein in suppressing early lung cancer.     

血红素加氧酶-1对肝癌细胞细胞周期调控因子的影响

目的观察血红素加氧酶-1（heme oxygenase 1,HO-1）对人肝癌细胞HepG2细胞周期调控因子的影响。方法构建含有野生型和突变型HO-1基因的重组载体pcDNA3．1（＋）-wtHO-1和pcDNA3．1（＋）-mHO-1G143H。利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组。通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系。经半定量RT—PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平。在HO-1表达改变的稳转细胞系中,利用Western印迹检测转染细胞系中P21、P27蛋白表达水平。结果成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达。结论HO．1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关。HO-1可能通过其它机制调节p21和p27的表达。     

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.     

Genetic interactions among genes involved in the STT4-PKC1 pathway of Saccharomyces cerevisiae

Loss of yeast protein kinase C function results in three distinct phenotypes: staurosporine sensitivity, cell lysis and blockage of cell cycle progression at the G2/M boundary. Genetic analysis of the PKC1/STT1 protein kinase C gene and its interactions with STT4, encoding an upstream phosphatidylinositol 4-kinase, and BCK1, encoding a downstream protein kinase, reveal that they form part of a single pathway. However, the BCK1-20 mutation (a gain-of-function mutation of BCK1) or overexpression of PKC1 cannot suppress all of the phenotypes caused by the loss of STT4 function, strongly suggesting the existence of a branch point between STT4 and PKC1. We also describe the MSS4 gene, a multicopy suppressor of the temperature-sensitive stt4-1 mutation. MSS4 is predicted to encode a hydrophilic protein of 779 amino acid residues and is essential for cell growth. Based on genetic and biochemical data, we suggest that MSS4 acts downstream of STT4, but in a pathway that does not involve PKC1. GenBank accession number: The accession number for the MSS4 sequence reported in this paper is D13716.     

P15INK4B高表达对人黑色素瘤细胞G1/S进程的阻滞及与MAPK信号相关性之初探

利用TdR-N_2O同步法分别获得P15高表达的MLIK6和表达空载体的MLC2的M期细胞和G_1期细胞,~3H-TdR掺入结果显示,与对照组细胞MLC2相比,实验组细胞MLIK6从G_1期进入S期时间延长2h,并且掺入强度明显减弱,DNA合成被抑制。进一步观察了P15~(INK4B)对G_1/S相关调控蛋白的影响,在M期细胞释放8h(晚G_1期细胞)后,与对照组MLC2细胞相比,实验组MLIK6细胞中CyclinD1,CyclinE,Cdk4,C-Myc蛋白水平均降低。相反,P27~(KIP1)的表达却上升。同时探讨了MAPK信号在P15~(INK4B)阻抑A375细胞G_1/S转换中的作用与相关性,结果显示晚G_1期的MLIK6细胞中ERK1,ERK2水平变化不大,而具有活性的P-ERK1和P-ERK2均表现出下降。上述实验表明,P15~(INK4B)可能通过作用于G_1期相关的周期调节蛋白和抑制ERK1和ERK2活性,阻滞G_1/S转换与抑制DNA合成。     

苜蓿银纹夜蛾核型多角体病毒的感染对Sf9细胞周期的影响

病毒的感染导致细胞内部发生一系列变化.应用流式细胞仪FACS的荧光检测,测出Sf9细胞完成整个周期循环大约需要18h,G\-1、S、G\-2/M各时相的时间间隔约为6h;AcNPV感染Sf9细胞12-18h,细胞被抑制于G\-2/M期;Sf9细胞同步于G\-1/S期后释放细胞并用AcNPV感染,12h后,2/3的细胞处于G\-2/M期,1/3的细胞处于S期.     

Synthesis and biological evaluation of 1,2,3-triazole linked aminocombretastatin conjugates as mitochondrial mediated apoptosis inducers

A series of 1,2,3-triazole linked aminocombretastatin conjugates were synthesized and evaluated for cytotoxicity, inhibition of tubulin polymerization and apoptosis inducing ability. Most of the conjugates exhibited significant anticancer activity against some representative human cancer cell lines and two of the conjugates 6d and 7c displayed potent cytotoxicity with IC₅₀ values of 53 nM and 44 nM against A549 human lung cancer respectively, and were comparable to combretastatin A-4 (CA-4). SAR studies revealed that 1-benzyl substituted triazole moiety with an amide linkage at 3-position of B-ring of the combretastatin subunit are more active compared to 2-position. G2/M cell cycle arrest was induced by these conjugates 6d and 7c and the tubulin polymerization assay (IC₅₀ of 1.16 μM and 0.95 μM for 6d and 7c, respectively) as well as immunofluorescence analysis showed that these conjugates effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Colchicine competitive binding assay suggested that these conjugates bind at the colchicine binding site of tubulin as also observed from the docking studies. Further, mitochondrial membrane potential, ROS generation, caspase-3 activation assay, Hoechst staining and DNA fragmentation analysis revealed that these conjugates induce cell death by apoptosis.     

URI基因稳定表达对肝癌SMMC-7721细胞增殖能力的影响

目的:瞬转以及筛选出能够稳定表达URI(RPB5-mediating protein)基因的SMMC-7721细胞株,以其为模型研究URI基因对人肝癌SMMC-7721细胞增殖的影响.方法:首先,抽提URI的重组质粒并转染到SMMC-7721细胞中,在G418药物的筛选下选出能够稳定表达URI基因的细胞株,RT-PCR法和酶切检测该稳定细胞中URI基因的表达效率以确认URI是否稳定表达,MTT法和克隆形成实验检测URI基因对SMMC-7721细胞增殖的影响.结果:成功建立URI基因过表达的稳定细胞株.与SMMC-7721细胞对照组比较,其URI mRNA表达水平显著上调,能稳定表达URI细胞株的增殖,克隆形成率明显升高.结论:pFLAG-CMV-4-URI重组质粒能使URI在肝癌SMMC-7721细胞内稳定表达,过表达URI基因有可能帮助细胞通过G2/M期检验点来提高肝癌细胞SMMC-7721的增殖能力.     

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.     

The cell cycle and development: redundant roles of cell cycle regulators

The existence of families of cell cycle regulators reflects the need by a developing organism to precisely control proliferation of its cells and also suggests that family members may play redundant roles. Recent advances have shown redundancy to be a theme in development.