Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

STAT3反义核酸对HL-60细胞周期及c-myc mRNA表达的影响

实验分反义组、无义组、脂质体组、空白组,转染后,应用四唑盐（MTT）比色法分析细胞增殖率,流式细胞仪检测细胞周期,RT-PCR检测细胞中STAT3 mRNA和c-myc mRNA的表达。探讨STAT3反义寡核苷酸对白血病细胞HL-60细胞周期及c-myc的影响。STAT3 ASODN抑制HL-60细胞增殖呈时间和浓度依赖性;反义寡核苷酸作用后,G0/G1期细胞明显减少,S期细胞增多,细胞周期进程受到明显阻滞;反义寡核苷酸作用48h后细胞内STAT3mRNA及c-myc mRNA的表达水平下降,与各对照组比较有显著性差异（P〈0.05）。STAT3 ASODN能够明显抑制HL-60细胞增殖,并能阻滞HL-60细胞于G0/G1期、并下调c-myc mRNA的表达。     

片仔癀对人卵巢癌OVCAR-3细胞增殖、凋亡及周期的影响

目的:研究片仔癀对人卵巢癌细胞株OVCAR-3增殖抑制作用,及其对细胞周期、细胞凋亡的影响。方法:采用MTT法观察片仔癀对人卵巢癌细胞株OVCAR-3细胞的增殖抑制率,流式细胞仪检测细胞凋亡及细胞周期,western-blot检测相关蛋白的表达。结果:片仔癀以剂量依赖式抑制人卵巢癌细胞株OVCAR-3细胞增殖,片仔癀250、500、1000μg·mL-1作用于OVCAR-3细胞24 h后,其早期凋亡率分别为6.6%、30.9%、43.2%,而对照组为0%,其诱导凋亡作用呈现剂量依赖性;细胞积聚在G0/G1期,同时S期细胞比例减少;Akt、PARP、CDK6表达下调。结论:片仔癀可以抑制OVCAR-3细胞增殖及诱导细胞凋亡作用,并能阻滞细胞于G0/G1期,有望成为卵巢癌治疗药。     

抑制VEGF基因表达对乳腺癌细胞细胞周期的影响

目的：研究针对VEGF基因的siRNA（small interferenceRNA）对乳腺癌MCF-7细胞细胞周期的影响。方法：依据Promega公司在网上提供的设计软件,设计针对VEGF基因的siRNA,合成DNA模板,体外转录合成siRNA。脂质体转染法将合成的siRNA转染入MCF-7细胞,以未转染细胞以及错义序列siRNAscr转染细胞为对照。用细胞计数法检测siRNA对MCF-7细胞增殖的影响：流式细胞法检测细胞周期变化,RT—PCR法比较转染前后p21、CyclinDl表达水平的变化,Westemblot检测转染前后磷酸化ERK的表达。结果：细胞计数法结果显示,转染24h后siRNA明显抑制MCF-7细胞增殖,转染48h后,抑制效率稳定。siRNA转染后能有效地抑制MCF-7细胞的增殖,阻滞细胞周期于G0／G1期,S期细胞明显减少,G0／G1期细胞比例逐渐增多;p21mRNA表达显著上调,抑制CyclinD1mRNA及磷酸化ERK蛋白的表达。结论：体外转录合成的siRNA可能通过上调细胞周期蚤白激酶抑制剂p21的表达,下调CyclinDl及磷酸化ERK的表达,将细胞周期阻滞于G0／G1期,从而显著抑制MCF-7细胞的增殖。     

蟾毒灵可抑制人红白血病细胞增殖并下调肾母细胞瘤1基因表达

已有研究证实蟾毒灵具有抑制肿瘤细胞增殖及诱导细胞凋亡的作用,在白血病治疗中疗效显著,然而其机制尚未阐明。本研究试图探讨蟾毒灵对人红系白血病(HEL)细胞增殖,肾母细胞瘤基因1 (Wilms'tumor 1 gene, WT1)甲基化的影响及其可能的作用机制。本研究采用不同浓度的蟾毒灵处理HEL细胞,观察细胞形态、增殖情况和细胞周期,采用RT-PCR、Western blotting和免疫细胞化学法检测WT1的mRNA和蛋白表达水平,并用甲基化特异性分析WT1的DNA甲基化和DNA甲基转移酶3a (DNMT3a)的蛋白表达水平。研究结果表明,蟾毒灵对HEL细胞的增殖抑制作用呈剂量依赖性,抑制率为23.13%～84.62%。在蟾毒灵处理的HEL细胞中观察到典型的凋亡形态特征;细胞周期增殖指数由75.45降至49.67;WT1 mRNA及其蛋白表达水平随着蟾毒灵剂量的增加而逐渐降低,同时WT1基因的甲基化状态由未甲基化状态变为部分或完全甲基化状态。而蟾毒灵处理后DNMT3a蛋白的表达水平逐渐增加,呈剂量依赖性。我们的研究初步说明蟾毒灵不仅能显著抑制HEL细胞增殖,阻滞G0/G1期细胞周期,还能诱导细胞凋亡,下调WT1的表达水平。     

茅苍术提取物在肺癌A549细胞中的抗增殖作用机制

目的:研究茅苍术提取物对肺癌A549细胞的肿瘤增殖抑制效果及机制。方法:MTT比色法检测茅苍术提取物对A549细胞增殖的抑制效果;流式细胞术分析茅苍术提取物对肺癌A549细胞周期的影响;Western Blot检测茅苍术刺激前后细胞周期蛋白D1(cyclin D1)蛋白表达量变化。结果:茅苍术提取物能有效抑制肺癌A549细胞增殖,且抑制效果与茅苍术提取物浓度呈现依赖性,茅苍术提取物刺激48h后,抑制效果最佳,IC50约为77.12μg/ml;细胞周期分析显示:茅苍术提取物可将细胞周期阻滞在G0/G1期,50μg/ml茅苍术提取物刺激48h后,G1期上升至53.41%;cyclin D1蛋白含量因茅苍术作用而下调。结论:茅苍术提取物能通过有效抑制cyclin D1的表达,进而将细胞周期阻滞于G1期,最终实现其抗增殖作用机制。     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G_1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.     

丙戊酸钠抑制A549细胞生长

目的研究丙戊酸钠对肺癌A549细胞增殖和细胞周期的影响。方法MTT检测生长抑制,流式细胞仪检测细胞周期和凋亡,Western blot检测p21WAF1/CIP1蛋白表达。结果丙戊酸钠以剂量依赖性方式抑制A549细胞生长;丙戊酸钠上调G0/G1期比例,下调S期和G2/M期,不影响细胞凋亡;丙戊酸钠上调p21WAF1/CIP1蛋白表达。结论丙戊酸钠上调p21WAF1/CIP1表达,使细胞阻滞于G0/G1期,抑制A549细胞生长。     

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.     

AS-mCLB1重组质粒体外抗肿瘤及化疗增敏作用

研究反义全长小鼠细胞周期蛋白B1重组质粒(pAS-mCLB1)体外抗肿瘤及化疗增敏作用。扩增后少量抽提并纯化pAS-mCLB1,通过脂质体将其转染入小鼠露易丝肺癌(LL/2)细胞,RT-PCR和Western印迹测定细胞内细胞周期蛋白B1的表达,观察转染后细胞形态变化,MTT法检测细胞增殖活性,流式细胞仪检测细胞周期及凋亡。细胞转染48h后,用化疗药物健择(gemcitabine;0.2μmol/L)处理24h,MTT法测定健择对细胞的杀伤作用。研究提示pAS-mCLB1转染后LL/2细胞形态明显异常,细胞内细胞周期蛋白B1表达显著下调,细胞周期阻滞于G1期,增殖受抑,凋亡增加;健择对pAS-mCLB1转染后LL/2细胞的杀伤作用显著增强。重组质粒pAS-mCLB1体外具有明显的抗肿瘤作用,并能增强肿瘤细胞对化疗药物的敏感性,估计上述作用与其下调肿瘤细胞内细胞周期蛋白B1表达,从而诱导细胞周期阻滞及凋亡等作用相关。     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.     

Characterization of Chrysanthemum ClSOC1-1 and ClSOC1-2, homologous genes of SOC1

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.