A novel mitosis-associated lncRNA,MA-linc1, is required for cell cycle progression and sensitizes cancer cells to Paclitaxel

Long noncoding RNAs (lncRNAs) are major regulators of many cellular processes including cell cycle progression and tumorigenesis. In this study, we identify a novel lncRNA, MA-linc1, and reveal its effects on cell cycle progression and cancer growth. Inhibition of MA-linc1 expression alters cell cycle distribution, leading to a decrease in the number of G1 cells and a concomitant increase in all other stages of the cell cycle, and in particular G2/M, suggesting its involvement in the regulation of M phase. Accordingly, knock down of MA-linc1 inhibits M phase exit upon release from a mitotic block. We further demonstrate that MA-linc1 predominantly functions in cis to repress expression of its neighboring gene, Purα, which is often deleted in human cancers and whose ectopic expression inhibits cell cycle progression. Knock down of Purα partially rescues the MA-linc1 dependent inhibition of M phase exit. In agreement with its suggested role in M phase, inhibition of MA-linc1 enhances apoptotic cell death induced by the antimitotic drug, Paclitaxel and this enhancement of apoptosis is rescued by Purα knockdown. Furthermore, high levels of MA-linc1 are associated with reduced survival in human breast and lung cancer patients.Taken together, our data identify MA-linc1 as a novel lncRNA regulator of cell cycle and demonstrate its potential role in cancer progression and treatment.     

Pretreatment with 5-FU enhances cisplatin cytotoxicity in head and neck squamous cell carcinoma cells

PURPOSE: In the treatment of head and neck malignancy, cisplatin and 5-FU have been used the most as chemotherapeutic agents. The difference in efficacies of these is unclear and controversial. To investigate more effective schedule, we analyzed the cytotoxicity in different treatment sequence with two agents in vitro and the mechanism for different effectiveness. METHODS: UM-SCC-23 and UM-SCC-81B, head and neck squamous cell carcinoma cell lines, were analyzed for cellular killing in alternative sequence treatment with cisplatin and 5-FU. The treatment schedule was designed based on the clinical regimen. To determine the mechanism for the difference of cytotoxicity with each schedule, cell cycle distributions of both cells after 5-FU treatment with various durations were analyzed by flow-cytometry and immunostaining with anti-PCNA and anti-BrdU. RESULTS: 5-FU pretreatment followed by cisplatin treatment showed higher cell killing in both types of cells than the reverse treatment schedule. In the cell cycle analysis and immunostaining after the treatment of 5-FU, the rate of PCNA-positive cells was increased from 24 to 144 h in both cells. The rate of BrdU-positive cells of UM-SCC-81B in flow-cytometry was also increased, while that of UM-SCC-23 was gradually decreased. These data suggested that the cells treated with 5-FU for more than 144 h were still in the S-phase with or without DNA synthesis. CONCLUSIONS: In head and neck carcinoma cells, we showed 5-FU pretreatment enhanced cisplatin cytotoxicity. The result of cell cycle analysis and immunostaining showed S-phase arrest by treatment of prolonged 5-FU treatment. The very long arrest in S-phase might be a mechanism to enhance cisplatin cytotoxicity by 5-FU pretreatment. We thus suggest pretreatment with 5-FU to enhance the effectiveness of cisplatin-based chemotherapy.     

shRNA靶向沉默Eag 1钾通道对骨肉瘤细胞增殖、细胞周期及cyclin D1／E表达的影响

目的 探讨短发夹RNA（shRNA）靶向沉默Ether-go-go 1（Eag1）钾通道对骨肉瘤MG-63细胞增殖、细胞周期及cyclin D1／E表达的影响。 方法 使用成功构建的Ad5-Eag1-shRNA表达载体转染MG-63细胞（抑制组）,同时设转染无义序列（Ad5-Control-shRNA）的空转染组及不进行任何处理的对照组。采用逆转录聚合酶链式反应和Western blotting检测Ad5-Eag1-shRNA转染骨肉瘤MG-63细胞后的Eag1 mRNA和蛋白水平,分别采用CCK-8法和克隆形成实验检测各组的细胞增殖情况,流式细胞仪和Western blotting分别检测各组的细胞周期变化和细胞周期蛋白（cyclin D1和cyclin E）的蛋白水平。建立裸鼠骨肉瘤异种移植模型并测量各组裸鼠肿瘤体积的变化情况。结果 抑制组的Eag1 mRNA和蛋白水平均低于空转染组和对照组,差异有统计学意义（P＜0.05）;与其余两组相比,抑制组的细胞增殖、克隆形成数、S期细胞比例及细胞周期蛋白水平均降低,而G0／G1期细胞比例升高,以上差异均有统计学意义（P＜0.05）。抑制组裸鼠的瘤体积小于空转染组和对照组（P＜0.05）。结论 通过shRNA抑制Eag1基因表达可抑制MG-63细胞的增殖并诱导G0／G1期阻滞,可能与调控cyclin D1／E通路有关,有望成为骨肉瘤治疗和诊断的新靶点。     

鼻咽癌相关基因NAG7对鼻咽癌细胞周期及凋亡的影响

背景与目的NAG7基因是我室克隆的鼻咽癌相关的肿瘤抑制候选基因,其功能与作用机制目前尚不清楚.研究鼻咽癌相关的潜在抑瘤基因NAG7对鼻咽癌细胞系(HNE1)细胞周期和细胞凋亡的影响,并探讨其作用机制.方法采用脂质体转染技术将NAG7基因导入HNE1细胞,建立稳定表达NAG7的细胞株,Northernblot分析转染细胞NAG7基因的表达,并采用流式细胞技术检测细胞周期、细胞周期素及细胞凋亡的改变,并用westernblot验证.结果NAG7基因重表达的HNE1细胞与空载体转染的HNE1和HNE1细胞相比G0/G1期细胞数增加(P<0.05),S期细胞数减少;细胞凋亡数目增加(P<0.05);细胞周期素A、D1、E表达明显降低(P<0.05),细胞周期素B1表达降低,Westernblot检测亦证实cyclinD1和cyclinE表达明显下调.结论NAG7的重表达导致细胞周期素表达下调,从而延缓细胞经G1期进入S周期及诱导细胞凋亡增加进而抑制NPC细胞的过度增殖.     

饥饿诱导肿瘤细胞自噬对细胞周期的影响

背景与目的:无血清饥饿方法可以诱发细胞自噬,同时也可以引起细胞周期阻滞。虽然研究者们对细胞自噬与细胞周期已经进行了深入的研究,但是对于两者之间的关系还是知之甚少。本研究旨在通过研究细胞周期素(Cyclin)在饥饿诱导的细胞发生自噬时表达的变化,来探讨自噬对细胞周期的影响。方法:对照组:d-Hanks液代替培养基饥饿诱导处于对数生长期的HeLa细胞,收获饥饿0、3、6、12h的细胞。实验组:用d-Hanks液开始饥饿的同时加入3-甲基腺嘌呤(3-methyladenine,3-MA),收获饥饿0、3、6、12h的细胞,用流式细胞术和Western blot法检测细胞周期素和特异性标记自噬的兔抗人微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1 light chain 3,LC-3)。结果:饥饿3h对照组HeLa细胞中有LC-3表达,并随饥饿时间延长而逐渐增加。Cyclin E、Cyclin D3在饥饿3h开始减少,在饥饿6h降低至最低水平,而Cyclin A、Cyclin B1在饥饿6h开始下降。实验组HeLa细胞在饥饿3h时LC-3的表达水平较低,随饥饿时间的延长LC-3表达基本不变化,Cyclin D3、Cyclin E、Cyclin A、Cyclin B1表达的下降速度也较对照组减慢。结论:自噬参与饥饿诱导的Cyclin降解的水解过程,Cyclin D3、Cyclin E水解比Cyclin A、Cyclin B1出现早,降解速度快。     

姜黄素对白血病细胞的细胞周期阻断与细胞周期蛋白的关系

目的研究姜黄素（Cur）对K562细胞和HL-60细胞的细胞周期阻断与细胞周期蛋白的关系。方法用流式细胞光度术对K562细胞和HL-60细胞进行细胞周期检测,用蛋白免疫印迹检测细胞周期蛋白水平。结果姜黄素0—10mg／L作用24h可将K562细胞和HL-60细胞阻滞于G0／G1期,该期细胞比例增加;阻断细胞从S期向G2／M期转化,G2／M期细胞比例减少;姜黄素作用24h减少K562细胞和HL-60细胞Cyclin D1、Cyclin B1的蛋白水平,但几乎不影响K562细胞和HL-60细胞Cyclin A的蛋白水平。结论姜黄素扰乱K562细胞和HL-60细胞的细胞周期进程与Cyclin D1和Cyclin B1的蛋白水平减少有一定关系。     

Growth inhibition and sensitization to cisplatin by zoledronic acid in osteosarcoma cells

Since osteosarcoma is a drug-resistant disease, the aim of the present study was to explore the possible interest of therapeutic approaches including nitrogen-containing biphosphonate zoledronic acid using osteosarcoma cell lines with different genetic backgrounds. Parental p53⁺/pRb⁺ U2-OS, p53-mutant U2-OS (U2-OS/175) and p53⁻/pRb⁻ SAOS were sensitive to zoledronic acid with no significant differences in IC₅₀ values. Analysis of cell cycle distribution revealed a time-dependent shifting of U2-OS cells towards G2 phase with cell cycle arrest in G2 phase at 96 h of exposure to the compound. Conversely, U2-OS/175 and SAOS cells responded to treatment with transient cell accumulation in S phase up to 48–72 h, respectively. Cell lines were exposed to increasing concentrations of cisplatin alone or combined with sub-toxic doses of zoledronic acid. A growth inhibitory effect was seen after combined treatment in U2-OS, otherwise resistant to cisplatin up to 100 ng/ml. Zoledronic acid did not efficiently sensitized U2-OS/175 and SAOS to cisplatin, thereby suggesting that different behavior may depend on p53 mutation. This data was confirmed in U2-OS cells where p53 expression was downregulated by RNA interference. Present findings indicate occurrence of sensitization to cisplatin by zoledronic acid in wild-type p53 osteosarcoma cells but not in p53-null cells nor in cells expressing a dominant-negative form of p53, supporting that wild-type p53 is required for synergistic interaction of cisplatin and zoledronic acid.     

TNF与U937细胞的bcl—2基因表达及细胞周期的关系

目的：探讨肿瘤坏死因子（ＴＮＦ）与白血病Ｕ９３７细胞的ｂｃｌ－２基因表达和细胞周期的关系。方法：首先用ＴＮＦ处理Ｕ９３７细胞，提取小片段ＤＮＡ进行ＤＮＡ断裂分析，继而用ＲＴ－ＰＣＲ和Ｓ－Ｐ免疫组化染色法检测Ｕ９３７细胞的ｂｃｌ－２基因表达变化，并通过流式细胞术分析ＴＮＦ对Ｕ９３７细胞的ＤＮＡ含量和细胞周期的影响。最后，用磷酸钙沉淀法将ｂｃｌ－２基因转染到Ｕ９３７细胞，经Ｇ４１８筛选稳定转染子，用不     

Overexpression of Phosphatidylinositol Synthase Enhances Growth and G1 Progression in NIH3T3 cells

Phosphatidylinositol (PI) turnover is thought to play an important role in the regulation of cell growth. PI synthase (PIS, cytidine diphosphate (CDP)-diacylglycerol (DG): myo -inositol 3–phos–phatidyltransferase, EC 2.7.8.11) acts at the last step in the de novo biosynthesis of PI by catalyzing the condensation of CDP-DG and myo -inositol. To study the physiological role of PIS, we established murine NIH3T3 fibroblasts that stably overexpress PIS, by transfection with PIS cDNA (NIH-PIS cells). In immunofluorescence assays, the constitutively overexpressed PIS was found to be localized in the endoplasmic reticulum, as previously reported for the native enzyme activity. NIH-PIS cells showed an increase in PI synthesis in vitro and in vivo , as well as increased cellular levels of PI–4,5–P₂ and PI–3,4,5–P₃. They also displayed a decrease in their doubling tune and accelerated G1 progression. Overexpression of PIS increased cellular levels of the cyclin D1 and E proteins and Akt kinase activity in serum-stimulated quiescent NIH3T3 cells. Moreover, PIS Overexpression potentiated the colony formation of NIH3T3 cells in soft agar. These results suggest that PIS accelerates G1 progression and stimulates growth by increasing cellular levels of cyclins D1 and E.     

重组人p53腺病毒注射液联合顺铂对卵巢癌细胞生长及凋亡的影响

目的:探讨重组人p53基因腺病毒(recombinant adenovirus-p53,rAd-p53)注射液联合顺铂(cisplatin, DDP)对卵巢癌细胞株生长及细胞凋亡的影响.方法:利用MTT、FCM和Western 印迹法比较单独采用rAd-p53、DDP及2者联合用药对卵巢癌细胞株SKOV-3和CAOV-3细胞生长抑制、细胞周期、细胞凋亡以及p53蛋白表达的影响.结果: rAd-p53注射液对卵巢癌细胞株的生长有抑制作用,并呈时间、剂量依赖性关系;联合用药对卵巢癌细胞株生长抑制作用更显著(P<0.01),联合用药时不同的用药顺序对卵巢癌细胞株生长抑制无明显差异(P>0.05).联合用药72 h后,卵巢癌细胞株细胞周期明显阻滞于G0/G1期,S期比例明显减少,且细胞凋亡率显著升高(P<0.01).rAd-p53作用于卵巢癌细胞株72 h后,有明显的p53蛋白表达.结论:rAd-p53能将外源性野生型p53基因导入卵巢癌细胞基因组,使之表达p53蛋白,从而使肿瘤细胞的细胞周期阻滞于G0/G1期,抑制细胞生长,并促进细胞凋亡.rAd-p53与DDP联合应用,其抗肿瘤效应较单药用药更加显著.     

P53, cell cycle control and apoptosis: Implications for cancer

Summary Cellular proliferation depends on the rates of both cell division and cell death. Tumors frequently have decreased cell death as a primary mode of increased cell proliferation. Genetic changes resulting in loss of programmed cell death (apoptosis) are likely to be critical components of tumorigenesis. Many of the gene products which appear to control apoptotic tendencies are regulators of cell cycle progression; thus, cell cycle control and cell death appear to be tightly linked processes. P53 protein is an example of a gene product which affects both cell cycle progression and apoptosis. The ability of p53 overexpression to induce apoptosis may be a major reason why tumor cells frequently disable p53 during the transformation process. Unfortunately, the same genetic changes which cause loss of apoptosis during tumordevelopment, may also result in tumor cellresistance to anti-neoplastic therapies which kill tumor cells by apoptosis. Elucidation of the genetic and biochemical controls of these cellular responses may provide insights into ways to induce cell death and thus hopefully suggest new targets for improving therapeutic index in the treatment of malignancies.     

顺铂对人肺腺癌细胞SLC-89细胞周期、p21及c-myc表达的影响

 目的　观察顺铂对SLC 89细胞增殖、细胞周期、p2 1及c myc蛋白表达的影响 ,以揭示顺铂抗癌的机理。方法　运用体外细胞培养技术 ,以不同浓度的顺铂作用于培养的SLC 89细胞 72h ,分别运用MTT法测定细胞增殖和流式细胞术观察细胞周期的变化及p2 1和c myc蛋白的表达。结果　顺铂能显著抑制SLC 89细胞生长 ,72h的顺铂IC50 为 18.4 7μg/ml。顺铂能以浓度依赖方式减少S期细胞而阻滞细胞于G0 /G1期 ,并诱导细胞p2 1及c myc蛋白的表达。 结论　顺铂能显著抑制SLC 89细胞生长增殖、改变细胞周期分布并诱导细胞 p2 1及c myc蛋白的表达 ,这可能是顺铂抗癌作用的重要机理之一。     

稳定表达Cyclin E细胞系的建立及Cyclin E高表达对人乳腺癌MCF 7细胞生长及细胞周期的影响

 目的 观察cyclin E 的高表达对乳腺癌细胞MCF 7 生长及周期的影响。方法 构建cyclin EcDNA真核表达载体并采用lipofectAMINE 转染方法将其导入MCF 7 细胞,获得稳定表达cyclin E的细胞系。通过对细胞生长曲线绘制、3H TdR测定及细胞周期分布等的分析,观察其对细胞生长、增殖的影响。结果 cyclin E的高表达可以使细胞的生长速度加快（ 约为对照1-52 倍） 及DNA 掺入增加（4-2 倍）,G1 S移行加速;pRB的磷酸化形式的增多（3 倍）.结论 cyclin E的高表达可明显影响MCF 7 细胞的生长、增殖,并可能通过pRB 磷酸化,影响细胞周期G1 S期移行而实现其对生长的调节。     

高温合并顺铂对人肺腺癌细胞株H1299的协同杀伤作用

目的:观察高温合并顺铂对人肺腺癌细胞株H1299的协同杀伤作用。方法:用CCK-8法检测高温处理或未处理的细胞对顺铂的敏感性。单独42℃作用2h、顺铂10μg/ml作用24h和两者联合作用后,采用CCK-8法检测细胞抑制率和流式细胞仪分析细胞各时相DNA含量,观察两者对细胞毒性和细胞周期的影响。结果:高温作用后,细胞对顺铂的半数抑制浓度(IC50)从9·51μg/ml降低至6·07μg/ml;高温和顺铂联合作用对细胞有明显的协同杀伤作用,流式细胞检测也显示高温对细胞周期有明显的影响,G0/G1期细胞比例明显增加,S期细胞比例明显下降。结论:高温可以明显提高H1299细胞对顺铂的敏感性。     

槲皮素对人乳腺癌裸鼠移植瘤细胞周期的影响

目的　研究槲皮素对人乳腺癌细胞株MCF 7裸鼠移植瘤细胞周期的影响。方法　 2 4只MCF 7细胞株移植成功的裸鼠分为对照组、槲皮素组、阿霉素组及联合用药组 ,每组 6只。用流式细胞仪分析细胞周期分布 ,用免疫组化测定cyclinD1表达水平。结果　G0 /G1期占细胞周期比例对照组明显低于槲皮素组 ,阿霉素组及联合用药组 (P <0 .0 1) ,S期占细胞周期比例对照组明显高于槲皮素组 ,阿霉素组及联合用药组 (P <0 .0 1) ,cyclinD1表达水平对照组明显高于槲皮素组、阿霉素组及联合用药组 (P <0 .0 1)。结论　槲皮素可作用于MCF 7移植瘤的G1/S节点 ,可抑制移植瘤细胞增殖及cyclinD1表达 ,延缓肿瘤生长。     

华蟾素注射液对人肝癌HepG-2细胞增殖及周期的影响

Objective: The aim of our study was to investigate the effect of Cinobufacini injection on the proliferation and cell cycle of human hepatoma HepG-2 cells. Methods: Cell proliferation was assessed by MTT assay, cell cycle distribution was detected by the flow cytometry (FCM). The expression of Cyclin A, CDK2 mRNA levels were examined by RT-PCR. Quantitative colorimetric assay was used to analyze Cyclin A/CDK2 activity in HepG-2 cells. Results: Cinobufacini injection significantly inhibited HepG-2 cells prol...     

雷帕霉素抑制人骨肉瘤MG-63细胞周期的实验研究

目的观察雷帕霉素对人骨肉瘤细胞株MG-63增殖、周期及Cyclin D1基因表达的影响,探讨雷帕霉素抑制骨肉瘤细胞周期的可能机制。方法体外培养骨肉瘤MG-63细胞,用不同浓度的雷帕霉素（1、10、25nmol／L）干预MG-63细胞。采用水溶性四唑盐WST-8比色法检测MC-63细胞增殖的变化;流式细胞仪检测MG-63细胞周期;RT—PCR及免疫细胞化学SABC法检测MG-63细胞Cyclin D1基因表达的变化。结果雷帕霉素能显著抑制MG-63细胞增殖,呈时间依赖性,差别有显著性意义（P〈0．05）;流式细胞分析显示雷帕霉素能显著抑制细胞周期,使GO／G1期细胞增多（P〈0．05）;RT—PCR检测显示雷帕霉素对MG-63细胞Cyclin D1 mRNA表达无明显影响（P〉0．05）;免疫细胞化学分析雷帕霉素能显著抑制Cyclin D1蛋白的表达,差别有显著性意义（P〈0．05）。结论雷帕霉素能下调MG-63细胞Cyclin D1蛋白的表达,抑制MG-63细胞增殖及细胞周期。     

9-顺式维甲酸对胃癌细胞周期及周期因子表达的影响

目的：探讨9-顺式维甲酸（9-cisRA）抑制胃癌细胞生长及其机制。方法：RT-PCR检测9-cisRA作用MGC80-3细胞前后细胞周期蛋白D1（CyclinD1）和周期蛋白依赖性激酶4（CDK4）mRNA表达的变化；流式细胞术和MTT法检测其对细胞周期和生长抑制的作用；电镜观察细胞形态学改变。结果：10μmol／L9-cisRA作用MGC80-3细胞96h时，CyclinD1和CDK4mRNA表达均显著下降（P〈0．01）；9-cisRA对MGC80-3细胞生长抑制作用呈时间和剂量依赖性；9-cisRA诱导MGC80-3细胞阻滞于细胞周期的G1期，并呈典型的凋亡特征性的“亚G1峰”和形态学改变。结论：9-cisRA对MGC80-3细胞有显著的生长抑制和诱导凋亡作用，与抑制CyclinD1和CDK4表达将细胞周期阻滞于G1期有关。     

抑癌基因PTEN对肝癌细胞增殖的抑制及作用机制

目的探讨抑癌基因PTEN对肝癌细胞增殖和细胞周期的调控作用。方法构建野生型PTEN基因和突变型PTEN基因的真核表达载体pEGFP—WT—PTEN和pEGFP—PTEN;G129R。采用脂质体介导的基因转染法,分别将上述载体转染不表达PTEN蛋白的人肝细胞肝癌细胞系HHCC,经G418筛选,获得稳定表达PTEN蛋白的细胞克隆。以流式细胞仪测定细胞周期,以Western blot法分析稳定表达PTEN蛋白的肝癌细胞内源性的磷酸化AKT表达水平,同时与未进行基因转染和转染空载体pEGFP-C1的HHCC细胞进行对照。结果稳定表达野生型PTEN蛋白的肝癌细胞,细胞生长受到明显抑制,与转染空载体的HHCC细胞比较,G1期细胞比例显著增高,G2期和S期细胞比例显著降低,且差异有统计学意义（P〈0．05）;而转染突变型PTEN基因的HHCC细胞,与转染空载体的HHCC细胞比较,差异无统计学意义（P〉0.05）。与转染空载体的HHCC细胞比较,稳定表达野生型PTEN蛋白的HHCC细胞,其内源性磷酸化AKT水平明显减低;而转染突变型PTEN基因的HHCC细胞,其AKT水平无明显变化。结论野生型PTEN基因对肝癌细胞周期具有调控作用,而突变型PTEN基因丧失对肝癌细胞周期的调控作用;野生型PTEN基因可能通过降低AKT的活化而实现对肝癌细胞周期的调控。