Effects of CDK4 antisense RNA on Human breast cancer cell proliferation and expression of cyclinD1, cyclinE and CDK2

The role of CDK4 in human breast cancer cell proliferation and expression of cyclinD1, cyclinE and CDK2 has been investigated using inhibition of CDK4 expression by antisense RNA. When CDK4 expression was inhibited, the rate of cell proliferation and tumorigenecity decreased apparently. This indicates that CDK4 plays an important role in formation and development of breast tumor. The results of Northern blot analysis showed that the levels of cyclinDl and CDK2 mRNAs changed slightly whereas the level of cyclinE mRNA decreased obviously. It is suggested that the expression of CDK4 is necessary for imction of cyclinE expression. Thus, inhibition of CDK4 expression affects not only the role of CDK4 itself but also the role of other genes.     

Effects of PKCαantisense RNA on human breast cancer cell proliferation and expression of cyclinDl and CDK4

The role of PKCα in human breast cancer cell proliferation and expression of cyclinD1 and CDK4 has been investigated using inhibition of PKCα expression by its antisense RNA. When PKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels of cyclinD1 and CDK4 mRNA were lower than the control. The results showed that PKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression of cyclinD1 and CDK4 which control start of cell cycle.     

Effects ofPKCα antisense RNA on human breast cancer cell proliferation and expression ofcyclinD1 andCDK4

The role of PKCα in human breast cancer cell proliferation and expression ofcyclinD1 andCDK4 has been investigated using inhibition ofPKCα expression by its antisense RNA. WhenPKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels ofcyclinD1 andCDK4 mRNA were lower than the control. The results showed thatPKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression ofcyclinD1 andCDK4 which control start of cell cycle.     

Effects of enhancing p21 waf1 on proliferation and expression of G1cyclins andCDKs in human breast cancer cells

The cyclin-dependent kinase inhibitor p21( waf1/cip1/sdil) is an important negative regulator in control of cell cycle. Its functions of inhibiting cancer cell growth and its effects on expression of G1 phase cyclins and related CDKs are a worthy topic for study. The plasmid expressing p2l with high level was transformed to human breast cancer cells, and the expression of p2l in cells was enhanced, then the cell growth rate, anchorage-independent growth and tu-morigenecity were tested, at the same time the expression levels of cyclinD1, CDK4, cyclinE and CDK2 were analyzed by Northern blot. The results showed that since the expression of p21 was enhanced in the cell, the rate of cell growth and anchorage-independent growth was inhibited, tumorigenecity was suppressed, the level of expression of cyclinE and CDK2 decreased while that of cyclinDl and CDK4 was not affected. It is suggested that the enhanced expression of p21 markedly inhibits the proliferation and lessens the tumorigenecity of breast cancer cells, and that p2l expression is not related to that of cyclinDl and CDK4, but affects the expression of cyclinE and CDK2 .     

CDK2在非小细胞肺癌组织中的表达

探讨CDK2在非小细胞肺癌组织中的表达与肺癌转移关系。将50例非小细胞肺癌组织分为转移组和非转移组,采用免疫组织化学和Western blot检测癌组织中CDK2蛋白的表达。结果表明:CDK2蛋白在肺癌细胞中主要位于细胞核。CDK2蛋白在肺癌组织中的表达水平显著高于癌旁组织(P0.05)。CDK2蛋白高水平表达与肺癌淋巴结转移呈正相关(P0.05),但与肿瘤类型无关(P0.05)。CDK2的过表达可能与肺癌的形成有关,并与淋巴转移有关。     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

CDK16在食管鳞癌中的表达及其对肿瘤发生发展的影响

目的研究发现,细胞周期素依赖性激酶(cyclin dependent kinases,CDKs)家族成员与食管鳞癌的发病密切相关,但细胞周期素依赖性激酶16(CDK16)对食管鳞癌发病的影响尚不清楚.本研究旨在探讨CDK16在人食管鳞癌组织中的表达及生物学意义.方法应用组织芯片技术结合免疫组织化学技术检测45例食管鳞癌组织、45例癌旁组织中CDK16蛋白的表达情况,并使用统计分析软件研究CDK16蛋白的表达与食管鳞癌临床病理特征之间的关系.结果 CDK16在食管鳞癌组织中呈阳性表达,且表达强度与肿瘤分化程度呈负相关.另外,CDK16在食管鳞癌细胞中的表达位置与肿瘤的分化程度紧密相关.CDK16的表达与食管鳞癌的分化程度、淋巴结转移及TNM临床分期均具有显著的相关性(P<0.05),而与患者的年龄、性别以及肿瘤的部位、最大径、病理形态没有显著的相关性(P>0.05).结论 CDK16在食管鳞癌中的高表达与食管鳞癌的发生发展及转移密切相关.检测CDK16的表达有助于食管鳞癌的临床诊断和预后评估.     

人突变型CDK2(F80A)稳定细胞系的建立

将CDK2激酶第80位的Phe突变成Ala,使该激酶特异地利用ATP类似物N6-(2-苯乙基)-ATP(PE-ATP)筛选CDK2激酶的体内特异性底物.将pCMV-CDK2(F80A)-myc载体转染人宫颈癌细胞(HeLa),经持续G418选择和克隆化获得6株抗G418细胞系.Immunoblotting分析发现,挑选的6株细胞系中有4株表达带有myc标签的人突变CDK2蛋白质,其中2株表达量较高,可以作为筛选CDK2底物的细胞系。     

Induction of autophagy and inhibition of tumorigenesis by beclin 1

The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.     

Driving the cell cycle with a minimal CDK control network

Control of eukaryotic cell proliferation involves an extended regulatory network, the complexity of which has made it difficult to understand the basic principles of the cell cycle. To investigate the core engine of the mitotic cycle we have generated a minimal control network in fission yeast that efficiently sustains cellular reproduction. Here we demonstrate that orderly progression through the major events of the cell cycle can be driven by oscillation of an engineered monomolecular cyclin-dependent protein kinase (CDK) module lacking much of the canonical regulation. We show further that the CDK oscillator acts as the primary organizer of the cell cycle, imposing timing and directionality to a system of two CDK activity thresholds that define independent cell cycle phases. We propose that this simple core architecture forms the basic control of the eukaryotic cell cycle.     

皮蛋模拟胃肠道消化物对HepG2细胞的影响

皮蛋是中国传统食品,其水解物具有抗氧化、抗炎和抗癌等功效,具有良好的医学前景,但目前皮蛋发挥抗癌功效的途径及作用机制尚不清楚。探究了皮蛋模拟胃肠道消化物(preserved eggs simulated gastrointestinal digests,PESD)对人肝癌HepG2细胞增殖和细胞周期的影响。将皮蛋经体外模拟胃肠道消化处理后,进行细胞实验,用CCK-8法进行细胞增殖分析,研究PESD对HepG2细胞存活率的影响,采用碘化丙啶(propidium iodide, PI)染色标记的方法,通过流式细胞术测定PESD对HepG2细胞周期的影响,用western blot法测定细胞周期相关调控蛋白的表达。研究发现:PESD以剂量依赖性方式对HepG2细胞增殖进行抑制,IC₅₀为4.17mg/mL;PESD处理显著增加了S期细胞的占比(P<0.05),使HepG2细胞阻滞于S期;4mg/mL的PESD处理HepG2细胞24h后,细胞中cyclin A、cyclin E、ATM、chk2和CDC25A的蛋白表达水平上升(P<0.05),而CDK2蛋白表达水平下降(P<0.05)。研究认为,皮蛋模拟胃肠道消化物是以剂量依赖性的方式来抑制人肝癌HepG2细胞的增殖,主要是通过ATM-chk2-CDC25A信号通路,上调cyclin A和cyclin E蛋白的表达,下调CDK2蛋白的表达来实现人肝癌HepG2细胞阻滞于S期,影响人肝癌HepG2细胞周期进程,抑制癌细胞的增殖。研究结果旨在为皮蛋抗癌作用机制的阐明提供一定的理论基础。     

MSH2 is required for cell proliferation, cell cycle control and cell invasiveness in colorectal cancer cells

We have investigated the role of MSH2,a mismatch repair gene in cell proliferation,cell cycle control and cell invasiveness in the SW480 human colorectal cancer cell line.RNAi-mediated inhibition of MSH2 expression was achieved using MSH2 shRNA lentiviral expression vectors.Effective knockdown of endogenous MSH2 expression was determined by real-time PCR analysis.The most efficient MSH2 knockdown vector was selected for subsequent studies using SW480 cells.Endogenous MSH2 mRNA levels decreased after lentiviral delivery of the MSH2-RNAi,indicating efficient silencing of MSH2 expression in SW480 cells.Cell proliferation,cell cycle progression and cell invasiveness were quantified by MTT assays,flow cytometry and transwell assays,respectively.RNAi-mediated inhibition of MSH2 expression in SW480 cells resulted in decreased cell proliferation,cell cycle arrest at the G0/G1 phase and decreased cell invasiveness.Taken together,these results provide evidence that MSH2 stimulates cell proliferation,promotes cell cycle progression and positively regulates cell invasiveness.     

P48L和D74N突变影响P16基因功能

应用ＰＣＲ技术，对Ｐ１６抑癌基因进行体外定突变。在Ｐ１６ｃＤＮＡ中引入第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）和第７４位密码子ＧＡＣ（Ａｓｐ）→ＡＡＣ（Ａｓｎ）突变，构建了ｐ１６－Ｐ４８Ｌ和ｐ１６－Ｄ７４Ｎ突变体，并把它们导入纯合缺失Ｐ１６基因的人肺癌细胞株Ｈ４６０。     

环境中有机磷酸酯阻燃剂（TDCP、TECP、TCPP）对人胚肾细胞（HEK293）的影响(英)

水环境中的微污染物有机磷酸酯如三(2-氯乙基)磷酸酯(TCEP)、三(2-氯-异丙基)磷酸酯(TCPP)和三(1,3-二氯丙基)磷酸酯(TDCP)主要用作聚氨酯泡沫塑料的阻燃剂。物理、化学和生物处理很难完全消除这些污染物。本研究的目的是研究阻燃剂在环境水平和较高浓度下对人细胞系的细胞毒性和细胞周期效应。结果表明,在浓度为0.001 mg/L和0.01mg/L时,只有TDCP具有轻微的细胞毒性,而当这三种化学物质浓度100 mg/L以上时对细胞毒性有显著的诱导作用。TCEP和TCPP的EC50分别为276.8 mg/L和58.4mg/L。三种药物均能抑制CDK 4的表达,TDCP能增加CDK 2和cyclin E的表达,而TCEP和TCPP在CDK 2和cyclin E的表达上表现出自差现象。TDCP和TCEP使细胞数量减少,细胞形态发生改变。可见三种化学物质对HEK 293细胞的杀伤作用可能是通过抑制CDK 4调节蛋白而产生的。     

人CDK4基因的原核表达及重组蛋白的纯化和复性

将人CDK4基因克隆入原核表达载体pET28a（+）中, 经 酶切和测序鉴定正确的重组质粒pET28a-CDK4, 转化E.coliBL21(DE3)后获得表达菌株. 该表达菌株经IPTG诱导后, 高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白, 表达量占菌体总蛋白的52.6%, 包涵体经过洗涤、 尿素变性溶解、 His Trap HP Kit柱纯化、 稀释复性, 获得纯度达98%以上的蛋白. SDS-PAGE及Western blot分析表明, 在分子量34 000处有一特异性蛋白条带. 结果表明, 已成功的表达和纯化纯度达98%的重组人CDK4蛋白.