p150 ADAR1 isoform involved in maintenance of HeLa cell proliferation

Background  

RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of null mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood.     

Effects of luzopeptins on protein B23 translocation and ribosomal RNA synthesis in HeLa cells

Localization of protein B23 in HeLa cells after treatment with luzopeptin A and its analogues was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with luzopeptin A (50 ng/ml), luzopeptin B (500 ng/ml), or luzopeptin D (10 ng/ml) for 2 h, uniform nucleoplasmic rather than specific nucleolar fluorescence was observed. Luzopeptin C had no effect on protein B23 translocation. Luzopeptin D, A, and B inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with 50% inhibitory concentration values of 3.7 +/- 1.1 (SD), 10.8 +/- 2.1, and 122.0 +/- 34.0 ng/ml, respectively. Less than 10% inhibition of [3H]uridine incorporation was found with luzopeptin C (500 ng/ml and 2 h incubation). Ribosomal RNAs (28 and 18S) were isolated from HeLa cells treated with luzopeptin D (50 ng/ml; 2 h). They were then separated and analyzed in 1% agarose gel electrophoresis. There were 90.1 +/- 1.38 and 95.0 +/- 1.04% inhibition of [3H]uridine incorporation into 28 and 18S ribosomal RNA, respectively. The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was luzopeptin D greater than luzopeptin A greater than luzopeptin B much greater than luzopeptin C, which correlates with the order of their 50% inhibitory concentration values for inhibition of [3H]uridine incorporation. With 34-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. With 70-85% inhibition of RNA synthesis, a uniform nucleoplasmic fluorescence was observed. These results indicate that translocation of protein B23 as observed by indirect immunofluorescence may be a rapid and simple screening test for the selection of antitumor agents which inhibit ribosomal RNA synthesis.     

Survivin短发夹状RNA对宫颈癌细胞系HeLa增殖和凋亡的影响

目的:观察survivin基因短发夹状RNA(short hairpin RNA,shRNA)对宫颈癌细胞系HeLa增殖和凋亡的影响。方法:通过脂质体介导的细胞转染技术将含人survivin基因shRNA的重组真核表达质粒pSilenc-er2.1-s2转染宫颈癌细胞系HeLa,G418筛选阳性克隆,半定量PCR、Western blot分别检测survivin mRNA和蛋白表达,四甲基偶氮唑蓝(MTT)比色法绘制细胞生长曲线,流式细胞仪检测细胞周期变化,流式细胞仪、Hoechst染色观察细胞凋亡情况。结果:G418筛选34天后出现阳性克隆,与转染阴性对照质粒(HeLa-NC)、空载质粒(HeLa-U6 neo)及未转染(HeLa)细胞比较,转染pSilencer2.1-s2质粒者survivin mRNA和蛋白表达明显下降,表达抑制率分别为:62.8%和60.1%;细胞增殖受到抑制,最高细胞生长抑制率为:(57.8±2.1)%(P<0.05);细胞周期发生显著变化,细胞被阻滞于G0/G1期,占(72.7±3.1)%(P<0.05),G2/M期明显减少,占(5.1±2.9)%(P<0.05);细胞凋亡率为:(29.2±1.4)%,明显升高(P<0.05)。结论:Survivin基因shRNA可通过下调HeLa细胞中survivin的表达,抑制细胞增殖并诱导细胞凋亡。     

TRICHOSTATIN A INHIBITS PROLIFERATION, INDUCES APOPTOSIS AND CELL CYCLE ARREST IN HELA CELLS

Objective： The histone deacetylase inhibitors （HDACIS） have been shown to inhibit cancer cell proliferation, stimulate apoptosis, an induce cell cycle arrest. Our purpose was to investigate the antiproliferative effects of a HDACI, trichostatin A （TSA）, against human cervical cancer cells （HeLa）. Methods： HeLa cells were treated in vitro with various concentrations of TSA. The inhibitory effect of TSA on the growth of HeLa cells was measured by MTT assay. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of TSA. Induction of cell cycle arrest was studied by flow cytometry. Changes in gene expression of p53, p21wafl and p27Kipl were studied by semiquantitative RT-PCR. Results： TSA inhibited cell growth in a time- and dose-dependent manner. Hoechst 33258 staining assay showed that TSA induced apoptosis. Cell cycle analysis indicated that treatment with TSA decreased the proportion of cells in S phase and increased the proportion of cells in G0/G1 and/or G2/M phases of the cell cycle. This was concomitant with overexpression of genes related to malignant phenotype, including an increase in p53, p21wall and p27Kipl. Conclusion： These results suggest that TSA is effective in inhibiting growth of HeLa cells in vitro. The findings raise the possibility that TSA may prove particularly effective in treatment of cervical cancers.     

Overexpression of ribosomal protein L15 is associated with cell proliferation in gastric cancer

Background  

Ribosomal proteins are the components of ribosome, which also exhibit various secondary functions in DNA repair, apoptosis, drug resistance and proliferation. In our previous study of microarray, ribosomal protein L15 (RPL15) was identified as an upregulated gene in gastric cancer.     

腺苷酸活化蛋白激酶介导多柔比星诱导的抗人类乳腺癌MCF-7细胞增殖作用

 目的　研究多柔比星对乳腺癌MCF-7细胞增殖的影响,并探讨腺苷酸活化蛋白激酶（AMPK）在其中的作用。方法　分别用AMPK激活剂AICAR、siRNA敲低AMPK表达（AMPK siRNA）、AMPK抑制剂复合物C（AMPKi）联合多柔比星（DOX）对MCF-7细胞进行处理,在不同时间通过Western blot方法检测AMPK、乙酰辅酶A羧化酶（ACC）、p38的活化,MTT检测细胞存活率间接反应细胞增殖。结果　DOX可诱导MCF-7细胞AMPK的活化,AICAR单独或联合DOX可诱导AMPK的激活及增加MCF-7细胞增殖抑制率,AICAR+ DOX组与DOX组细胞存活率分别为（17.7±1.6） %和（71.4±1.8） %（P＜0.001）;AMPKi或AMPK siRNA联合DOX后,p-AMPK及p-ACC表达明显下降,p38活化水平不受影响,MCF-7细胞增殖抑制率下降,AMPKi+DOX组与DOX组细胞存活率分别为（72.7±1.8）%和（96.3±1.7） %（P＜0.001）,AMPK siRNA +DOX组与错义siRNA+DOX组细胞存活率分别为（76.9±2.2） %和（95.9±1.8） %（P＜0.001）。结论　AMPK介导DOX诱导的抗乳腺癌细胞增殖。     

应用可调控Notch1干扰载体诱导HeLa细胞增殖抑制

背景与目的:Notch信号转导途径与细胞增殖与分化的调控密切相关,它的功能失调在肿瘤细胞的增殖分化中发挥着重要的作用.本研究构建了针对Notch1的可调控RNA干扰载体,并从分子水平及细胞水平上研究其对HeLa细胞的影响.方法:利用受CRE重组调控的RNA干扰载体pSico和pSicoR构建针对GAPDH和Notch1的shRNA表达载体,以pBS185-CRE作为CRE蛋白的表达载体,将HeLa细胞分为pSico、pSico/CRE、pSicoR和pSicoR/CRE 4组,分别转染相应质粒,RT-PCR和Western blot法检测干扰效率,CBF-1荧光素酶报告载体检测细胞内活性Notch信号水平,MTS实验检测细胞增殖状态.结果:各组细胞在转染重组质粒pSico(R)-GAPDH和pSico(R)-Notch1后,EGFP的表达均表现出明显的CRE依赖性;RT-PCR和Western blot实验显示,pSico(R)-Notch1载体能在CRE蛋白的调控下抑制Notch1基因的表达,CBF-1荧光素酶报告载体实验可见,Notch1的干扰作用使细胞内Notch信号水平下降;MTS实验可见Notch1干扰引起的HeLa细胞增殖抑制.结论:由可调控RNA干扰载体介导的CRE依赖性Notch1表达降低能降低细胞内Notch信号水平并抑制HeLa细胞增殖.     

p53 is involved in the inhibition of cell proliferation mediated by activin A in cultured human prostate cancer LNCaP cells

Large tumor size is a negative prognostic variable for attaining complete regression (CR) with local hyperthermia (HT) and radiotherapy (RT). Such poor prognosis lesions (i.e., >7 cm(2) or >14 cm(3)) have an expected CR rate of similar to 30+/-8%. To improve on this result we added cisplatin to HT and RT with standard fractionation (std Fx) in an earlier study, and observed a 19% CR rate in head and neck (H&N) patients. We now report the results of a second generation trial combining HT, cisplatin (40 mg/m(2)) and hyperfractionated RT in a series of 13 pretreated poor prognosis H&N patients. Therapy encompassed 44 triple modality sessions and was well tolerated: toxicity included one episode of grade-3 skin reaction and one grade 1 leukopenia. Although the overall remission rate was 92%, the CR rate was only 8%; this resulted in early closure of this trial concluding that hyperfractionated RT had no (over std Fx RT) benefit in this combined modality approach.     

CD30 is involved in inhibition of T-cell proliferation by Hodgkin's Reed-Sternberg cells

CD30 is expressed on Hodgkin's Reed-Sternberg (H-RS) cells, the tumor cells in Hodgkin's disease. Increased levels of serum CD30 are observed in Hodgkin's disease patients and are a good marker for predicting a poor prognosis and a poor response to therapy. In this study, we addressed the effect of CD30 on T cells. We showed that CD30, either as a membranous protein on H-RS cells and Chinese hamster ovary cells or as a plate-bound chimeric protein, inhibited T-cell proliferation. Anti-CD3-stimulated T cells in the presence of CD30 failed to increase tritium uptake and failed to express CD25 and CD26 and to produce interleukin 2. The inhibition of T-cell proliferation was, however, reversed with addition of exogenous interleukin 2 or pretreatment of H-RS cells with anti-CD30. Inability of T cells to express CD25 and CD26 in cocultures with H-RS cells or a plate-bound CD30 chimeric protein is in accordance with the results of immunohistochemistry on disease-involved tissues. We conclude that H-RS cells are able to inhibit the proliferation and activation of T cells through CD30-related interaction. The outcome of CD30-related interaction is an ineffective antitumor immunity, which is clearly in favor of the growth and survival of the tumor cells.     

ckshs expression is linked to cell proliferation in normal and malignant human lymphoid cells

Cyclin kinase sub‐units (CKS) are known to interact with cyclin‐dependent kinases (CDKs), but their functions are not completely understood and their expression in human tissues is not documented. For analyzing relationships of CKS with cell proliferation and/or with differentiation, we investigated the expression of ckshs1 and ckshs2 in normal and malignant human lymphoid cells. ckshs1 and ckshs2 expression appeared to be related to cell proliferation: (i) mRNAs increased with stimulation of normal peripheral‐blood lymphocytes, and from the G₁ to the SG₂M phase in elutriated cells; (ii) P9 proteins were also induced by lymphocyte stimulation and were localized in nucleus where phosphorylated forms of CDK1 were also found; (iii) in vitro, the phosphorylated forms of CDK1 and CDK2 were preferentially linked to CKS. Among 45 patients presenting acute or chronic lymphoid malignancy, ckshs1 and ckshs2 mRNAs varied in a similar way and were significantly correlated to cell proliferation (p < 0.0001). When analysis was restricted solely to acute lymphoblastic leukemia (ALL) this correlation was still found and ckshs1 and ckshs2 were significantly more expressed in T‐cell ALL than in B‐cell‐lineage ALL. These results confirm relationships between ckshs expression and cell proliferation, and pose the question of a link with cell differentiation. Int. J. Cancer 82:98–104, 1999. © 1999 Wiley‐Liss, Inc.     

TSPY potentiates cell proliferation and tumorigenesis by promoting cell cycle progression in HeLa and NIH3T3 cells

Background  

TSPY is a repeated gene mapped to the critical region harboring the gonadoblastoma locus on the Y chromosome (GBY), the only oncogenic locus on this male-specific chromosome. Elevated levels of TSPY have been observed in gonadoblastoma specimens and a variety of other tumor tissues, including testicular germ cell tumors, prostate cancer, melanoma, and liver cancer. TSPY contains a SET/NAP domain that is present in a family of cyclin B and/or histone binding proteins represented by the oncoprotein SET and the nucleosome assembly protein 1 (NAP1), involved in cell cycle regulation and replication.     

siRNA抑制Ku80表达后对宫颈癌HeLa细胞增殖的影响

背景与目的:Ku80为细胞受辐射后DNA双链断裂(DNA double strand break,DSB)的主要修复蛋白.现阶段关于Ku80的研究主要集中在DSB修复方面,其他方面研究较少.本研究建立利用siRNA抑制Ku80表达的宫颈癌HeLa细胞模型,以此探讨Ku80在细胞增殖方面的作用.方法:构建靶向抑制Ku80的siRNA表达载体,转染HeLa细胞,筛选稳定表达siRNA的转化克隆;Western blot检测Ku80表达变化:克隆形成实验、MTT法和裸鼠皮下瘤形成实验分别检测细胞克隆形成率及细胞在体外和体内的增殖情况.结果:构建表达质粒pKu80-siRNA与pNeg-siRNA转染HeLa细胞,G418筛选后获得稳定转染克隆,Western blot分析表明转染pKu80-siRNA后的细胞Ku80蛋白表达受到明显抑制,将其命名为HeLa/Ku80-siRNA;转染pNeg-siRNA的HeLa细胞克隆形成率为0.62±0.02,而HeLa/Ku80-siRNA细胞的克隆形成率为0.46±0.05,明显低于对照细胞(t=5.11,P＜0.01);MTT显示细胞培养48 h和72 h时,HeLa/Ku80-siRNA细胞的增殖率均显著低于对照细胞(P＜0.05);裸鼠皮下瘤生长实验示种植25天时,HeLa/Ku80-siRNA细胞种植瘤的平均体积为(18.92±3.60)mm3,明显低于对照细胞种植瘤体积(194.88±30.61)mm3,(t=12.69,P＜0.01).结论:稳定转染及siRNA技术建立的Ku80表达抑制克隆可以成为简单实用的细胞模型:Ku80-siRNA抑制Ku80的表达后可以在体内外抑制HeLa细胞的增殖.