Inactivation and sensitization of tumor cells after transfection with gene Bax

Experiments on the transfection of cultured SKOV3 tumor cells of human ovarian adenocarcinoma and HeLa cells of human cervical carcinoma with gene Bax have demonstrated that SKOV3 cells are highly sensitive to the protein product of this gene, whereas the sensitivity of HeLa cells is substantially lower. HeLa cells obtained as a result of Bax transfection and subsequent selection are characterized by an extremely high Bax protein content and a hypersensitivity to doxorubicin. All Bax-transfected SKOV3 cells with an increased Bax content have died. In the SKOV3 cell line, a Bax exon 3 mutation has been found that corresponds to genotype G7/G9, whereas the native type of the Bax gene corresponds to genotype G8/G8. The results suggest that the G7/G9 mutation in Bax exon 3 deprives the Bax protein of proapoptotic activity.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 40–47.Original Russian Text Copyright © 2005 by Rodina, Sladkova, Obuchova, Vezirkhanova, Moskaleva, Prusakova, Beletskii, Belushkina, Strelnikov, Ivanov, S. Severin, E. Severin.     

Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.     

Role of ATP5G3 in sodium nitroprusside-induced cell death in cervical carcinoma cells

We identified a gene, subunit C3 (ATP5G3) of mitochondrial ATP synthase, that displayed changes in gene expression under oxidative stress. We examined the role of ATP5G3 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using ATP5G3 small interfering RNA (siATP5G3)-transfected HeLa cells. A significant increase in cytotoxicity was observed in the transfected cells treated with SNP, which suggests a protective role of ATP5G3 in SNP-induced cytotoxicity in the cells. The transfected cells treated with photodegraded SNP showed equal cytotoxicity to SNP, and pretreatment with deferoxamine (DFO) completely inhibited this cytotoxicity. Further, cytotoxicity was significantly inhibited by pretreatment with a p38 inhibitor and was accentuated by the p38 activator in cells. Pretreatment with the Bcl-xL inhibitor also significantly accentuated cytotoxicity. The increase in p38 phosphorylation was significantly higher in siATP5G3-transfected cells treated with SNP in immunoblotting, which was inhibited by pretreatment with DFO. The increase in cytotoxicity with siATP5G3 transfection was completely blocked by cotransfection with sip38, and the blocking effect disappeared by cotransfection with additional siBcl-xL, which suggests that the protective role of ATP5G3 is mediated by Bcl-xL via the inhibition of p38 activity. Cytotoxicity was completely blocked by the cotransfection of siATP5G3 with siBax. No change in apoptotic parameters was observed during cytotoxicity. However, pretreatment with lysosomal inhibitors significantly inhibited cytotoxicity and increased p62 protein levels. These findings suggest that ATP5G3 plays a protective role in autophagic cell death/lysosome-associated cell death induced by SNP via the sequential signaling of ROS/p38/Bcl-xL/Bax in HeLa cells.     

Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents

This study compared six commercially available reagents (Arrest-In, ExpressFect, FuGENE HD, jetPEI, Lipofectamine 2000, and SuperFect) for gene transfection. We examined the efficiency and cytotoxicity using nine different cell lines (MC3T3-E1 mouse preosteoblasts, PT-30 human epithelial precancer cells, C3H10T1/2 mouse stem cells, MCF-7 human breast cancer cells, HeLa human cervical cancer, C2C12 mouse myoblasts, Hep G2 human hepatocellular carcinoma, 4T1 mouse mammary carcinoma, and HCT116 human colorectal carcinoma), and primary cells (HEKn human epidermal keratinocytes) with two different plasmid DNAs encoding luciferase or β-galactosidase in the presence or absence of serum. Maximal transfection efficiency in MC3T3-E1, C3H10T1/2, HeLa, C2C12, Hep G2, and HCT116 was seen using FuGENE HD, in PT-30, 4T1, and HEKn was seen using Arrest-In, and in MCF-7 was seen using jetPEI. Determination of cytotoxicity showed that the largest amount of viable cells was found after transfection with jetPEI and ExpressFect. These results suggest that FuGENE HD is the most preferred transfection reagent for many cell lines, followed by Arrest-In and jetPEI. These results may be useful for improving nonviral gene and cell therapy applications.     

Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the ΔΨ_m. These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.     

LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制

摘要 目的：探讨狐猴酪氨酸激酶2（LMTK2）基因沉默对人上皮性卵巢癌(EOC)细胞生长和转移的抑制作用及其可能的机制。方法：通过RT-qPCR和Western-blot检测了人正常卵巢上皮细胞IOSE80和人上皮性卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的表达,使用Lipofectamine 3000转染试剂将LMTK2的短发夹RNA（shRNA）、阴性对照shRNA、LMTK2过表达重组pcDNA3.1质粒或阴性对照质粒转染到SKOV3细胞中,并分为LMTK2-shRNA组、NC-shRNA组、LMTK2-pcDNA3.1组或NC-pcDNA3.1组。另外,使用PI3K/Akt抑制剂LY294002处理SKOV3细胞1 h。通过CCK-8法测定细胞增殖,Annexin V-FITC/PI染色法测定细胞凋亡,划痕实验评价细胞迁移,Transwell实验评价细胞侵袭。对BALB/c雌性裸鼠皮下注射转染NC-shRNA或LMTK2-shRNA的SKOV3细胞建立体内移植瘤模型,并记录接种28 d内的肿瘤体积。结果：与人正常卵巢上皮细胞IOSE80相比,卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的mRNA和蛋白表达水平均显著升高,其中SKOV3的LMTK2 mRNA和蛋白表达水平最高（P<0.05）。与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞活力、相对迁移面积、侵袭细胞数均显著降低,而细胞凋亡率显著升高（P<0.05）。此外,与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞中Bax的蛋白表达水平显著升高,而Bcl-2、MMP2、MMP9、p-Akt的蛋白表达水平显著降低（P<0.05）。LY294002处理逆转了上调LMTK2对SKOV3细胞生长和转移的影响（P<0.05）。在接种第21天和28天时,与NC-shRNA组相比,LMTK2-shRNA组裸鼠的肿瘤体积显著降低（P<0.05）。结论：LMTK2基因沉默通过抑制PI3K/Akt信号通路降低了人上皮性卵巢癌细胞的生长和转移能力。     

Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53.

Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37 degrees C and a wild-type like p53 at 32 degrees C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfection of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfection experiments utilizing the neomycin resistance gene only (SKN). Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that is associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32 degrees C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32 degrees C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32 degrees C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not in SKN cells. In both clones Gas1 mRNA was not detected either at 37 degrees C or 32 degrees C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37 degrees C) or wild type p53 (SK23a and SK9 kept at 32 degrees C) on the mechanism of response of cancer cells to DNA damaging agents.     

RNA interference-mediated silencing of the PAR gene inhibits the growth of PC3 cells via the induction of G2/M cell cycle arrest and apoptosis

BACKGROUND: The prostate androgen-regulated (PAR) gene is ubiquitously overexpressed in prostate cancer (PCa) cells and is involved in proliferation of PCa. However, the mechanism by which the modulation of PAR gene expression elicits its biological effects on PCa cells is not well documented. Here, we investigate the mechanism of PAR depletion inhibiting PCa cell growth. METHODS: PAR expression was depleted by small interfering RNA (siRNA) and its subsequent effects on proliferation of PC3 cells were determined by the trypan blue exclusion assay. Flow cytometric analysis provided the evidence for the progression of cell cycle and the induction of apoptosis which was further confirmed by the observation of cleavage of poly(ADP-ribose) polymerase. Western blot analysis was performed to investigate the involvement of critical molecular events known to regulate the cell cycle and the apoptotic machinery. RESULTS: siRNA transfection results in a dose-dependent inhibition of cell growth in PC3 cells by causing G2/M phase cell cycle arrest and apoptosis. The G2/M arrest by PAR depletion was associated with decreased levels of cyclin B1, pCdc2 (Tyr15), Cdc2 and Cdc25C. PAR depletion also was found to result in inhibition of procaspases 9, 8, 6 and 3 with significant increase in the ratio of Bax : Bcl-2. CONCLUSIONS: Our data indicate that PAR depletion induces G2/M arrest via the Cdc25C-Cdc2/cyclin B1 pathway. Furthermore, the results of the present study point toward involvement of pathways mediated by both caspase 8 and caspase 9 in apoptosis induction by PAR depletion.     

CEA启动子控制HSV—TK基因表达对人结直肠癌细胞的杀伤作用

An expression plasmid pCEA-TK, in which HSV-TK gene was under the control of CEA promoter, was constructed. The human colorectal carcinoma cell line LoVo or the human uterine cervical cancer cell line HeLa was co-transfected with pSV2-neo and pCEATK, respectively. After G418 selection, both transgenic cell clones (LoVo/CEATK and HeLa/CEATK) were obtained. LoVo/CEATK cells were 1300 times more sensitive to the cytotoxicity of ganciclovir than LoVo cells. However, the elevation of GCV sensitivity induced by pCEATK gene in HeLa line was only 8 times. Injection of GCV resulted in significant regression of HSV-TK transfected LoVo tumor in nude mice. These data suggested that the expression of TK gene driven by CEA promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electromicroscopy and DNA fragmentation assay demonstrated that GCV could induce apoptosis in LoVo/CEATK cells. The possibility of the CEATK/GCV system in the treatment of human colorectal carcinoma was discussed.     

CEA启动子控制HSV-TK基因表达对人结直肠癌细胞的杀伤作用

构建了以CEA启动子控制的HSV-TK基因表达质粒pCEA-TK。转染pCEA-TK的人结肠癌细胞LoVo对GCV的敏感性提高了1300倍。同样条件下,人宫颈癌细胞HeLa对GCV的敏感性仅提高8倍,且对低于血药浓度(20μmol/L)的GCV不敏感。以上结果显示在GCV存在时,CEA启动子控制下HSV-TK基因的表达使CEA阳性的人结直肠癌细胞获得专一性杀伤。此外,DNA片段分析和电镜观察表明GCV诱导转染pCEA-TK的LoVo细胞发生凋亡可能是这个系统杀死肿瘤细胞的机制之一。本工作还讨论了癌胚抗原(CEA)基因启动子用于人结直肠癌专一性自杀基因治疗的可能性。     

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1对卵巢癌细胞增殖的影响

目的: 探讨miR-335 靶向Rho相关卷曲螺旋形成蛋白激酶1(rho associated coiled-coil forming protein kinase 1,ROCK1)对卵巢癌细胞系SKOV3增殖的调控作用。方法:(1)选取卵巢癌细胞系SKOV3及人正常卵巢上皮细胞系IOSE80,采用RT-PCR检测各组细胞中miR-335表达;采用Western blot检测各组细胞中ROCK1蛋白表达;(2)选取卵巢癌细胞系SKOV3,分别转染miR-335 mimic及mimic control,采用RT-PCR检测细胞中miR-335表达;(3)选取卵巢癌细胞系SKOV3,将SKOV3荧光素酶报告载体与miR-335 mimic共转染,采用荧光素酶活性实验验证miR-335对SKOV3的靶向作用;(4)选取卵巢癌细胞系SKOV3,分为3组,即SKOV3组(转染mimic control)、miR-335 mimic组(转染miR-335 mimic)及miR-335 mimic+ROCK1组(共转染miR-335 mimic+ROCK1),采用MTT法检测各组细胞增殖活性,采用Western blot检测各组细胞中ROCK1蛋白表达,采用RT-PCR检测细胞中Cyclin D1表达。结果: (1)RT-PCR结果显示,卵巢癌细胞SKOV3中miR-335表达显著低于人正常卵巢上皮细胞IOSE80(P < 0.05);Western blot结果显示,卵巢癌细胞SKOV3中ROCK1蛋白表达显著高于人正常卵巢上皮细胞IOSE80(P < 0.05);(2)RT-PCR结果显示,转染miR-335 mimic可使卵巢癌细胞SKOV3中miR-335表达上调,与转染mimic control相比较差异具有统计学意义(P < 0.05);(3)双荧光素酶活性检测结果显示,miR-335 mimic可显著抑制野生型ROCK1-Wt报告载体的荧光素酶活性,但对突变型ROCK1-Mut报告载体的荧光素酶活性并无显著抑制作用;(4)转染miR-335mimic后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,卵巢癌细胞SKOV3增殖活性及Cyclin D1表达较单纯转染miR-335 mimic组显著提高(P < 0.05),但仍显著低于阴性对照组(P < 0.05)。Western blot检测结果显示,转染miR-335mimic后,卵巢癌细胞SKOV3中ROCK1蛋白表达较阴性对照组显著降低(P < 0.05);而转染miR-335 mimic+ROCK1后,ROCK1蛋白表达较单纯转染miR-335mimic组显著增高(P < 0.05),且显著高于阴性对照组(P < 0.05)。结论: miR-335可通过靶向ROCK1抑制卵巢癌细胞系SKOV3增殖。     

TBP-like Protein（TLP）通过G2／M期阻滞抑制HeLa细胞的增殖

TBP—likeprotein（TLP）是真核细胞中一种常见的转录因子,在调节生长发育方面起着重要的作用。该实验构建重组质粒pEGFP—N1．TLP,研究TLP对人宫颈癌细胞HeLa增殖的影响。利用流式细胞仪检测质粒的转染效率,通过激光共聚焦显微镜观察外源TLP蛋白的亚细胞定位。经过MTT检测、RNAi—TLP诱导的基因沉默7LHoechst33258染色研究TLP对HeLa细胞的增殖抑制作用。流式细胞术、Westernblot和RT-PCR实验结果表明,TLP将HeLa细胞周期阻滞于G2／M期,并抑制周期相关基因CDKl和CyclinBl的转录和翻译。研究表明,外源TLP在HeLa细胞的细胞核中表达,通过降低细胞周期相关基因CDKl和CDKl的表达水平,将HeLa细胞的细胞周期阻滞于G2／M期,从而抑制细胞的增殖。     

稳定表达乙型肝炎病毒G145R变异表面抗原细胞系的建立

构建可以在真核细胞表达G145R HBsAg的重组质粒PCI-HBs145。用此质粒转染Hela细胞,经克隆化培养 以及G418筛选,建立稳定表达G145R HBsAg的细胞系。ELISA检测表明表达G145R HBsAg与野生型HBsAg 在免疫反应性上有较大的差异。生物学性状研究结果表明稳定表达G145R HBsAg的2A8细胞纯度好,遗传性状 稳定。     

Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.     

瘦素对卵巢癌细胞 SKOV3 增殖及凋亡的影响

目的:探讨瘦素对人卵巢癌SKOV3细胞增殖及凋亡的影响及其作用机制。方法:用不同浓度的瘦素(0、50、100、200 ng/m L)处理人卵巢癌SKOV3细胞48 h后,采用MTT法检细胞的生长;以血清饥饿诱导细胞凋亡,同时给予瘦素刺激,Annexin V/PI双染法检测细胞凋亡的变化;western blotting分析p21、cyclin D1、Bcl-2、Bax蛋白的表达水平和ERK1/2通路的活化情况。结果:瘦素以剂量依赖性的方式促进人卵巢癌SKOV3细胞的增殖,同时抑制血清饥饿诱导的细胞凋亡。瘦素处理可下调p21和上调cyclin D1的表达,抑制促凋亡分子Bax的表达和上调抗凋亡分子Bcl-2的表达。瘦素可诱导细胞中ERK1/2通路的活化,其抑制剂PD98059可明显抑制瘦素诱导的促细胞增殖和抗凋亡作用,同时伴随有cyclin D1、Bcl-2蛋白表达的下调和Bax的上调。结论:瘦素可能通过活化ERK1/2通路调节细胞有丝分裂进程,进而促进卵巢癌细胞的增殖;同时通过调节凋亡相关蛋白Bcl-2和Bax的表达抑制卵巢癌细胞的凋亡。     

Hepatitis B virus sensitizes hepatocytes to TRAIL-induced apoptosis through Bax

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.