Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.     

DNA错配修复、染色体不稳定和肿瘤的关系

DNA错配修复系统可以识别并纠正DNA复制过程中出现的错误.保证基因组的稳定性和完整性.错配修复系统缺陷可能导致遗传物质发生突变,引发恶性肿瘤.肿瘤患者经常表现出染色体不稳定,具体表现为微卫星不稳定性和杂合性缺失.本文就DNA错配修复、染色体不稳定和肿瘤之间的联系予以综述.     

Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81–88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.     

DNA错配修复与癌症的发生及治疗

DNA错配修复是细胞复制后的一种修复机制,具有维持DNA复制保真度,控制基因变异的作用。DNA错配修复缺陷使整个基因组不稳定,最终会导致肿瘤和癌症的发生。DNA错配修复系统不仅通过矫正在DNA重组和复制过程中产生的碱基错配而保持基因组的稳定,而且通过诱导DNA损伤细胞的凋亡而消除由突变细胞生长形成的癌变。错配修复缺陷细胞的抗药性也引起了癌症化疗研究方面的关注。大多数情况下,错配修复健全型细胞对肿瘤化疗药物敏感,而错配修复缺陷细胞却有较高的抗性。DNA错配修复系统通过修复和诱导细胞凋亡维护基因组稳定的功能,显示了错配修复途径在癌症生物学和分子医学中的重要性。     

反义bcl—2在胃癌细胞中的诱导表达及促凋亡作用

为了观察ｂｃｌ－２反义ＲＮＡ对胃癌细胞ＳＧＣ７９０１的促凋亡作用及ＭＴ－１１启动子的启动诱导活性,用分子克隆方法构建载体,以电泳术,电镜术,流式细胞术,原位杂交,裸鼠肿瘤抑制试验及化学发光分析等方法观察,结果表明,可诱导载体ｐＭＴｂＮ具有良好的诱导启动转录活性,Ｂｃｌ－２蛋白的表达下降使胃癌细胞ＳＧＣ７９０１更易发生凋亡,致瘤性也发生改变。     

DNA错配修复系统研究进展

DNA错配修复(mismatch repair, MMR)系统广泛存在于生物体中.从原核生物大肠杆菌到真核生物及人类,MMR系统有不同的组成成分和修复机制.人体内MMR基因缺陷会造成基因组的不稳定并诱发遗传性非息肉型直肠癌以及其他自发性肿瘤.大肠杆菌MMR系统中的MutS蛋白可特异识别错配或未配对碱基,目前已经发展了多种基于MutS蛋白的基因突变/多态性检测技术.     

miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

An IPTG Inducible Conditional Expression System for Mycobacteria

Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of β-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.     

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.     

Evidence for an Inducible Repair-Recombination System in the Female Germ Line of Drosophila Melanogaster. III. Correlation between Reactivity Levels, Crossover Frequency and Repair Efficiency

We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system.     

四环素诱导可控表达细胞周期素B1单克隆的筛选

目的筛选四环素诱导细胞周期素B1（CyclinB1）可控表达的293单克隆细胞株（tetracycline-regulated expression 293 cells,T-REx^TM-293）。方法从人胎肝cDNA文库中PCR带有酶切位点的CyclinB1基因全长,将其酶切后插入到pcDNA4／TO／myc-HisB载体中。然后用测序正确的质粒转染T-RExTM-293细胞,加杀稻瘟菌素（Blasticidin）和腐草霉素（Zeocirt）双药物筛选两周后,传96孔板,等单个细胞扩增出单克隆。然后用Western印迹和流式细胞仪检测CyclinB1的诱导表达情况。结果构建好的pcDNA4／TO／myc-HisB—CyclinB1载体,经鉴定序列正确。筛选出的单克隆细胞株,在未加四环素时没有外源性的CyclinB1表达,在加入四环素后,3h就有表达,随时间的增加,CyclinB1表达量也增加,48h最多。结论筛选出的T-REx^TM-293单克隆细胞株能可控表达CyclinB1。     

MiR-200c Increases the Radiosensitivity of Non-Small-Cell Lung Cancer Cell Line A549 by Targeting VEGF-VEGFR2 Pathway

MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT), is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research.     

Cytokine Induction of Inducible Nitric Oxide Synthase in an Oligodendrocyte Cell Line

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and interferon-γ (IFNγ), had either minimal (TNFα) or no (IL-1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNFα and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.     

Control of Large Chromosomal Duplications in Escherichia Coli by the Mismatch Repair System

Excessive recombination between repeated, interspersed, and diverged DNA sequences is a potential source of genomic instability. We have investigated the possibility that a mechanism exists to suppress genetic exchange between these quasi-homologous (homeologous) sequences. We examined the role of the general mismatch repair system of Escherichia coli because previous work has shown that the mismatch repair pathway functions as a barrier to interspecies recombination between E. coli and Salmonella typhimurium. The formation of large duplications by homeologous recombination in E. coli was increased some tenfold by mutations in the mutL and mutS genes that encode the mismatch recognition proteins. These findings indicate that the mismatch recognition proteins act to prevent excessive intrachromosomal exchanges. We conclude that mismatch repair proteins serve as general controllers of the fidelity of genetic inheritance, acting to suppress chromosomal rearrangements as well as point mutations.     

Impairment of Lysosomal Activity as a Therapeutic Modality Targeting Cancer Stem Cells of Embryonal Rhabdomyosarcoma Cell Line RD

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing’s sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.     

Selection and Characterization of a Peptide Specifically Targeting to Gastric Cancer Cell Line SGC-7901 Using Phage Display

Peptides selected from phage display have great potential to become probes for the imaging detection of the cancer. To develop the peptide probe for diagnosis of GC, a 12-mer phage display library was used to select peptides that bind specifically to the human GC cell line SGC-7901. After four rounds of in vitro selection, five phage clones that bound specifically to the SGC-7901 cells were selected. The phage clone GP-5 had a particularly high affinity and specificity for SGC-7901 cells. This clone was identified using a series of methods. The peptide GP-5 that was displayed on phage GP-5 exhibited high specificity to SGC-7901 cells and gastric tissues. Thus, the peptide GP-5 displays excellent potential for imaging detection of human gastric cancer.     

可诱导的稳定表达Spata3细胞系的建立及特征

spata3是一个在睾丸中特异性表达的基因,可能与精子发生或生精细胞凋亡相关.为了进一步研究Spata3的功能,将spata3克隆入经修饰的pcDNA5/FRT/TO表达载体,应用Flp-InTMT-RExTM-293 细胞系作为拗飨赴? 成功地构建了可被四环素或 Doxycline 诱导的稳定表达 Flp-InTMT-RExTM-sptat3 的细胞系.该细胞系在spata3基因 的3'端有2×FLAG tag和2×Histag,在缺乏可利用的spata3或其抗体的情况下,也能够很容易地应用商品化的FLAG抗体检测到spata3全长蛋白的表达.这种可诱导的稳定表达Flp-InTMT-Rex TM-spata3 的细胞系的建立,不仅有利于spata3的分析鉴定和功能研究,而且对于其他蛋白质的分离纯化和功能研究也有很好的借鉴作用.     

Establishment,Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

Background

Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.

Methodology

MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.

Principal Findings

Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras^wt, B-raf^mut, HROC87: K-ras^wt, B-raf^mut), whereas the HROC113 cell line (K-ras^mut, B-raf^wt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM⁺) tumor cells, showing surface tumor marker expression (CEACAM⁺). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.

Conclusions

These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.