Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.     

Stable luciferase transfected cells for studying steroid receptor biological activity

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in Intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.     

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.     

DNA repair of clustered uracils in HeLa cells

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.     

An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems

BACKGROUND: Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery. METHODS: In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells. RESULTS: Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells. CONCLUSION: Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.     

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes

Maintenance of genome stability relies on the accurate repair of DNA double-strand breaks (DSBs) that arise during DNA replication or introduced by DNA-damaging agents. Failure to repair such breaks can lead to the introduction of mutations and chromosomal translocations. Several pathways, homologous recombination, single-strand annealing and nonhomologous end-joining, are known to repair DSBs. So far in the silkworm Bombyx mori, these repair pathways have been analyzed using extrachromosomal plasmids in vitro or in cultured cells. To elucidate the precise nature of the chromosomal DSB repair pathways in cultured silkworm cells, we developed a luciferase-based assay system for measuring the frequency of chromosomal homologous recombination and SSA. An I-SceI-induced DSB, within a nonfunctional luciferase gene, could be efficiently repaired by HR. Additionally, the continuous expression of the I-SceI endonuclease in the HR reporter cell allowed us to investigate the interrelationship between HR, SSA and NHEJ. In this study, we demonstrated that chromosome DSBs were mainly repaired by NHEJ and HR, whereas SSA was unlikely to be a dominant repair pathway in cultured silkworm cell. These results indicate that the assay system presented here will be useful to analyze the mechanisms of DSB repair in insect cells.     

DNA polymerase β and PARP activities in base excision repair in living cells

To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14 h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair capacity for uracil-DNA in wild type mouse fibroblasts was very strong, whereas the repair capacity for THF-DNA, although strong, was slightly weaker. Repair capacity in DNA polymerase β (Pol β) null cells for uracil-DNA and THF-DNA was reduced by approximately 15% and 20%, respectively, compared to that in wild type cells. In both cases, the repair deficiency was fully complemented in Pol β null cells expressing recombinant Pol β. The effect of inhibition of poly(ADP-ribose) polymerase (PARP) activity on repair capacity was examined by treatment of cells with the inhibitor 4-amino-1,8-naphthalimide (4-AN). PARP inhibition decreased the repair capacity for both lesions in wild type cells, and this reduction was to the same level as that seen in Pol β null cells. In contrast, 4-AN had no effect on repair in Pol β null cells. The results highlight that Pol β and PARP function in the same repair pathway, but also suggest that there is repair independent of both Pol β and PARP activities. Thus, before the BER capacity of a cell can be predicted or modulated, a better understanding of Pol β and PARP activity-independent BER pathways is required.     

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3′-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.     

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.     

In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells

We describe a rapid and highly efficient method for the assembly, recombination, targeted chromosomal integration and regulatable expression of mosaic metabolic pathways by homeologous recombination in DNA repair deficient yeast cells. We have assembled and recombined 23 kb pathways containing all the genes encoding enzymes for the production of flavonoids, a group of plant secondary metabolites of nutritional and agricultural value. The mosaic genes of the pathways resulted from pair-wise recombination of two nonidentical (homeologous) wild-type genes. The recombination events occurred simultaneously in the cell. Correctly assembled mosaic gene clusters could only be observed in DNA repair deficient strains. Thus, libraries of intragenic mosaic pathways were generated. Randomly isolated clones were screened for their ability to produce flavonoids such as kaempferol, phloretin and galangin. Thus, the functionality of the recombinant pathways was proven. Additionally, significant higher concentrations of metabolites such as naringenin, pinocembrin and dihydrokaempferol were detected. Further analysis also revealed the production of different aromatic compounds such as styrene, hydroxystyrene, phloretic acid and other molecules. We show that the in vivo homeologous recombination strategy can generates libraries of intragenic mosaic pathways producing a high diversity of phenylpropanoid compounds.     

Differential regulation of expression of cytosolic and mitochondrial thioredoxin reductase in rat liver and kidney

Adequate supply of selenium (Se) is critical for synthesis of selenoproteins through selenocysteine insertion mechanism. To explore this process we investigated the expression of the cytosolic and mitochondrial isoenzymes of thioredoxin reductase (TrxR1 and TrxR2) in response to altered Se supply. Rats were fed diets containing different quantities of selenium and the levels of TrxR1 and TrxR2 protein and their corresponding mRNAs were determined in liver and kidney. Expression of the two isoenzymes was differentially affected, with TrxR1 being more sensitive to Se depletion than TrxR2 and greater changes in liver than kidney. In order to determine if the selenocysteine incorporation sequence (SECIS) element was critical in this response liver and kidney cell lines (H4 and NRK-52E) were transfected with reporter constructs in which expression of luciferase required read-through at a UGA codon and which contained either the TrxR1 or TrxR2 3'UTR, or a combination of the TrxR1 5' and 3'UTRs. Cell lines expressing constructs with the TrxR1 3'UTR demonstrated no response to restricted Se supply. In comparison the Se-deficient cells expressing constructs with the TrxR2 3'UTR showed considerably less luciferase activity than the Se-adequate cells. No disparity of response to Se supply was observed in the constructs containing the different TrxR1 5'UTR variants. The data show that there is a prioritisation of TrxR2 over TrxR1 during Se deficiency such that TrxR1 expression is more sensitive to Se supply than TrxR2 but this sensitivity of TrxR1 was not fully accounted for by TrxR1 5' or 3'UTR sequences when assessed using luciferase reporter constructs.     

Apoptosis induced by DNA uptake limits transfection efficiency

Electrotransfection is an effective method for transfecting lymphoid cells. However, the transfection efficiency of certain lymphoid cells is low. L1210 subclones and NFS-70 pro-B cells, which are highly refractory to various transfection methods, were used to identify the limiting factors. Cells were electrotransfected with plasmids coding for green fluorescence protein or luciferase. The luciferase expression of L1210 subclone 3-3 was found to increase 6-12 h after electroporation, but decreased significantly from 12 to 48 h. The lower level of luciferase activity at later time periods correlated with decreases in cell viability, which was shown to be due to apoptosis, as determined by propidium iodide/acrindine orange staining, DNA laddering, and prevention of cell death by addition of caspase inhibitors. Similar results were observed with NFS-70 pro-B cells and select L1210 subclones. In contrast, L1210 parental and L1210 subclone 7-15.6 cells undergo only low levels of apoptosis (< or = 5%). Apoptosis occurred only when DNA (plasmids or salmon sperm DNA) was present during electroporation, but was not dependent on the conformation of the DNA used or the expression of transgenes. Cells pulsed in the presence of dextran sulfate (MW 500,000) did not apoptose. Similar results were observed when L1210 subclone 3-3 was transfected using the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane, although the transfection efficiency and corresponding rate of apoptosis were significantly lower. Applying the caspase inhibitor fluoromethyl ketone (Boc-ASP-FMK) dramatically improved cell viability and transgene expression of select L1210 subclones and NFS-70 pro-B cells.     

雄激素应答元件假冒DNA对PSA基因启动子的抑制作用

研究雄激素应答元件假冒DNA(AREdecoy)对前列腺特异抗原 (PSA)基因启动子的抑制作用 .联合运用报告基因和假冒DNA策略 ,构建了含PSA基因 5′侧启动子区 6 40bpDNA的萤光素酶表达载体pGL3 PSA ,与人工合成的双链硫代ARE假冒DNA共转染前列腺癌细胞株PC3 M并作用不同的时间 (2 4h、4 8h、72h) .应用双萤光素酶测定系统 ,检测萤光素酶的表达活性 .结果显示 :AREdecoyDNA显著抑制报告基因萤光素酶的表达 ,抑制率可达 95 % ,而对照decoyDNA无此作用 .作用不同的时间对萤光素酶活性的抑制无显著性差异     

cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester

The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells. PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA. PMA increased luciferase activity in transfected HT-29 cells. A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells. Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA. There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells. Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines. As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA. These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells. In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.