DNA In Mammalian Cells Research Articles

Development of an on-line liquid chromatography-electrospray tandem mass spectrometry assay to quantitatively determine 1,N(2)-etheno-2'-deoxyguanosine in DNA.

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysate. Using the newly developed technique, basal levels of 1,N(2)-etheno-2'-deoxyguanosine were determined in commercial calf thymus DNA (1.70 +/- 0.09 adducts/10(7) dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 +/- 0.4 adducts/10(7) dGuo), and in untreated female rat liver DNA (5.22 +/- 1.37 adduct/10(7) dGuo). The mutagenicity of 1,N(2)-etheno-2'-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N(2)-etheno-2'-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.

Morphology of human embryos and apoptosis, as measured by P53 expression, a protein marker

Objective: The current method to determine embryo quality for transfer to the uterus during in-vitro fertilization (IVF) procedures is based on morphologic criteria. The morphology of a poor quality embryo is consistent with the changes a cell undergoes during apoptosis, or programmed cell death. The expression of p53, a tumor suppressor marker and component in the apoptotic cascade, represents damaged DNA, arrested DNA, or RNA synthesis in mammalian cells. These cellular changes are the result of a cascade of molecular interactions in developing embryos. A limited number of studies on mouse and human embryos have shown that morphology is indicative of apoptosis. In addition, mouse embryo studies have demonstrated the relationship between p53 expression and embryo quality. The objective of this study was to evaluate a molecular marker (p53 protein expression) for apoptosis in human pre-implantation embryos and its relationship with embryo morphology.

Double strand break rejoining after irradiation of human fibroblasts with X rays or alpha particles: PFGE studies and numerical models.

When a charged-particle track intercepts the chromatin fibre in DNA of mammalian cells, clustered damage is induced depending on the DNA conformation, local environment and track structure. Intra-track correlated DNA damage may have a higher probability of being mis-repaired or left un-repaired. Fragment size-distributions of DNA double strand breaks (DSBs) induced in primary human fibroblasts by 240 kVp X rays and 238Pu alpha particles (110 keV.micron-1) were resolved using pulsed-field gel electrophoresis (PFGE). By monitoring DSB rejoining kinetics and changes in the fragment size distribution with repair time, the relevance of spatial association of DSBs in determining rejoining kinetics was investigated. Rejoining kinetics appeared bi-phasic and independent of the size of the DNA fragments for both radiation qualities, with high LET radiation-induced DSBs repairing more slowly. Results suggest that local complexity of individual DSBs, rather than spatial association with other breaks is more significant in the determination of rejoining kinetics.

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 μg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

Human cytomegalovirus terminase as a target for antiviral chemotherapy.

Herpesviral DNA packaging is a complex process involving binding and cleavage of DNA containing the specific DNA-packaging motifs, pac1 and pac2, and packaging of the resulting unit-length genomes into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of human cytomegalovirus the terminase consists of the proteins pUL56 and pUL89. While pUL56 (i) mediates the specific binding to pac sequences on the concatamers, (ii) provides energy for the translocation of the DNA to the procapsids and (iii) associates itself with the capsid for enabling the entry of the DNA into the procapsid, pUL89 is mainly required to effect DNA cleavage. Based on the limited efficacy of the current drugs ganciclovir, cidofovir and foscarnet, new antiviral therapeutics appear to be in demand. Inhibitors targeting pUL56 and/or pUL89 may offer an attractive alternative since mammalian cell DNA replication does not involve cleavage of concatameric DNA. Drugs targeted to terminase-like proteins should therefore be safe and highly selective.

Methyl CpG-binding proteins and transcriptional repression.

Since its discovery, methylation of DNA in mammalian cells has been correlated with transcriptional repression and with specialized chromatin structures. Recently, considerable progress has been reported in the identification of protein factors with a highly conserved DNA interaction surface, termed the methyl CpG-binding domain or MBD. A subset has been biochemically linked to histone deacetylases, suggesting a molecular mechanism for the functional properties of methylated DNA. Despite several obvious attractions, the connection between MBD proteins and histone deacetylases fails to explain all the existing data. In fact, the biochemistry and DNA-binding properties of most MBD family members have not been adequately described and considerable evidence exists for alternative mechanisms in the repression of methylated loci. Null mutations have been generated in mice for several MBD family members, the phenotypes of the mutant animals raise important questions regarding the functions of the MBD family. Here, I review the biochemistry, DNA-binding properties, and genetics of the MBD proteins that are linked to transcriptional repression, namely, MeCP2, MBD1, MBD2, and MBD3. Several models to account for the functional properties of methylated DNA are presented.

Induction of T → G and T → A transversions by 5-formyluracil in mammalian cells

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5′-CFTAAG-3′ and 5′-CTFAAG-3′ (F represents 5-fU), the recognition site for the restriction enzyme AflII (5′-CTTAAG-3′). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01–0.04%. The T → G and T → A transversions were the mutations found most frequently, suggesting the formation of 5-fU·C and 5-fU·T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.

Can a mixed damage interfere with DNA-protein cross-links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo, by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.

Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members

The BAC and PAC cloning systems allow investigators to propagate large genomic DNA fragments up to 300 kb in size in E. colicells.We describe a new PAC shuttle vector that can be propagated in both bacterial and human cells. Specifically, the P1 cloning vector pAd10sacBII was modified by the insertion of a puromycin-resistance gene ( pac), the Epstein-Barr Virus (EBV) latent replication origin oriP,and the EBV EBNA1 gene. Transfection studies in HEK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a loxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre -mediated deletion events. The transposon vector pTnPGKpuro/loxP was modified to make this system amenable to propagation in human cells by inserting pac, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C., 1997. Isolating large nested deletions in PACs and BACs by in vivo selection of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2205–2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an individual library member to demonstrate that all of the deletions still contain the required eukaryotic elements and that they were nested. All library members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid in delineating the boundaries of genes and other cis-acting elements.

Chromosomal integration of transduced recombinant baculovirus DNA in mammalian cells.

Our group and others have recently demonstrated the ability of recombinant baculoviruses to transduce mammalian cells at high frequency. To further characterize the use of baculovirus as a mammalian gene delivery system, we examined the status of transduced DNA stably maintained in Chinese hamster ovary (CHO) cells. Four independent clones carrying two introduced markers, the genes for neomycin resistance (Neo) and green fluorescent protein (GFP), were selected. PCR analysis, Southern blotting, and DNA sequencing showed that discrete portions of the 148-kb baculovirus DNA were present as single-copy fragments ranging in size from 5 to 18 kb. Integration into the CHO cell genome was confirmed by fluorescent in situ hybridization (FISH) analysis. For one clone, the left and right viral/chromosomal junctions were determined by DNA sequencing of inverse PCR products. Similarly, for a different clone, the left viral/chromosomal junction was determined; however, the right junction sequence revealed the joining to another viral fragment by a short homology (microhomology), a hallmark of illegitimate recombination. The random viral breakpoints and the lack of homology between the virus and flanking chromosomal sequences are also suggestive of an illegitimate integration mechanism. To examine the long-term stability of reporter gene expression, all four clones were grown continuously for 36 passages in either the presence or absence of selection for Neo. Periodic assays over a 5-month period showed no loss of GFP expression for at least two of the clones. This report represents the first detailed analysis of baculovirus integrants within mammalian cells. The potential advantages of the baculovirus system for the stable integration of genetic material into mammalian genomes are discussed.

DNA methylation influences the decatenation activity of topoisomerase II

Methylation of cytosine in CpG sequences of the DNA in mammalian cells is involved in the regulation of events like gene expression, genomic imprinting, transcription, and DNA replication. Changes in the DNA methylation pattern influence the DNA conformation. Therefore, certain proteins are disturbed in their interaction with DNA. In this paper, we investigate the influence on the decatenation activity of topoisomerase II, an essential enzyme that modulates DNA topology. We compare the decatenation activity of topoisomerase II for a mitochondrial parasite DNA, that we had methylated to different degrees, and found the decatenation to be methylation dependent. Thus, changes in the methylation pattern are a mechanism for the disturbance of certain topoisomerase II activities and may, therefore, be an event in topoisomerase II related mutagenicity and carcinogenicity.

Glutathione peroxidase and viral replication: implications for viral evolution and chemoprevention.

It is likely that several of the biological effects of selenium are due to its effects on selenoprotein activity. While the effects of the anti-oxidant selenoprotein glutathione peroxidase (GPx) on inhibiting HIV activation have been well documented, it is clear that increased expression of this enzyme can stimulate the replication and subsequent appearance of cytopathic effects associated with an acutely spreading HIV infection. The effects of GPx on both phases of the viral life cycle are likely mediated via its influence on signaling molecules that use reactive oxygen species, and similar influences on signaling pathways may account for some of the anti-cancer effects of selenium. Similarly, selenium can alter mutagenesis rates in both viral genomes and the DNA of mammalian cells exposed to carcinogens. Comparisons between the effects of selenium and selenoproteins on viral infections and carcinogenesis may yield new insights into the mechanisms of action of this element.

Cell growth and gene modulatory activities of Yunzhi (Windsor Wunxi) from mushroom Trametes versicolor in androgen-dependent and androgen-insensitive human prostate cancer cells.

The incidence of prostate cancer varies greatly throughout the world; it is highest in African-Americans and lowest in the Asian populations of China, India, and Japan. Geographical differences in both prevalence of latent prostate cancer and mortality have been postulated to be influenced by diverse tumor-promoting and protective factors, both environmental and dietary. Prostate cancer is a tumor with an extremely long latency; the pattern of prostate tumorigenesis, in terms of the display and sequence of appearance of particular molecular or biochemical features, or morphological changes, characterizing different stages of the carcinogenic process, is expected to be heterogeneous. Some insights into tumor heterogeneity and progression can be obtained from studies using cell lines, particularly those derived from different anatomical sites. The present study aims to investigate whether hormone-responsive LNCaP and androgen-refractory JCA-1, PC-3, and DU-145 prostate cancer cells are responsive to Yunzhi (YZ), a proprietary dietary supplement prepared from extracts of Trametes versicolor, also known as Coriolus versicolor (a mushroom consumed by Chinese for its purported health benefits), and to elucidate its mechanism of action. Ethanolic extracts (70%) of YZ significantly reduced LNCaP cell growth, down-regulated the levels of secreted PSA, but had less effects on the expression of intracellular PSA and did not affect levels of the androgen receptor. In androgen-unresponsive prostate cancer cells, YZ had a much less pronounced suppressive effect on proliferation of PC-3 and DU-145 cells, compared to LNCaP, and was inactive against JCA-1 cells. Western blot analyses show that the expression of Rb, a key regulatory protein in G1/S transition, and PCNA, integrally involved in mammalian cell DNA replication, were significantly reduced by treatment with YZ in PC-3 and DU-145 cells, respectively. In contradiction, none of these biochemical parameters were affected in JCA-1 cells under identical treatment conditions. Further analysis shows that YZ increased the levels of signal transducer and activator family of transcription factors STAT 1 and STAT 3 in JCA-1 and not LNCaP cells. The greater sensitivity of LNCaP cells to this polysaccharopeptide raises the possibility that YZ may be considered as an adjuvant therapy in the treatment of hormone responsive prostate cancer; additionally, it may have chemopreventive potential to restrict prostate tumorigenic progression from the hormone-dependent to the hormone-refractory state.

In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to C-raf.

The use of antisense oligodeoxynucleotides (AS-ODN) remains a viable method to downregulate selected gene function. However, limitations to the antisense approach remain, such as (1) difficulties in delivery of the AS-ODN into target tissues, (2) instability of AS-ODN in vivo, (3) uncertanties about the precise mode of action, and (4) toxic effects in animal and human studies. To circumvent some of these difficulties, we designed a vector set that directs the in vivo production of single-stranded DNA (ssDNA) of a desired target sequence with limited extraneous vector nucleotide sequences. One plasmid was designed to express Moloney murine leukemia virus (MoMuLV) reverse transcriptase (RT). Another expression plasmid contains the MoMuLV primer binding site at the 3'-end of its RNA transcript so that an ssDNA would be synthesized by RT when both plasmids are cotransfected into cells. To test this expression system, we constructed a plasmid set, pssXA/pssXB that produces ssRNA-cleaving DNA 10-23 enzyme (Santoro, S.W., and Joyce, G.F. [1997]. Proc. Natl. Acad. Sci. USA 37, 13330-13342). The DNA enzyme sequence was placed between two oligonucleotide arms that are complementary and able to specifically target C-raf kinase mRNA. These plasmids were transfected into the A549 lung carcinoma cell line. Reduced C-raf mRNA levels by up to 34%-36%, as determined by Northern blot analysis, were observed in the transfected cells. Our results demonstrate the feasibility of using this novel ssDNA expression system to generate any sequence of interest in vivo for antisense, RNA-cleavage DNA enzyme, or triplex-forming strategies.

Over Production of Recombinant Protein from Transfected Plasmid DNA in CHO-k1 Mammalian Cells

Over Production of Recombinant Protein from Transfected Plasmid DNA in CHO-k1 Mammalian Cells

Improvement of RNA-DNA oligonucleotide design by using mammalian nuclear extracts

An RNA-DNA oligonucleotide (RDO) has been shown to either correct or cause a specific point mutation in episomal or genomic target DNA in mammalian cells. The original design of the RDO consists of a double-hairpin capped duplex comprising a 25 nucleotide-long DNA stretch (DNA-containing strand) paired to a fully complementary 2'-O-methyl RNA stretch with a pentameric DNA interruption that carries a mismatch to target DNA (RNA-containing strand). In order to improve the gene conversion activity of the RDO, several oligonucleotides with structural and chemical modifications were synthesized and compared in their gene correction activity. Previously, we established an in vitro system capable of RDO-mediated gene correction of a point mutation (G → A) in the E. coli β-galactosidase gene by using mammalian nuclear extracts. Conversion frequencies among six mammalian cell types and the chicken DT40 cell line were compared by using a convenient bacterial assay that score blue or white colonies. This in vitro reaction with DT40 nuclear extract is now used to study the structure and activity relationship of the RDO. Modifications of the original RDO design including a complete sequence complementarity of the RNA-containing strand to target DNA, a replacement of the central five DNA residues with 2'-O-methyl RNA, and chemical modification of the hairpin loops result in a ten-fold increase in gene correction activity. Moreover, we show that the single-base correction in the target DNA is preferentially driven by the DNA-containing strand of the RDO by comparing two RDOs that carry a mismatch to target DNA either on the RNA- or DNA-containing strand. Thus, the highly sensitive and convenient assay utilizing E. coli β-galactosidase is not only useful to compare the gene correction frequency among different cell types but also to optimize the RDO structure.

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5′ side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.

Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-β- d-arabinofuranosylcytosine triphosphate (ara-CTP)

The antimetabolite 1-β- d-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-β- d-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases α, δ, and ϵ, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases α, δ, and ϵ are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase α increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase δ activity was not inhibited significantly by ara-CTP at 100 μM. Recent work has shown that the synthesome-associated DNA polymerase ϵ does not function in in vitro SV40 DNA replication, suggesting that only polymerases α and δ drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase α. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.

No sex please, we're mitochondria: a hypothesis on the somatic unit of inheritance of mammalian mtDNA

In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely. This model can account for the very slow rates of mitotic segregation observed in cultured, heteroplasmic cell-lines, and also for the apparently poor complementation observed between different mutant mtDNAs co-introduced into ρ0 cells (cells that lack endogenous mtDNA). It also provides a potential system for maintaining the mitochondrial genetic fitness of stem cells in the face of a presumed high somatic mutation rate of mtDNA and many rounds of cell division in the absence of phenotypic selection. BioEssays 22:564–572, 2000. © 2000 John Wiley & Sons, Inc.

No sex please, we're mitochondria: a hypothesis on the somatic unit of inheritance of mammalian mtDNA.

In this article we develop a model for the organization and maintenance of mitochondrial DNA (mtDNA) in mammalian somatic cells, based on the idea that the unit of genetic function comprises a group of mtDNA molecules that are semi-permanently associated as a mitochondrial nucleoid. Different mtDNA molecules within a nucleoid need not be genetically identical. We propose that nucleoids replicate faithfully via a kind of mitochondrial mitosis, generating daughter nucleoids that are identical copies of each other, but which can themselves segregate freely. This model can account for the very slow rates of mitotic segregation observed in cultured, heteroplasmic cell-lines, and also for the apparently poor complementation observed between different mutant mtDNAs co-introduced into rho(0) cells (cells that lack endogenous mtDNA). It also provides a potential system for maintaining the mitochondrial genetic fitness of stem cells in the face of a presumed high somatic mutation rate of mtDNA and many rounds of cell division in the absence of phenotypic selection. BioEssays 22:564-572, 2000.