Effect of split doses of N-methyl-N-nitrosourea on DNA repair synthesis in cultured mammalian cells

DNA repair was measured in human fibroblasts, mouse C3H 10 T 1/2 fibroblsts and rat hepatocytes by the non-semi-conservative incorporation of [3H]-TdR during DNA repair synthesis using liquid scintillation techniques. Confluent monolayers of these cells grown on cover slips were exposed to split doses (125 or 250 microgram/ml) of the mutagenic and carcinogenic alkylating agent MNU and DNA repair synthesis compared with that produced by a single dose (500 microgram/ml). No significant difference in DNA repair capacity was detected in the three cell lines treated with a single dose or split doses of MNU.     

Monodispersed circular DNA in mammalian cells

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units.mg-1.     

Oligomerization of expanded-polyglutamine domain fluorescent fusion proteins in cultured mammalian cells

Six inherited neurologic diseases, including Huntington's disease, result from the expansion of a CAG domain of the disease genes to produce a domain of more than 40 glutamines in the expressed protein. The mechanism by which expansion of this polyglutamine domain causes disease is unknown. Recent studies demonstrated oligomerization of polyglutamine-domain proteins in mammalian neurons. To study oligomerization of polyglutamine proteins and to identify heterologous protein interactions, varying length polyglutamine-green fluorescent protein fusion proteins were expressed in cultured COS-7 cells. The 19- and 35-glutamine fusion proteins (non-pathologic length) distributed diffusely throughout the cytoplasm. In contrast, 56- and 80-glutamine fusion proteins (pathologic length) formed fibrillar arrays resembling those previously observed in neurons in Huntington's disease and in a transgenic mouse model. These aggregates were intranuclear and intracytoplasmic. Intracytoplasmic aggregates were surrounded by collapsed intermediate filaments. The intermediate filament protein vimentin co-immunoisolated with expanded polyglutamine fusion proteins. This cellular model will expedite investigations into oligomerization of polyglutamine proteins and their interactions with other proteins.     

Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system

Mean field analysis of FKBP12 complexes with FK506 and rapamycin has been performed by using structures obtained from molecular docking simulations on a simple, yet robust molecular recognition energy landscape. When crystallographic water molecules are included in the simulations as an extension of the FKBP12 protein surface, there is an appreciable stability gap between the energy of the native FKBP12-FK506 complex and energies of conformations with the "native-like" binding mode. By contrast, the energy spectrum of the FKBP12-rapamycin complex is dense regardless of the presence of the water molecules. The stability gap in the FKBP12-FK506 system is determined by two critical water molecules from the effector region that participate in a network of specific hydrogen bond interactions. This interaction pattern protects the integrity and precision of the composite ligand-protein effector surface in the binary FKBP12-FK506 complex and is preserved in the crystal structure of the FKBP12-FK506-calcineurin ternary complex. These features of the binding energy landscapes provide useful insights into specific and nonspecific aspects of FK506 and rapamycin recognition.     

A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.     

Effect of mimosine on DNA synthesis in mammalian cells

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitivity growth-suppressed accumulated the mRNA of IgG1 which is reported stable, and IgG1 production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM lambda 1 chain and cultured at nonpermissive temperature to enhance production of lambda 1. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.     

Circadian rhythm of mitoses and number of cells synthesizing DNA following adrenalectomy in mice

The diurnal changes in the number of mitoses and DNA-synthesizing cells were studied in intact and adrenalectomized mice. In the corneal epithelium of adrenalectomized mice the diurnal rhythm of DNA-synthesizing cells became biphasic, and the variation amplitude of the 24-hour mitotic activity was sharply intensified. In the liver the diurnal rhythm of DNA-synthesizing cells was similar in the intact and the operated on mice; as to the mitotic rhythm - it became biphasic in the operated on animals. Thus, adrenalectomy deranged the phasic structure of the mitotic and DNA-synthesizing rhythms in the tissues under study and led to dissociation in the entrance of cells into the DNA-synthesis and mitosis.     

A hepatocyte-targeted conjugate capable of delivering biologically active colchicine in vitro

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.     

Targeted recombination with single-stranded DNA vectors in mammalian cells

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

DNA transfection to study translational control in mammalian cells

Mammalian cells respond to changes in their environment by rapid and reversible covalent modification of the translational machinery. In most cases, these modifications involve the phosphorylation and dephosphorylation of translation initiation factors (for review see Ref. 1). The modification of translation initiation factors may affect translational activity of either specific mRNAs or general cellular mRNAs. To study the effect of a particular factor or its modification on the translational capacity of an mRNA, there are a number of potential approaches that include in vitro translation reactions as well as in vivo experiments. Generally, experiments initially report a covalent modification that correlates with altered translational capacity of either a specific or a general class of mRNAs. The modification and the particular amino acid residue involved are then identified. Then mutations are made at the modified residue to prevent modification (for example, a serine-to-alanine mutation to prevent phosphorylation) and the effect of the mutant factor on the translation of a target mRNA is tested. The most convenient method for monitoring the effect of a mutant translation factor on translation is the use of transient DNA transfection. However, in certain situations it is desirable to isolate stably transfected cell lines to study the effect of overexpression, underexpression, or expression of a particular mutant translation factor. This article reviews two methods that are routinely used to study translational control that involve either transient or stable DNA transfection.     

Interference of DNA sequence divergence with precise recombinational DNA repair in mammalian cells

Studies done in prokaryotes and eukaryotes have indicated that DNA sequence divergence decreases the frequency of homologous recombination. To determine which step(s) of homologous recombination is sensitive to DNA sequence divergence in mammalian cells we have used an assay that does not rely on the recovery of functional products. The assay is based on the acquisition by homologous recombination of endogenous LINE-1 sequences by exogenous LINE-1 sequences. In parallel experiments, we introduced into mouse cells two gapped exogenous LINE-1 sequences, one from the mouse, L1Md-A2, and the other from the rat, L1Rn-3. Although L1Rn-3 is on average less than 85% homologous to the LINE-1 elements of the mouse, the frequency of homologous recombination with endogenous LINE-1 elements obtained with L1Rn-3 was the same as the one obtained with L1Md-A2 which is on average 95% homologous to the LINE-1 elements of the mouse. The endogenous LINE-1 sequences rescued by L1Rn-3 were 8-18% divergent from L1Rn-3 sequences, whereas those rescued by L1Md-A2 were 2-5% divergent from L1Md-A2 sequences. The gap which had been introduced into the exogenous LINE-1 sequences had been precisely repaired in 50% of the recombinants obtained with L1Md-A2. None of the L1Rn-3 recombinants showed precise gap repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells

We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.     

Circadian rhythms in cultured mammalian retina

Many retinal functions are circadian, but in most instances the location of the clock that drives the rhythm is not known. Cultured neural retinas of the golden hamster (Mesocricetus auratus) exhibited circadian rhythms of melatonin synthesis for at least 5 days at 27 degrees celsius. The rhythms were entrained by light cycles applied in vitro and were free-running in constant darkness. Retinas from hamsters homozygous for the circadian mutation tau, which shortens the free-running period of the circadian activity rhythm by 4 hours, showed a shortened free-running period of melatonin synthesis. The mammalian retina contains a genetically programmed circadian oscillator that regulates its synthesis of melatonin.     

Induction of oxidative DNA damage by ferric iron in mammalian cells

Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were compared with respect to their potential to induce oxidative DNA damage in V79 Chinese hamster cells. DNA base modifications, including 8-hydroxyguanine (7,8-dihydro-8-oxoguanine), were quantified by the frequency of lesions recognized by the bacterial Fpg protein (formamidopyrimidine-DNA glycosylase) in combination with the alkaline unwinding assay. Fe-NTA induced oxidative DNA damage in a time- and dose-dependent manner, yielding significant increases in Fpg-sensitive sites above background after incubation for 24 or 48 h with 500 and 250 microM respectively. At both time points the frequency of DNA base modifications exceeded the number of DNA strand breaks. In contrast, neither DNA strand breaks nor Fpg-sensitive sites were detected after treatment with Fe-citrate at concentrations up to 2 microM for 24 or 48 h; this inactivity of Fe-citrate was independent of the molar ratio of iron to ligand (1:1, 1:2, 1:10 or 1:20). The results indicate that the cellular damage induced by ferric iron depends strongly on the actual complex applied, possibly due to differences in the intracellular distribution, which in turn may affect the availability of iron for redox reactions at or in close proximity to the DNA.     

DNA damage produced by povidone-iodine in cultured human diploid cells

Povidone-iodine is capable of selective altering the DNA of human diploid cells growing in culture. This finding extends to eukaryotic cells, the previously reported DNA-modifying activity of this agent for bacteria. In view of the known relationship between DNA-modifying activity and potential carcinogenicity, the results suggest that the potential hazards posed by the widespread use of this agent be evaluated.