5-azacytidine induces transgene silencing by DNA methylation in Chinese hamster cells

The cytosine analog 5-azacytidine (5-AzaC) is a demethylating agent that is also known to induce mutagenesis in mammalian cells. In this study, the mutagenic potential of this drug was tested in the G10 and G12 transgenic Chinese hamster cell lines, which have a single bacterial gpt gene integrated into the genome at different sites, with its expression driven by a simian virus 40 (SV40) promoter. We show that the mutation frequencies following a 48-h exposure to different concentrations of 5-AzaC were 10 to 20 times higher than those of any of the other numerous mutagens that have been tested in the G10-G12 system. Moreover, the mutation frequencies were much higher in the G10 cell line than in the G12 cells. Detailed molecular analysis of the 6-thioguanine (6-TG)-resistant variants demonstrated that transgene silencing by de novo DNA methylation and increased chromatin condensation in the SV40 promoter was the major factor responsible for this high level of 6-TG resistance. As would be expected, exposure to 5-AzaC lowered the overall genomic DNA methylation levels, but it unexpectedly caused hypermethylation and increased chromatin condensation of the transgene in both the G10 and G12 cell lines. These results provide the first evidence that 5-AzaC may also induce transgene-specific DNA methylation, a phenomenon that can further be used for the elucidation of the mechanism that controls silencing of foreign DNA.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.     

SCE induction and harlequin staining in mycoplasma-contaminated Chinese hamster cells

Chinese hamster V79 and CHO cells infected with Mycoplasma hyorhinis show elevated sister-chromatid exchange (SCE) levels but normal cell proliferation and levels of chromosomal aberrations when compared with uninfected cells. Harlequin staining patterns differ from those seen with uninfected cells at similar levels of bromodeoxyuridine (BrdUrd), indicating that BrdUrd is rapidly depleted from the medium by the mycoplasmal uridine phosphorylase and therefore becomes unavailable over the two cell cycles necessary for harlequin staining. Continuous treatment with the antibiotic minocycline restores the SCE level and harlequin staining to that seen in uncontaminated cells. The results suggest that mycoplasma infection should be suspected if harlequin staining patterns indicate a sudden decrease in incorporation of BrdUrd in cells grown in normal levels of BrdUrd.     

DNA methylation levels in porcine fetal fibroblasts induced by an inhibitor of methylation, 5-azacytidine

Removal of the somatic DNA methylation pattern from donor cells and remodeling of embryonic status have been suggested as integral processes for successful nuclear transfer (NT) reprogramming. This study has investigated the effects of 5-azacytidine (5-azaC), a DNA methylation inhibitor, on global methylation changes in porcine fetal fibroblasts (PFF); this may improve NT attributable to the potential reprogramming of the methyl groups. PFF in 5th passage cultures were treated with 0, 0.5, 1.0, 2.0, and 3.0 μM 5-azaC for 96 h; 5-azaC inhibited the growth at all tested concentrations. At the higher concentrations of 5-azaC used, cells appeared to exhibit morphological changes and to become apoptotic as observed by TUNEL assay. Thus, cells were negatively affected by 5-azaC. Differences in cellular ploidy were also observed at higher concentrations. Analysis showed no considerable changes in the proportion of cells at the G₁-phase of the cell cycle with 5-azaC concentrations. The fractional part of the methylated DNA of these cells was significantly reduced by 5-azaC treatment. Confocal microscopy confirmed the inhibition of methylation levels in PFF with increased concentrations of 5-azaC. Exposure to 5-azaC altered the expression of genes involved in imprinting (IGF2) or pro-apoptosis (BAX), whereas there was a reduction in the expression of the main enzyme responsible for replicating the DNA methylation pattern (DNMT1) and anti-apoptosis (BCL2L1). Therefore, 5-azaC induces a relative reduction in methylation in PFF, and cells treated with 0.5 μM 5-azaC may have enhanced potential for porcine NT.The financial support of BioGreen 21 (grant no. 100052004002000) and KOSEF (grant no. R05-2004-000-10702-0) in Korea is gratefully acknowledged.     

Human leukemic K562 cells: differential effects of 5-azacytidine on DNA methylation of epsilon-, gamma-globin and 7SL RNA genes

5 Azacytidine ribonucleoside (5 Aza CR), greatly enhances erythroid differentiation of the K562(h) cell line, with a sharp increase of embryonic and fetal globin gene expression. This phenomenon is correlated with the undermethylation of gamma-globin but not of epsilon-globin, as the epsilon-globin gene is already extensively undermethylated before 5AzaCR induction. By contrast no variations in both DNA methylation and expression are observed in 7SL RNA genes.     

Differential effects of 5-azacytidine and 5-azadeoxycytidine on cytotoxicity, DNA-strand breaking and repair of X-ray-induced DNA damage in HeLa cells

The cytotoxicity, DNA-strand breaking ability, and effects on repair of X-ray-induced DNA damage by short treatments with 5-azacytidine (azaCyd) and 5-azadeoxycytidine (azadCyd) were examined in HeLa cells. azaCyd was shown to be an effective inhibitor of the repair of X-ray-induced DNA single-strand breaks whereas azadCyd did not have this effect. At high doses, both compounds also induced DNA damage by themselves. The cytotoxicity, inhibition of repair, and drug-induced DNA damage associated with azaCyd treatment were all reversed by the concurrent addition to the cells of cytidine or uridine but not by thymidine, deoxycytidine or deoxyuridine. Cytotoxicity and drug-induced strand breaks associated with azadCyd treatment were reversed to varying degrees with all nucleosides and deoxynucleosides. These results support the notion that these two antileukemic cytidine analogs may have different mechanisms of action in exerting their antiproliferative activity.     

The effects of 4CMB, 4HMB and BC on SCE,chromosome aberration and point mutation in cultured Chinese hamster ovary cells

Cultured Chinese hamster ovary cells (CHO-KI-BH₄) were treated for 2 h with 4CMB, 4HMB and BC, in the absence of any exogenous metabolic activation system. The cells were subjected to tests for survival, sister-chromatid exchange, chromosome aberration and mutation to thioguanine resistance.4HMB had no effect in any test at concentrations up to 100 μg/ml. 4CMB was slightly more toxic than BC. Both 4CMB and BC induced SCE and chromosome aberrations, but the effects were more marked with BC. With 4CMB, SCE increased with dose only up to about 7 μg/ml and then levelled off. A weak mutagenic effect was observed with both BC and 4CMB, but in each case, the response reached a peak and was not evident at higher doses.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Recovery from effects of heat on DNA synthesis in Chinese hamster ovary cells

The hyperthermic inhibition of cellular DNA synthesis, i.e., reduction in replicon initiation and delay in DNA chain elongation, was previously postulated to be involved in the induction of chromosomal aberrations believed to be largely responsible for killing S-phase cells. Utilizing asynchronous Chinese hamster ovary cells heated for 15 min at 45.5 degrees C, an increase in single-stranded regions in replicating DNA (as measured by BND-cellulose chromatography) persisted in heated cells for as long as replicon initiation was affected. Alkaline sucrose gradient analyses of cells pulse-labeled immediately after heating with [3H]thymidine and subsequently chased at 37 degrees C revealed that these S-phase cells can eventually complete elongation of the replicons in operation at the time of heating, but required about six times as long relative to control cells which completed replicon elongation within 4 h. DNA chain elongation into multicluster-sized molecules was prevented for up to 18 h in these heated cells, resulting in a buildup of cluster-sized molecules (approximately 120-160 S) mainly because of the long-term heat damage to the replicon initiation process. Utilizing bromodeoxyuridine (BrdU)-propidium iodide bivariate analysis on a flow cytometer to measure cell progression, control cells pulsed with BrdU and chased in unlabeled medium progressed through S and G2M with cell division starting after 2 h of chase time. In contrast, the majority of the heated S-phase cells progressed slowly and remained blocked in S phase for about 18 h before cell division was observed after 24 h postheat. Our findings suggest that possible sites for where the chromosomal aberrations may be occurring in heated S-phase cells are either (1) at the persistent single-stranded DNA regions or (2) at the regions between clusters of replicons, because this long-term heat damage to the DNA replication process might lead to many opportunities for abnormal DNA and/or protein exchanges to occur at these two sites.     

Independence of centriole formation and initiation of DNA synthesis in Chinese hamster ovary cells

The relationship between centriole formation and DNA synthesis was investigated by examining the effect of taxol on the centriole cycle and the initiation of DNA synthesis in synchronized cells. The centriole cycle was monitored by electron microscopy of whole-mount preparations [Kuriyama and Borisy, J. Cell Biol., 1981, 91:814-821]. A short daughter centriole appeared in perpendicular orientation to each parent during late G1 or early S and elongated slowly during S to G2. Addition of 5-20 micrograms/ml taxol to a synchronous population of cells in S phase did not inhibit centriole elongation; rather, elongation was accelerated. In contrast, when taxol was added to M phase or early G1 cells, centriole duplication was completely inhibited. The taxol block was reversible since nucleation and elongation of centrioles resumed as soon as the drug was removed. Cells exposed to taxol progressed through the cell cycle and became blocked in mitosis, as indicated by an increase in the mitotic index, but eventually the mitotic arrest was overcome, resulting in formation of multinucleated cells. A peak in mitotic index was seen in the following generation, indicating that chromosomes duplicated in the presence of taxol. Incorporation of 3H-thymidine followed by autoradiography confirmed that DNA synthesis was initiated in the presence of taxol even though formation of daughter centrioles was inhibited. It seems, therefore, that centriole duplication is not a prerequisite for entry into S phase. Since DNA synthesis has already been demonstrated not to be necessary for centriole duplication, these two events, normally coordinated in time, appear to be independent of each other.     

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.     

Genotoxic effects and DNA photoadducts induced in Chinese hamster V79 cells by 5-methoxypsoralen and 8-methoxypsoralen

The induction of lethal effects and 6-thioguanine-resistant (6-TGr) mutants were studied in Chinese hamster V79 cells after treatment with the two bifunctional furocoumarins 5- and 8-methoxypsoralens (5-MOP, 8-MOP) in the presence of 365-nm radiation (UVA). The in vivo DNA-photobinding capacity of these two compounds was measured and in parallel the cross-linking capacities of 5-MOP and 8-MOP were determined using the alkaline elution technique. The results show that 5-MOP plus UVA was about 2.5 times more effective than 8-MOP plus UVA for inhibiting cell survival and for inducing the same frequency of 6-TGr mutants (10(-4]. The total number of photoinduced lesions by 5-MOP plus UVA was about 6 times higher than that induced by 8-MOP plus UVA. However, the cross-linking capacities of 5-MOP and 8-MOP were found to be within the same range at equal doses of UVA. At equal number of DNA photoadducts produced, the lesions induced by 5-MOP appeared to be less genetically active than those induced by 8-MOP. The apparently weaker genotoxicity of 5-MOP-induced lesions is likely to be due to the induction of a lower proportion of cross-links by 5-MOP at a given number of photoadducts.     

Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products.     

Effects of 5-azacytidine on methylation and expression of specific DNA sequences in C3H 10T1/2 cells.

The present study indicates that the transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine leads to extensive loss of methylation of the protooncogene c-mos and the beta-globin locus at the cell population level and in at least 40 isolated subclones. These changes persisted, even when the cells were serially passaged for many generations without further exposure to the drug. Even though the amount of demethylation, assessed through differential digestion by the restriction enzymes HpaII and MspI, was quite extensive, neither locus was transcribed at a detectable level. This nonselective analysis suggests, therefore, that loss of DNA methylation is not sufficient per se to induce the expression of certain loci. Presumably, the expression of these loci requires additional factors, some of which may be related to cell lineage and differentiation.