Mutagenesis by N-acetoxy-2-acetyl aminofluorene of chinese hamster V79 cells is unaffected by caffeine

8-Azaguanine (AZG)- and 6-thioguanine (TG)-resistant cells (mutants) were induced in Chinese hamster V79-4 cells by 0.1--2.5 microgram/ml N-acetoxy-2-acetyle aminofluorine (AcAAF) treatments in the presence of 5% fetal bovine serum (FBS). The frequency of resistant colonies increased from 1 to 47 per 10(5) survivors. The effect of caffeine (50--200 microgram/ml) during the mutagenesis expression period was determined by adding caffeine 1--24 h after AcAAF. The medium was replaced after 48 h exposures so that caffeine was absent during subsequent selection with AZG or TG. No significant change in the AcAAF-induced mutant frequency occurred with any treatment combination although caffeine greatly enhanced the lethality associated with AcAAF treatments. Thus, caffeine interferes with postreplication repair in V79-4 cells without affecting the probability of error of the repair process. These results were obtained with a quantitative mutagenesis assay in which the cells were reseeded prior to selection to achieve maximum expression without interference from metabolic crossfeeding. In contrast, the commonly used in situ assay is subject to serious interference from crossfeeding and yields an artifactual enhancement of AcAAF mutagenesis by caffeine.     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.     

Ploidy dependence of induced mutation frequency in transformed Syrian hamster cells

The ploidy dependence of the induced frequency of a phenotype can be used to determine the dominant or recessive nature of a somatic mutation to a given trait. To demonstrate this we induced mutations in diploid and spontaneously occurring tetraploid clones of Syrian hamster embryo cells by treatment with EMS (1.2 mg/ml, 4 h). Mutagenized cells were assayed for the recessive mutation to 6-thioguanine resistance (5 μg/ml) and the dominant mutation to ouabain resistance (1.2 mM). The frequency of induction of the dominant mutation was equal in the diploid and tetraploid clones (2.3 × 10^?4). The frequency of induction of the recessive mutation was greatly reduced in the tetraploid clone relative to the diploid clone (1.8 × 10^?4 vs. 1.2 × 10^?3).6TG^r mutant subclones from the tetraploid clone remain nearly tetraploid, or even increase in ploidy, but show a reduction in the number of X chromosomes from two to one, or in some cases none (based on chromosome morphology). The principle of ploidy dependence is now being used to study the induction of phenotypes related to neoplastic transformation.     

Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Facilitation by pyrimidine deoxyribonucleosides and hypoxanthine of mutagenic and cytotoxic effects of monofunctional alkylating agents in Chinese hamster cells.

Hypoxanthine (Hx), thymidine (TdR) and deoxycytidine (CdR), at concentrations of 10(-5) M increased the yield of 8-azaguanine-resistant (AzGr) mutants induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster V79 cells. The cytotoxicity of MNNG was increased 2-fold in the presence of Hx, and more than 10-fold in the presence of TdR. This cytotoxic effect of TdR was abolished by equal concentrations of CdR, which by itself did not increase the cytotoxicity of MNNG. However, the yield of MNNG-induced AzGr colonies was increased 2--10-fold in the presence of both CdR and TdR. The AzGr colonies displayed phenotypes characteristic of hypoxanthine: guaninephosphoribosyltransferase-deficient (HGPRT-) mutants, or indicative of mutant HGPRT with altered substrate affinities. The nucleosides did not affect the growth or expression time of the HGPRT- mutants; the same extent of alkali-labile DNA damage occurred in cells treated with alkylating agents in the presence and absence of TdR and CdR; and the increase in mutation frequency in the presence of these nucleosides occurred not only with MNNG, but also with ethylating agents. Nucleosides treated with MNNG were not mutagenic, and treatment of the cells with TdR and CdR only prior to treatment with MNNG or only during selection with AzG did not increase the induced mutation frequency. Therefore, the interpretation is proposed that CdR, TdR and Hx produce nucleotide-pool imbalances that increase lethal and mutagenic errors of replication of alkylated DNA.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

Structure of thymidine kinase gene introduced into mouse Ltk- cells by a new injection method

Pricking, a new injection method developed by Yamamoto et al. (1981), can be used to introduce DNA into cultured cells with high efficiency. Closed circular plasmid DNA containing the cloned HSV-TK gene (pTK-1) was introduced by this method and the structure of DNA in stable transformants was examined. In most clones, the introduced DNA was integrated into the mouse genome in a tandemly repeated form. The possibility of multiple integration via mouse middle repetitive sequences was also examined using the chimeric plasmid with TK genes and middle repetitive sequences (pMRTK-1). Digestion with restriction enzymes showed that the middle repetitive sequence used in this experiment had no effect on the efficiency of transformation, suggesting that this sequence is unable to mediate homologous recombination with mouse genomes.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Factors affecting the growth of Chinese hamster cells in halt selection media

Factors affecting the efficiency of selection of “reverants” of salvage pathway mutants in media containing amethopterin have been examined. Our V79 Chines hamster cell line was found to require a significantly higher level of thymidine for optimal growth in such media than has been reported for other cell lines. Hypoxanthine (but not glycine) was also required for reversal of amethopterin toxicity, but levels did not differ significantly from those reported elsewhere. Growth in HAT was also dependent on plating density and serum batch. Our modification (VHAT) was compared with published HAT recipies in back selection reconstruction experiments. A sharp fall in EOR (efficiency of recovery) of wild type cells from mixtures with mutants at plating densities greater than 3500 cells/cm² (10⁵ cells/6 cm dish) was observed for VHAT. EOR with other HAT recipes was lower still, and was affected also by the particular mutant used in the mixture.EMS induced “revertants” were isolated from three 8AZ^r mutants by plating in VHAT. All. revertants were however amethopterin resistant, they were also 8AZ resistant and the mobility of residual HGPRT (as measured by polyacrylamide gel electrophoresis) was similar to that of their 8AZ^r parents i.e. dissimilar from that in wild type. The modal chromosome number of V79 wild type cells was 21. No significant deviation from this mode was detected in any of the mutant lines examined. The data indicate that the recovery of colonies in HAT from 8AZ^r mutants does not necessarily indicate that a back mutation in the structural gene for HGPRT has occurred. Thus, the frequency of HAT⁺ colonies cannot be taken as a direct indication of reversion frequencies.     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

Phenotypic expression time of mutagen-induced 6-thioguanine resistance in Chinese hamster ovary cells (CHO/HGPRT system).

Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (referred to as the CHO/HGPRT system) can be quantitated by selection for the phenotype of resistance to 6-thioguanine (TG) under stringently defined conditions. The phenotypic expression time, that is, the time interval after mutagen treatment which is necessary befor all mutant cells are able to express the TG-resistant phenotype, has been found to be 7–9 days in this CHO/HGPRT system when the cells are subcultured every 48 h. Subculture in medium with or without hypoxanthine (HX) utilizing trypsin, ethylenediaminetetraacetic acid (EDTA), or ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for cell removal yields identical results. When subculture at intervals greater than 48 h is employed, a slight lengthening of the expression time is observed. An alternative method to regular subculture has also been achieved by maintaining the cells in a viable, non-dividing state in serum-free medium. This procedure yields a similar time course of phenotypic expression and thus shows that continued cell division is not essential to this expression process. In addition, this observation offers methodology which can significantly reduce the investment of time and money for mutation induction determinations in this mammalian cell gene mutation assay.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

The effect of explosive blast loading on human neuroblastoma cells

Diagnosis of mild to moderate traumatic brain injury is challenging because brain tissue damage progresses slowly and is not readily detectable by conventional imaging techniques. We have developed a novel in vitro model to study primary blast loading on dissociated neurons using nitroamine explosives such as those used on the battlefield. Human neuroblastoma cells were exposed to single and triple 50-psi explosive blasts and single 100-psi blasts. Changes in membrane permeability and oxidative stress showed a significant increase for the single and triple 100-psi blast conditions compared with single 50-psi blast and controls.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

PFKFB3 Regulates Oxidative Stress Homeostasis via Its S-Glutathionylation in Cancer

Whereas moderately increased cellular oxidative stress is supportive for cancerous growth of cells, excessive levels of reactive oxygen species (ROS) are detrimental to their growth and survival. We demonstrated that high ROS levels, via increased oxidized glutathione (GSSG), induce isoform-specific S-glutathionylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) at residue Cys206, which is located near the entrance to the 6-phosphofructo-2-kinase catalytic pocket. Upon this ROS-dependent, reversible, covalent modification, a marked decrease in its catalytic ability to synthesize fructose-2,6-bisphosphate (Fru-2,6-P₂), the key glycolysis allosteric activator, was observed. This event was coupled to a decrease in glycolytic flux and an increase in glucose metabolic flux into the pentose phosphate pathway. This shift, in turn, caused an increase in reduced glutathione (GSH) and, ultimately, resulted in ROS detoxification inside HeLa cells. The ability of PFKFB3 to control the Fru-2,6-P₂ levels in an ROS-dependent manner allows the PFKFB3-expressing cancer cells to continue energy metabolism with a reduced risk of excessive oxidative stress and, thereby, to support their cell survival and proliferation. This study provides a new insight into the roles of PFKFB3 as switch that senses and controls redox homeostasis in cancer in addition to its role in cancer glycolysis.