Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

Chromosomal aberrations induced by maleic hydrazide and related compounds in Chinese hamster cells in vitro.

The cytotoxic effects and chromosomal abnormalities induced by maleic hydrazide (MH) and its salts were investigated in cultured V79 cells. MH, and its potassium (K-MH) and diethanolamine (DEA-MH) salts were tested. MH was 5--14 times more cytotoxic than its salts and almost 4.5 times less toxic than the related compound, hydrazine dihydrochloride (HDC). MH salts had very weak cytotoxicity; the LD50 values on V79 cells on exposure for 3 h in vitro were (in microgram/ml) 1100 (MH), 12 000 (DEA-MH), 20 000 (K-MH), 230 (HDC) and 10 000 (NaCl). Both MH and its salts--but neither HDC nor NaCl--caused chromosomal aberrations in cultured V79 cells. The maximal frequencies of aberrant cells in cultures exposed to the compounds for 3 h in vitro were 18% (mh at 100 microgram/ml), 18% (K-MH at 20 000 microgram/ml) and 13% (DEA-MH at 20 000 microgram/ml). Maximal frequencies observed in cultures treated with HDC or NaCl were 10% (HDC at 400 microgram/ml) and 5% NaCl at 10 000 microgram/ml). Those of positive groups were 97% (N-methyl-N'-nitro-N-nitrosoguanidine, MNNG, at 5 microgram/ml) and 16% (ethyl methanesulfonate, EMS, at 400 microgram/ml). These frequencies of MH and its salts were 3.25--4.5 times those in untreated control cells. These results suggested that MH and its salts had weak inducibili     

Accuracy of UV-induced DNA repair in V79 cells with imbalance of deoxynucleotide pools

Chemical mutagens generally cause nucleotide pool imbalance. We postulated that this effect might enhance the mutagenic effect by reducing the accuracy of DNA-repair synthesis. We used an inducer of DNA repair which causes minor pool modifications, namely UV light, and imbalanced the nucleotide pools by incubating UV-irradiated V79 cells with thymidine or deoxycytidine (10(-5)-10(-2) M) during the early phases of repair. The effects on pool sizes of the incubation with deoxynucleosides were determined by directly measuring the 4 deoxynucleoside triphosphates in cell extracts. The impairment of repair accuracy was evaluated by comparing the frequency of mutations at the HGPRT locus (induction of resistance to 6TG) in irradiated cells incubated with deoxynucleosides or allowed to carry on repair synthesis in nucleoside-free medium. Despite the marked imbalance of pyrimidine nucleotide pools, an increase of mutations was observed only with the highest concentrations of thymidine and deoxycytidine. Such an increase was much lower than that reported in the case of facilitation by excess nucleosides of chemically induced mutagenesis. The results indicate that UV-induced repair is scarcely affected by precursor biases.     

Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation.

The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.     

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.     

Caffeine inhibits excision of 7-bromomethylbenz (a) anthracene-DNA adducts from exponentially growing but not from stationary phase Chinese hamster cells.

Excision of 7-bromomethylbenz(a)anthracene (7-BMBA)-DNA adducts from exponentially growing cultures of Chinese hamster V79-379A cells followed logarithmic kinetics with a half of approximately 20 hrs. Post-treatment incubation in the presence of a sub-toxic concentration of caffeine markedly reduced this loss. Caffeine brought about a concomitant increase in overall DNA synthetic rate in treated exponential cultures. Excision in stationary, non-DNA-replicating cultures, was slower and caffeine did not affect this reduced rate of excision. These findings lend support to a previous proposition that the caffeine-induced inhibition of elongation of nascent DNA on a template containing chemical lesions results in an interference with the excision repair mechanism that removes these lesions.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

Potentially lethal damage, deficient repair in X-ray-sensitive caffeine-responsive Chinese hamster cells

Further studies are described with a radiation-sensitive clone of V79 Chinese hamster cells, designated V79-AL162/S-10. Extended postirradiation treatment with caffeine causes V79-AL162/S-10 cells to respond like repair-competent V79 cells, but both kinds of cells suffer enhanced killing by caffeine, in a similar fashion, when the postirradiation treatment period is relatively brief. Extended postirradiation treatment of repair-competent cells causes them to respond like sensitive cells without caffeine post-treatment. Treating irradiated V79-AL162/S-10 cells with hypertonic saline appreciably reduces the survival rescue which can be effected by caffeine. This latter observation leads to the inference that the sectors of damage affected by anisotonic shock and caffeine post-treatment overlap. From these and other results we propose that the DNA replicational machinery of the cell is the locus of action of these radiation damage/repair processes.     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

Inhibition of benzo[a]pyrene-induced mutagenesis in Chinese hamster V79 cells by hemin and related compounds

The inhibitory effects of hemin and related compounds on the mutagenicity of benzo[a]pyrene (BP) were investigated in Chinese hamster V79 cells co-cultivated with X-irradiated hamster embryo cells. Mutant V79 cells were selected by their resistance to ouabain. The mutation frequency induced by BP was substantially inhibited dose dependently by hemin. The mutagenicity of BP (1 microgram/ml) on V79 cells was reduced to 6.5% by hemin, 52% by biliverdin, 73% by protoporphyrin and 85% by chlorophyllin at the highest concentration of the compounds tested (15 microM).     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.     

Defect in the caffeine-dependent postreplication DNA repair in UV-sensitive Chinese hamster clone cells

The postreplication repair of DNA in the presence of caffeine was investigated in the Chinese hamster clones cells of different UV-sensitivity. Caffeine (10(-2)M) inhibits the repair of daughter DNA (PRR of DNA) in the UV-light irradiated cells of UV-resistant clones CHO-K1, 14-2C-1 and V79, but does not influence the PRR of DNA in cells of UV-sensitive clones CHS1 and CHS2. Thus, deficiency of PRR of DNA in cells of UV-sensitive clones (the repair of daughter DNA is significantly retarded) is associated with the defect of the caffeine-dependent component of this repair process.     

Sublethal and potentially lethal damage repair on thermal neutron capture therapy

Tonicity shock or caffeine postirradiation treatment makes evident fast-type potentially lethal damage (PLD). Caffeine expresses fast-type PLD more efficiently than tonicity shock in X-irradiated B-16 mouse melanoma cells, compared with V79 Chinese hamster cells. The survival curves of thermal neutrons for either V79 or B-16 cells exhibit no shoulder. Neither V79 nor B-16 cells show the sublethal damage (SLD) repair of thermal neutrons. Caffeine-sensitive fast-type PLD repairs exist in X-irradiated B-16 cells, as well as V79 cells. The fast-type PLD repair of B-16 cells exposed to thermal neutrons alone is rather less than that of X-irradiated cells. Furthermore, an extremely low level of fast-type PLD repair of B-16 cells with 10B1-paraboronophenylalanine (BPA) preincubation (20 hours) followed by thermal neutron irradiation indicated that 10B(n,alpha)7Li reaction effectively eradicates actively growing melanoma cells. The plateau-phase B-16 cells are well able to repair the slow-type PLD of X-rays. However, cells can not repair the slow-type PLD induced by thermal neutron irradiation with or without 10B1-BPA preincubation. These results suggest that thermal neutron capture therapy can effectively kill radioresistant melanoma cells in both proliferating and quiescent phases.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Activation of a well-behaved cell cycle in araC-treated V79 cells by caffeine

.3-1.0 microM araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1-2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 microM araC-treated cells. The cell cycle activated by caffeine in 0.3 microM araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0 microM araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with less than G1 DNA content.     

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Mitomycin C-induced mutagenesis

The mutagenic effect of mitomycin C (MC) has been shown in the S phase of Crepic capillaris cells. The repair ability of MC-induced DNA lesions proves exceedingly high, due to post-replicative and excision repair processes. In the experiments with MC-pretreatment of Crepic capillaris cells, nonmutagenic concentration of 1 microgram/ml provides inducible repair system--"adaptive response", which considerably decreases the levels of mutagenesis induced by MC at concentrations of 10, 20 and 40 micrograms/ml. Under adaptive response, the action of methyltransferase is possible.     

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m² to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.