Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Studies on DNA binding of caffeine and derivatives: evidence of intercalation by DNA-unwinding experiments

Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.     

Repair of human sperm chromosome aberrations in the hamster egg

Summary In order to study the repair capacity of fertilized hamster eggs for the lesions present or induced in human sperm, we have examined the potentiating effect of caffeine, a DNA repair inhibitor, on the frequency and types of sperm chromosome aberrations. Sperm samples were donated by an individual treated with chemotherapy for a testicular cancer 3 years previously. Exposure of spermatozoa and inseminated oocytes to caffeine led to an increase of sperm chromosome aberrations, indicating that the damage to human sperm can be repaired in untreated hamster egg cytoplasm. The potentiating effect of caffeine was mainly reflected in an increase of unrejoined aberrations, indicating that the formation of chromosomal rearrangements is also inhibited. Since both chromatid-type and chromosome-type aberrations increase after treatment with caffeine, damage to human sperm can probably be repaired inside the hamster egg cytoplasm by pre and post-replication repair mechanisms.     

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.     

The effect of caffeine upon cell survival and post-replication repair of DNA after treatment of BHK 21 cells with either UV irradiation or N-methyl-N-nitrosoguanidine

It has been found that in BHK 21 cells caffeine potentiates cell killing by both UV irradiation and N-methyl-N-nitrosoguanidine (MNNG). The potentiating effect is greater with UV than with MNNG. While non-toxic concentrations of caffeine inhibit the joining of newly-replicated DNA fragments into large molecular weight DNA (post-replication repair) after UV irradiation, they have no such effect after MNNG treatment. Furthermore, the joining of DNA fragments continues in cells treated with 3 μg/ml of MNNG, a dose which leads to less than 5% cell survival. While inhibition of the synthesis of large molecular weight DNA can explain the synergistic effect of caffeine upon cell survival after UV irradiation, it cannot explain the similar effect after MNNG treatment.     

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.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Contribution of a caffeine-sensitive recombinational repair pathway to survival and mutagenesis in UV-irradiated Schizosaccharomyces pombe

Summary Cells of wild-type Schizosacharomyces pombe exposed to UV radiation in either G1 or G2 phase show enhanced inactivation of colony-forming ability if plated in the presence of caffeine. This UV-sensitization by caffeine is abolished in both G1 and G2 phase cells by the rad1 mutation; since both caffeine and the rad1 mutation markedly reduce recombinational events, this suggests that a recombinational repair process is active in cells irradiated either in G1 or G2 phase. A prereplicative or sister chromatid exchange recombinational process appears to account for caffeine-sensitive repair of UV-damage in G2 cells (which possess at the time of radiation exposure the duplicated genome necessary for recombination), since caffeine-sensitive repair begins immediately and is completed before resumption of DNA synthesis. In contrast, since caffeine-sensitive repair of UV-damage in G1 cells displays a considerable lag and then occurs concomitantly with DNA synthesis, it appears that G1 cells must acquire a second genome in order to accomplish a caffeine-sensitive recovery process. Since a duplicated genome is required for caffeinesensitive repair, all such repair would seem to involve a recombinational mechanism. In G1 cells the process may be a post-replication recombinational mechanism. Since G2 phase cells are considerably more UV-resistant than G1 phase cells, the prereplicative recombinational process appears to be a much more efficient process for dealing with UV-induced damage than the post-replication mechanism.UV-induced mutagenesis was examined in wildtype and rad mutants using a forward mutation system. Rad mutants which show higher UV-induced mutation rates than wild-type retain UV-sensitization by caffeine (and thus presumably retain the recombinational mechanism). In contrast, rad strains which are relatively UV-immutable compared to wild-type do not possess the caffeine-sensitive UV-repair process. The recombinational process therefore may be the major pathway responsible for UV-induced mutation.AECL Reference No. 6251; NRC Publication No. 16999     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) Diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA.

Treatment of Chinese hamster lung V79-379A cells with the anti-tumour agent cis platinum (II) diamminedichloride, (cis Pt(II)), resulted in an immediate recuction in the rate of DNA synthesis. Sedimentation of newly synthesised DNA through alkaline sucrose gradients revealed it to be approximately the same size as that obtained from untreated cells. In contrast, in the presence of 0.75 mM caffeine, the rate of DNA synthesis rapidly returned to control levels, although sedimentation analysis showed the DNA synthesised in cis Pt(II)-treated cells to be of lower molecular weight than in untreated cells. The reduction in molecular weight was directly proportional to the initial dose of the platinum compound. Furthermore, the results of separate binding studies suggested that at several levels of reaction the new DNA was synthesised up to a size approximately equal to the interplatinum distance in the template strand. This has been interpreted as being the result of the formation of a gap in the daughter DNA strand opposite every DNA-platinum product in the template strand. If caffeine was removed from the culture medium, there was a rapid increase in the molecular weight of the nascent DNA strands. However, if caffeine remained in the medium, the DNA remained of lower molecular weight than in untreated cells. It is proposed that this effect of caffeine is the result of the inhibition of a post-replicative DNA repair process which allows the eventual synthesis of a continuous DNA strand on a template containing unexcised lesions. It is further proposed that inhibition of this post-replicative DNA repair process provides a molecular basis for the previously observed potentiation by caffeine of cis Pt(II)-induced chromosomal aberrations and lethality.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Anthramycin-induced sister-chromatid exchange and caffeine potentiation in the chromosomes of Indian muntjac

Cell-cycle kinetics, sister-chromatid exchange (SCE) and chromosome aberrations have been studied from the skin fibroblasts of the Indian muntjac after treatment with 100 micrograms/ml of caffeine and 0.05 microgram/ml of anthramycin. The cultures were incubated for a period which was sufficient for the completion of two consecutive cell cycles and both the drugs appeared to produce a slight inhibitory effect. When anthramycin-treated cells were however post-treated with caffeine, the cells did not proceed beyond one cycle and exhibited a mitotic block. The SCE frequency in the control and the experiments with caffeine and anthramycin was 8.63, 18.32 and 34.88 per cell respectively. The SCEs were randomly distributed amongst all chromosomes unlike a non-random distribution within the X chromosomes. Caffeine and anthramycin produced only 0.5% and 3.1 cells with chromosome aberrations respectively. Potentiation of chromosome aberrations was observed when the anthramycin-treated cells were post-treated with caffeine. Caffeine potentiation presumably results from an inhibition of the cells to cycle and a failure to repair the effect of the mutagen on DNA.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

The inhibition by strongly acidic polypeptides of the repair of double-stranded DNA breaks induced by the gamma irradiation of Chinese hamster fibroblast cells]

The ability of polypeptides consisted of aspartic and glutamic acids to inhibit the repair and to promote the formation of unrepaired double-strand DNA breaks and chromosomal aberrations in gamma-ray induced Chinese hamster cells was shown. A complete inhibition of the double-strand DNA breaks repair was observed at the concentrations of 20 mu M/l (polyglutamic acid with molecular weight 2000-15,000 daltons) and 100 mu M/l (aspartylglutamic acid with molecular weight 1500-4500 daltons). Both polypeptides were low toxic at the given concentrations.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Enhancement and reduction by methylated oxypurines of the frequencies of chromatid aberrations induced by camptothecin in root-tip cells of Vicia faba.

In root-tip cells of Vicia faba the frequencies of chromatid aberrations induced by 3-h treatments with 0.05 microM camptothecin were strongly modified when the treatments were carried out in the presence of caffeine at concentrations above 1 mM. Depending on the concentration of caffeine, the clastogenic effect of camptothecin was either enhanced or reduced. At concentrations between 1 and 6 mM, caffeine increased the camptothecin-induced chromosome damage, the strongest enhancement being obtained at 5 mM. A reduction of the chromosome damage was apparent at caffeine concentrations above 10 mM, and in the presence of 20 mM caffeine the clastogenic effect of camptothecin was almost completely suppressed. When present during the camptothecin treatment, theophylline, 8-chlorocaffeine and 1,3,7,9-tetramethyluric acid influenced the induced chromosome damage in a similar way as caffeine, although with varying efficiency. If the concentrations required to produce the two types of modifying effect are used as a criterion, 8-chlorocaffeine was the most effective and 1,3,7,9-tetramethyluric acid the least, whereas caffeine and theophylline were about equally effective.     

Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells: Evidence for excision repair as an error-free repair process

Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various nontoxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequency was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.