Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

Replicative DNA synthesis in permeable fibroblasts from normal individuals and ataxia-telangiectasia patients

Replicative DNA synthesis in normal human fibroblasts was inhibited by 50% when they were X-irradiated (8 Gy) and made permeable 30 min later, whereas only a slight inhibition (20%) was observed in similarly treated ataxia-telangiectasia cells. Treatment of irradiated normal cells with caffeine (2 mM) before permeabilization reversed the inhibitory effects of X-rays, buf caffeine had no effect on DNA synthesis in permeable ataxia-telangiectasia cells. Diadenosine tetraphosphate (0.1 mM) did not affect DNA synthesis in permeable normal fibroblasts.     

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21(Cip1/Waf1) post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase eta, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol eta protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

花生种子萌发早期胚轴细胞DNA合成的特点

花生种子吸胀6h后胚轴DNA中有~3H-胸苷掺入。咖啡因和羟基脲均对6～12h的~3H—胸苷掺入具强烈的抑制作用;当12～24h时,咖啡因的抑制作用较大;但30h以后,羟基脲的抑制作用超过咖啡因。双链DNA放射性从种子吸胀9h后迅速上升,单链DNA放射性在吸胀12h后出现一个明显的峰。但在吸胀12h后,单链DNA形成和存在的时间是短暂的。     

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.     

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.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea.     

Effects of caffeine and inhibitors of DNA synthesis on chromatid-type aberrations induced by acetaldehyde in root-tip cells

Root-tip cells of Allium cepa were exposed to acetaldehyde (AA) and post-treated with caffeine and 3 inhibitors of DNA synthesis, namely hydroxyurea (HU), 5-fluorodeoxyuridine (FdUrd), and arabinofuranosylcytosine (araC). Caffeine strongly potentiated the frequency of chromatid-type aberrations when given immediately after the AA treatment or as a 5-h treatment starting 10 h before the addition of colchicine. In contrast, no enhancement was observed when caffeine was present for the last 2.5 h, simultaneously with colchicine. The inhibitors of DNA synthesis were given following this last schedule. Both HU and FdUrd clearly enhanced the yield of AA-induced chromatid aberrations, while no enhancement fo chromosone damage was observed after exposure in araC.     

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.     

Caffeine inactivation of mammalian cells in the S phase of the mitotic cycle due to anomalous replicon initiation during the inhibition of the elongation of replicating DNA

Chinese hamster cells were treated with an inhibitor of DNA synthesis (hydroxyurea or arabinoside-cytesine) in non-toxic concentrations for 20 hours in the presence or absence of caffeine (2 mM). Under these conditions caffeine considerably inactivates the cells. If cells are synchronized by hydroxyurea (0.25 mM) in the S-phase of mitotic cycle, the addition of caffeine kills all the S-phase cells, while gamma-irradiation or novobiocine treatment markedly decreases the sensibilizing effect of caffeine. These findings permit us to conclude that cell inactivation is due to anomalous reinitiation of DNA synthesis stimulated by caffeine in the presence of drugs which inhibit the DNA chain elongation.     

Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.     

Properties of mer HeLa cells sensitive or resistant to the cytotoxic effects of MNU; effects on DNA synthesis and of post treatment with caffeine

A line of HeLa cells was shown to be particularly sensitive to N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but not to variety of other cytotoxic agents. A resistant line (designated HeLa/A22), was derived by treating Hela cells repeatedly with MNU. Both the sensitive (HeLa) and resistant (Hela/A22) cells have a mer⁻ phenotype based both on their reduced rates of loss of O⁶-methylguanine (O⁶-MeG) from DNA and their low levels of the enzyme O⁶-methylguanine methyltransferase (MT). HeLa cells are therfore sensitive to unrepaired O⁶-MeG in DNA while the Hela/A22 cells are resistant to unexcised O⁶-MeG and thus the A22 cells have the mer⁻ rem⁺ phentype. MNU produced an imediate dose-dependent inhibition of DNA synthesis in cultures of both sensitive resistant cells which increased with time until about 4 h after treatment. DNA synthesis then recovered to near control rates in both sensitive and resistant cells before then exhibiting a progressive decrease after 24 h. DNA synthesis was more depressed at these late times after treatment in cultures of sensitive cells than in those of similarly-treated resistant cells. DNA synthesis remained depressed in sensitive cells but recovered 3 days after treatment in resistant cells.

Post treatment of incubation of MNU-treated HeLa cells with caffeine did not increase the toxic action of MNU. In contrast, post treatment of the resistant HeLa/A22 cells with caffeine resulted in a dramatic increase in the toxic effects of a higher equitoxic dose of MNU. The depressed rate of DNA synthesis observed in both cell lines after doses of MNU was partially reversed by post treatment with caffeine in both sensitive and resistant cells. These observations can be interpreted in terms of the effects of caffeine on DNA replication in treated cells.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G₁ cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells were accompanied by a lack of inhibition of DNA synthesis (replicon initiation) neither γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative.     

Effects of irradiation on nuclear protein synthesis in G2 phase of the cell cycle

A method was developed to determine the synthesis of nuclear proteins throughout the cell cycle which was resolved into six compartments on the basis of DNA and nuclear protein content (i.e., early and late G1, early and late S, etc). Using this technique cell-cycle-specific synthesis of certain nuclear proteins was observed. Of particular interest was a 170-kDa protein(s) whose synthesis was initiated in early S phase and reached a maximum rate in late G2. Following irradiation with 6.8 Gy of 137Cs gamma rays the synthesis of the 170-kDa protein(s) declined in the G2 population with near total inhibition seen by 24 h. Synthesis of the 170-kDa protein(s) appeared to be slightly enhanced, and the postirradiation inhibition of its synthesis was reversed, in the presence of 3 mM caffeine. Also, the synthesis of 55-kDa nuclear protein(s) was stimulated throughout the cell cycle in the presence of 3 mM caffeine. These observations suggest new possibilities regarding the mechanism of the X-ray-induced G2 block and its reversal by caffeine. However, the exact role of these nuclear proteins in cellular events remains to be ascertained.     

Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.     

Effect of caffeine on DNA synthesis in irradiated and unirradiated mammalian cells

Caffeine increased the availability of replication origins, and consequently the number of growing points, in the DNA of Chinese hamster V79 and human (HeLa) cells. Caffeine also prevented the inhibition of replicon initiation normally caused by X-radiation and exposure to low doses of ultraviolet light. When caffeine was removed from the medium after irradiation, replicon initiation was inhibited. Caffeine also reversed the inhibition of replicon initiation caused by novobiocin, which is not a DNA-damaging agent. Because caffeine increases the number of growing points, it also partially reversed the inhibition of total DNA synthesis induced by hydroxyurea. It is proposed that caffeine alters the conformation of intracellular chromatin in such a way that the conformation usually induced by DNA-damaging agents is prevented.     

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.