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.     

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.     

DNA repair and chromosomal stability in the alkylating agent-hypersensitive Chinese hamster cell line 27-1

27-1 is a mutant of Chinese hamster ovary cells (CHO cells) that is hypersenstivie to the toxic effects of ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and other monofunctional alkylating agents. We show here that the enhanced MNNG sensitivity of these cells is not due to alterations in the amount of DNA methylation products introduced nor by a defect in the first step of removal of the main alkylation products 7-methylguanine and 3-methyladenine. However, these mutant cells perform more DNA repair synthesis after treatment with MNNG than normal CHO-9 cells. This observation might indicate a possible defect of a ligase involved in sealing DNA repair patches.27-1 cells did not show elevated frequencies of sister-chromatid exchange and chromosomal aberration induced by MNNG. The data show that MNNG-induced cell killing is not necessarily related to increased chromosomal instability.     

Mutagenic and epigenetic influence of caffeine on the frequencies of UV-induced ouabain-resistant Chinese hamster cells

Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive “error-free” postreplication repair process, allows an “error-prone” DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Relationship between DNA alkylation and specific-locus mutation induction by N-methyl- and N-ethyl-N-nitrosourea in cultured Chinese hamster ovary cells (CHO/HGPRT system)

Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43--13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per mumol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7--26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per mumol nucleotide per mM. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringently-defined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01--2.0 mM); mean values were 2800 and 840 mutants per 10(6) clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.     

Mechanism of comutagenesis of sodium arsenite withn-methyl-n-nitrosourea

Arsenic compounds are known carcinogens. Although many carcinogens are also mutagens, we have previously shown that sodium arsenite is not mutagenic at either the Na⁺/K⁺ ATPase orhprt locus in Chinese hamster V79 cells. It can, however, enhance UV-mutagenesis. We now confirm the nonmutagenicity of sodium arsenite in line G12, a pSV2gpt-transformed V79 (hprt ⁻) cell line, which is able to detect multilocus deletions in addition to point mutations and small deletions. The lack of arsenic mutagenicity has led to studies emphasizing its comutagenicity. Sodium arsenite at relatively nontoxic concentrations (5 μM for 24 h or 10 μM for 3 h) is comutagenic withN-methyl-N-nitrosourea (MMU) at thehprt locus in V79 cells. Using a nick translation assay, which measures DNA strand breaks by incorporating radioactive deoxyribonucleoside monophosphate at their 3′OH ends in permeabilized cells, we found that much more incorporation was seen in cells treated with MNU (4 mM, 15 min) followed by 3-h incubation with 10 μM sodium arsenite compared with cells exposed to the same MNU treatment followed by 3-h incubation without sodium arsenite. This result shows that in the presence of arsenite, strand breaks resulting from MNU or its repair accumulate over a 3-h period. We suggest that the repair of MNU-induced DNA lesions may be inhibited by arsenite either by affecting the incorporation of dNMPs into the MNU-damaged DNA template or by interfering with the ligation step.     

Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells

Studies were performed on the non-linear dose response for gene mutations induced by low doses of monofunctional methylating agents in V79 Chinese hamster cells. When treatment with methylnitrosourea was applied at the beginning of the S phase in synchronized cells, a linear dose-response curve was obtained, whereas application of the dose after gene replication resulted in a strong reduction of the number of induced mutations. Additional time for repair resulted in reduced dose response of MNU, indicating that an error-free repair process operates on methylated DNA in V79 Chinese hamster cells.     

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.     

Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage

Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O⁶-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N′-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced γH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.     

Characteristics of mutagenesis induced by nitrosomethylurea in a mammalian multilocus system in vitro

The mutagenic effect of short- and long-term MNU exposition causing alkylation and that combined with carbamoylation, correspondingly, at the four specific gene loci of the CHO-AT3-2 Chinese hamster line was studied. The increase both in MNU mutagenic effects and in the range of induced changes resulted from intensification of carbamoylating processes. True point mutations occurred mainly on alkylation. When carbamoylation is increased, the quantity and variety of mutations change towards accumulation of the number of other genetic lesions, for example, frameshift mutations and deletions.     

Repair studies on methylated DNA of CHO cells

DNA methylation pattern was investigated on Chinese hamster ovary (CHO) cells after treatment with N-(14C)-methyl-N-nitrosourea (14C-MNU). The main target was the N-7 position of guanine, exceeding the methylation in the O6 position of guanine by a factor of 8 and that in the N-3 position of guanine and adenine by a factor of 20. No DNA repair could be observed within 2 hours after methylation. Pretreatment of cells with gamma irradiation (7 rad) before application of MNU induced repair of N-7-methylguanine. This methylation product was decreased to about 50% within two hours, whereas the repair of the other methylated bases was not influenced. The analysis was carried out by high performance liquid chromatography after acid hydrolysis of isolated DNA. 14C-methylated products were determined by liquid scintillation counting.     

Relative mutagenicity of antineoplastic drugs and other alkylating agents in V79 chinese hamster cells,independence of cytotoxic and mutagenic responses

The mutagenic and cytotoxic effects of 4 antineoplastic drugs, vinblastine, vincristine, adriamycin and nitrogen mustard and of several monofunctional alkylating agents have been assayed in V79 Chinese hamster cells. Vincristine, vinblastine and nitrogen mustard did not significantly increase the frequency of TG^RHGPRT^? mutants but were all highly cytotoxic. Adriamycin and the monofunctional alkylating agents were all significantly mutagenic even at the lowest doses tested (approx. 70 % survival level). Induced mutant frequency increased linearly with increasing dose whereas dose-response curves for cytotoxicity for these effective mutagens invariably showed a shoulder followed by an exponential decline. At equitoxic doses the relative mutagenic effectiveness was MNU ENU EMS MMS ? DMS. MNU was approx. 20 times more effective than MMS and DMS.Measurement of the total amount of alkylation and the relative amounts of reaction with individual DNA bases at approx. equitoxic doses of MNU and DMS indicated a significantly higher O⁶/N⁷ ratio after MNU (0.15) than after DMS (0.005). However, approx. equal numbers of mutants/10⁵ cells/μM O⁶-Meguanine were induced by these 2 agents. These results support previous conclusions, that mutagenic and cytotoxic responses are independent in V79 cells.     

Protective effects of sodium selenite on killing and mutation by N-methyl-N''-nitro-N-nitrosoguanidine in E. coli.

Sodium selenite was found to protect Escherichia coli cells against killing and mutagenic effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such protective effects were not observed when cells were treated with N-methyl-N-nitrosourea (MNU). The protection by sodium selenite was not controlled by the ada gene, which is responsible for the repair of alkylated damage in DNA. A reduction of the amount of glutathione was found when cells were treated with sodium selenite, and glutathione is known to be involved in the methylation of DNA by MNNG, not by MNU. Reduced methylation by MNNG due to the reduction of the amount of glutathione caused by abundant sodium selenite was suggested to be the mechanism of protection.     

Correlation between specific DNA-methylation products and mutation induction at the HGPRT locus in Chinese hamster ovary cells

Suspension cultures of Chinese hamster ovary (CHO) cells were exposed to methyl methanesulfonate (MMS) or methylnitrosourea (MNU) and assayed for mutation induction (6-thioguanine resistance) and for specific DNA adducts. DNA methylation at the 1-, 3- and 7-positions of adenine, the 3-, O6- and 7-positions of guanine, and phosphate was detected in cultures exposed to MMS, while MNU produced 3- and 7-methyladenine, 3-methylcytosine, 3-, O6- and 7-methylguanine, O4-methylthymidine and methylated phosphodiesters. When mutations induced by MMS and MNU were compared by linear correlation analysis with levels of each of these adducts, only O6-methylguanine displayed a strong correlation with mutations (r = 0.879, p less than 0.001). The relationship between O6-methylguanine and induced mutations in CHO cells is similar to that previously reported in CHO cells for O6-ethylguanine and mutations (Heflich et al., 1982) and indicates that alkylation-induced mutations at the HGPRT locus in CHO cells are primarily associated with O6-alkylguanine formation.     

Methylation damage response in hematopoietic progenitor cells

The cellular response to methylation DNA damage was compared in multipotent CD34(+) hematopoietic stem cells and mature CD34(-) cells isolated from cord blood of the same donor. Cytofluorimetric analysis of freshly isolated cord blood cells indicated that both cell types were in the G0/G1 phase of the cell cycle. Quantitative RT-PCR identified a general trend towards high expression of several DNA repair genes in CD34(+) cells compared to their terminally differentiated CD34(-) counterparts. The overexpressed genes included members of the mismatch repair (MMR) (MSH2, MSH6, MLH1, PMS2), base excision repair (AAG, APEX), DNA damage reversal (O(6)-methylguanine DNA methyltransferase) (MGMT), and DNA double strand breaks repair pathways. These differences in gene expression were not apparent in CD34(+) and CD34(-) cells obtained following expansion of CD34(+) cells in a medium containing early acting cytokines. Early progenitor CD34(+) and early precursor CD34(-) cells form the two populations isolated under these experimental conditions, and both contain a significant proportion of cycling cells. The methylating agent N-methyl-N-nitrosourea (MNU) induced similar levels of apoptosis in these cycling CD34(+) and CD34(-) cells. Cytotoxicity required the presence of the MGMT inhibitor O(6)-benzylguanine and the timing of MNU cell death (48 and 72h) was similar in CD34(+) and CD34(-) cells. These data indicate that cycling CD34(+) and CD34(-) cells are equally sensitive to methylation damage. MGMT provides significant protection against MNU toxicity and MGMT and MMR play the expected roles in the MNU sensitivity of these cells.     

Effects of cis-platinum(II) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing HeLa cells in the absence and presence of caffeine

Synchronously growing HeLa cells demonstrated a different profile of DNA synthesis to that observed for Chinese hamster V79-379A cells after treatment with cis-Platinum(II) diamminedichloride (cis-Pt(II)) in the G₁ phase of the cell cycle. The progression of G₁ phase treated cells into the DNA synthetic phase was not affected. The peak rate of DNA synthesis in the first cycle was decreased in a dose dependent manner. However, no displacement in the time of appearance of this peak rate of DNA synthesis was observed in the first cycle as had been observed in Chinese hamster V79-379A cells. The timing of mitosis after the first cycle was delayed in a dose dependent manner and resulted in a concomitant delay in the appearance of the peak rate of DNA synthesis in the second cycle. The peak rate of DNA synthesis in the second cycle was reduced in a dose dependent manner. The ability of cells to divide after the first cycle was not related to their eventual ability to survive. Incubation of HeLa cells with caffeine after treatment with cis-Pt(II) did not increase the toxicity of cis-Pt(II). This was consistent with the lack of effect of caffeine posttreatment on the rate of DNA synthesis in cis-Pt(II) treated synchronously growing HeLa cells. HeLa cells did not show the characteristics of caffeine sensitive replication repair, nor did they show evidence for the presence of an inducible repair system. The rate of DNA synthesis, cell number and survival data were discussed in relation to a mechanism of cell death proposed for Chinese hamster cells.     

Enhanced survival and reduced mutation and aberration frequencies induced in 79 Chinese hamster cells pre-exposed to low levels of methylating agents

Exposure of V79 Chinese hamster cells to a single very low (sub-toxic and sub-clastogenic) dose of MNU or MNNG made these cells resistant to the toxic, mutagenic and clastogenic activities of the same agents given 6 h later. Cell survival was increased nearly 2-fold under optimal conditions when compared with the non-pretreated controls. Aberration frequencies were reduced to nearly half the control values (cells not pretreated). This was observed for a wide range of pretreatment concentrations and at different recovery times. The effect of mutagen pretreatment was most pronounced with respect to the induction of TG resistance, which became drastically reduced. The data indicate the existence of an adaptive repair pathway in V79 cells which may be induced by very low levels of methylating agents and which is error-free in handling lesions responsible, at least partially, for reproductive cell death, mutations and chromosomal aberrations.     

A relationship between nuclear DNA and RNA synthetic activities and the changes produced by n-methyl-n-nitroso urethane: A study using Amoeba proteus as a single cell model

Changes caused by a carcinogen generally vary from one cell to another even among similar types of cells. The following work investigates the degree to which damage (inhibition of division, lethality, or inherited cellular changes) caused by N-methyl-N-nitroso urethane (MNU) alters at different times during the cell cycle, and relates fluctuations in the sensitivity of cells to changes in their DNA and RNA synthetic activities—possibly in the configuration of their DNA—at the time of treatment.Studies on amoebae exposed to MNU for short periods at 50 different times in their cell cycle led to the following conclusions: amoebae are sensitive to MNU at all ages, but the dose needed to produce lethal damage to young and old cells varies by a factor of 3. Cells are most sensitive at the time of division and during the peak of DNA synthesis. Smaller changes are found during the G₂ phase, some of which occur at times of intensive RNA synthesis. Transfer of nuclei between treated and control cells proved that the changing sensitivity of the cells, as shown by both inherited changes and lethal damage, was dependent on changes in their nuclei. Though the cytoplasm could be affected directly by MNU, i.e. in the absence of a nucleus, supralethal doses 2–6 times whole cell dose were required to either kill the cell or to cause a recognizable change in the offspring of viable cells. Experiments with cells having altered nuclear/cytoplasmic ratios showed that the relative insensitivity of older cells was not due to the increased volume of their cytoplasm. However, a possible involvement of cytoplasm in the repair of nuclear damage is suggested by the ability of control cytoplasm to alleviate some nuclear damage, particularly in S phase cells.     

Cytotoxicity, mutations and DNA damage produced in Chinese hamster cells treated with streptozotocin, its analogs, and N-methyl-N'-nitro-N-nitrosoguanidine.

The activities of streptozotocin (SZ), three structural analogs of SZ, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in producing cytotoxicity, mutations to 8-azaguanine (8-AzG) resistance, and DNA damage (single-strand breaks) in V79 Chinese hamster cells have been examined. These three biological processes appear to be associated. MNNG was about 10(3) times more active on a molar basis than SZ, and the activities of the analogs fell within these extremes.