Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.     

Toxic effects of ozone on murine L929 fibroblasts. Damage to DNA.

Damage to DNA caused by exposure of L929 fibroblasts to ozone was reflected by the generation of strand breaks, DNA inter-strand cross-links and DNA-protein cross-links. Addition of propan-2-ol, a hydroxyl radical scavenger, did not affect the formation of strand breaks. In model experiments it appeared that both purines and pyrimidines were involved in DNA inter-strand and DNA-protein cross-links.     

Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK_cs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G₂/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G₂/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G₂/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.     

Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide.

Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores. Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations. The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation. The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation. During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired. In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance. These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance. Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.     

The Role of the Human Psoralen 4 (hPso4) Protein Complex in Replication Stress and Homologous Recombination

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G₂/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.     

DNA damage by C1027 involves hydrogen atom abstraction and addition to nucleobases

C1027 is a potent antitumor agent that damages DNA. It has the unusual ability to produce double strand breaks and interstrand cross-links (ICLs) intracellularly, which enable it to initiate concurrent ataxia-telangiestasia mutated (ATM) and Rad-3 related (ATR) independent damage responses. The latter form of damage is not well characterized. We have examined the effect of DNA sequence on C1027 reactivity and found it to be more diverse than previously thought. In addition, analysis of the chemical stability of ICLs suggests that they result from reaction with the deoxyribose ring on one strand but direct addition to a nucleobase on the opposite strand.     

A possible role of Ku in mediating sequential repair of closely opposed lesions

One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions. We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate. It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU. However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme. The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku. Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.     

Aphidicolin-induced topological and recombinational events in simian virus 40.

Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial stage, single strand DNA breaks caused loss of superhelicity in the 40S replication intermediates. This DNA breakage was accompanied by the formation of strong, permanent protein-DNA crosslinks which reached a maximum as nicking of the aberrant DNA replication intermediates was completed. These protein-associated DNA strand breaks were not repaired. In the slower second stage of breakdown, the aberrant DNA replication intermediates remained nicked and strongly associated with protein as they underwent DNA replication fork breakage and recombinational changes to produce high molecular weight forms.     

Protein-associated DNA breaks in cells treated with adriamycin or ellipticine

The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alkaline elution. Adriamycin and ellipticine were found to produce large numbers of single-strand breaks. These breaks appeared to be intimately associated with protein to the extent that enzymatic deproteinization of the DNA was necessary to reveal the breaks. The frequency of breaks and cross-links increased with concentration and time of exposure to the drugs. These data suggest that DNA single-strand scission may be a feature common to intercalators. The association with a cellular protein is previously undescribed and suggests possible mechanisms for the strand scission.     

Transient formation of DNA strand breaks during the induced differentiation of a human promyelocytic leukaemic cell line, HL-60.

During the induced differentiation of the human promyelocytic leukaemic cell line, HL-60, along the myelocytic lineage, DNA strand-breaks are formed. These breaks which are formed in the face of a proficient DNA repair mechanism, are only transiently maintained and subsequently become religated. The ligation of these breaks requires the activity of the nuclear adenosine diphosphoribosyl transferase (ADPRT). Inhibition of nuclear ADPRT, an enzyme totally dependent on the presence of DNA strand-breaks for its activity and required for efficient DNA repair in eukaryotic cells, blocks the religation of these breaks but not their formation. The inhibition of DNA strand ligation in the differentiating HL-60 cells results in loss of viability and cell death.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

Role of the Nfo (YqfS) and ExoA apurinic/apyrimidinic endonucleases in protecting Bacillus subtilis spores from DNA damage

The Bacillus subtilis enzymes ExoA and Nfo (originally termed YqfS) are endonucleases that can repair apurinic/apyrimidinic (AP) sites and strand breaks in DNA. We have analyzed how the lack of ExoA and Nfo affects the resistance of growing cells and dormant spores of B. subtilis to a variety of treatments, some of which generate AP sites and DNA strand breaks. The lack of ExoA and Nfo sensitized spores (termed alpha-beta-) lacking the majority of their DNA-protective alpha/beta-type small, acid-soluble spore proteins (SASP) to wet heat. However, the lack of these enzymes had no effect on the wet-heat resistance of spores that retained alpha/beta-type SASP. The lack of either ExoA or Nfo sensitized wild-type spores to dry heat, but loss of both proteins was necessary to sensitize alpha-beta- spores to dry heat. The lack of ExoA and Nfo also sensitized alpha-beta-, but not wild-type, spores to desiccation. In contrast, loss of ExoA and Nfo did not sensitize growing cells or wild-type or alpha-beta- spores to hydrogen peroxide or t-butylhydroperoxide. Loss of ExoA and Nfo also did not increase the spontaneous mutation frequency of growing cells. exoA expression took place not only in growing cells, but also in the forespore compartment of the sporulating cell. These results, together with those from previous work, suggest that ExoA and Nfo are additional factors that protect B. subtilis spores from DNA damage accumulated during spore dormancy.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

DNA damage and cytotoxicity of nitracrine in cultured HeLa cells

The effects of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine on DNA of cultured HeLa cells were studied. DNA strand breakage and interstrand cross-linking as well as DNA-protein cross-linking were measured by means of an alkaline elution technique and were compared with the cytotoxic effect of the drug. Interstrand cross-links were not detectable in the concentration range that inhibited cell growth up to 99%. DNA single-strand breaks were found when cells were treated with highly cytotoxic doses of the drug. DNA breakage was not reparable and exhibited a tendency to increase during incubation after drug removal. The only chromatin lesion induced by sublethal doses of nitracrine were DNA-protein cross-links which persisted for 24 h after drug treatment. It is concluded that DNA breaks represent degraded DNA from dying cells, whereas DNA-protein cross-links are specific cellular lesions, which may be responsible for the cell-killing effect of nitracrine.     

Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.