Detection of deoxyribonucleoside-triphosphate imbalance death-induced DNA double strand breakage in FM3A cells by orthogonal-field-alternation gel electrophoresis (OFAGE)

The mechanism of intracellular deoxyribonucleoside-triphosphate (dNTP) imbalance death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-fluorodeoxyuridine, deoxyadenosine, or 2-chlorodeoxyadenosine, dNTP pool imbalance resulted. The imbalance was followed by DNA double strand breaks and subsequent cell death. The DNA double strand breaks have been directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). Fragmented DNA band appeared to be approximately 100-200 kb in size.     

The dNTP imbalance death.

The mechanism of intracellular deoxyribonucleoside-triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A (F28-7) cells was studied. When the cells were treated with 5-fluorodeoxyuridine (FdUrd), deoxyadenosine, 2-chlorodeoxyadenosine, or alpha,alpha-bis(2-hydroxy-6-isopropyltropon-3-yl)-4-methoxytolu ene, an imbalance in the cellular dNTP pool was induced. The imbalance was followed by DNA double-strand breaks and subsequent cell death. Fragmented DNA appeared to be approximately 100-200 kbp in size. The base of 5'-termini in the DNA were adenine and thymine. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. We propose the new mechanism dNTP pool imbalance induced cell death named; dNTP Imbalance Death.     

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.     

Deoxyribonucleoside-triphosphate imbalance death: deoxyadenosine-induced dNTP imbalance and DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of deoxyadenosine (dAdo)-induced death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to dAdo at 3 mM, an imbalance of intracellular dNTP pool resulted: dATP concentration was elevated 100-fold and the dGTP concentration was reduced to less than 1% of the control values. The imbalance was followed by breakage of mature DNA. DNA double strand breaks were observed in the dAdo treated cells 12 hr after the administration. We assume that the double strand breaks play an important role in the process of the dAdo-mediated cell death, and that the intracellular dNTP imbalance is the trigger of these events.     

dNTP pool imbalance induced endonuclease: 5-fluorodeoxyuridine induced DNA double strand break in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleotide triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. When the cells were treated with 1 microM 5-fluorodeoxyuridine (FdUrd), the imbalance of the cellular dNTP pool was induced. The imbalance was followed by DNA double stranded breaks and subsequent cell death. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. SDS-polyacrylamide gel electrophoresis showed a major protein species of approximate 45 kDa. The endonuclease was revealed, using electrophoretic separation in SDS-polyacrylamide gels containing DNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzyme activity.     

The mechanism of dNTP-unbalanced cell death induced by 5-fluorouracil and its derivatives

The mechanism of 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (FdUR)-induced death of mouse mammary tumor FM3A cells was studied. When the cells were exposed to 5-FU or FdUR, an unbalance of intracellular dNTP pool resulted. The unbalance was followed by breakage of mature DNA. DNA double strand breaks were observed in the FdUR (1 microM) treated cells 16 hrs after the administration. We assume that the double strand breaks play an important role in the mechanism of the FdUR-mediated cell death. In addition, the activity that can induce DNA double strand breaks was detected in the lysate of FdUR treated FM3A cells. Since intracellular dNTP pool unbalance seems to be the trigger of these events, this phenomenon may be termed as dNTP-unbalanced cell death.     

细胞dNTP库的稳态维持与基因组稳定性

作为DNA合成的重要前体,细胞中4种脱氧核糖核苷三磷酸(dATP、dTTP、dGTP和dCTP)是DNA复制、重组和修复所必需的原材料,而DNA的正确合成及其完整性则是基因组稳定性的重要体现,因此dNTP库状态的稳定对维持基因组的稳定进而保证细胞的稳定至关重要.从dNTP库的质量上讲,一些异质dNTP如氧化的dNTP掺...     

The role of mitochondrial dNTP levels in cells with reduced TK2 activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

The Role of Mitochondrial dNTP Levels in Cells with Reduced TK2 Activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

The role of deoxyribonucleotide metabolism in 5-bromo-2′-deoxyuridine mutagenesis in mammalian cells

The effects of deoxyribonucleoside triphosphate (dNTP) pool imbalance on the induction of mutations and siste-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) in mammalian cells is reviewed. The INC BrdUrd mutagenesis protocol involves the incorporation of BrdUrd into DNA under conditions of specific dNTP pool imbalance, while the REP BrdUrd mutagenesis protocol involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of specific (but different) dNTP pool imbalance. Biochemical and genetic analyses of both the INC and REP mutagenesis protocols provided evidence that (1) INC mutagenesis resulted from errors of incorporation due to the mispairing of BrdUTP with a guanine residue in replicating DNA leading to GC to AT transitions and (2) REP mutagenesis resulted from errors of replication due to the mispairing of dGTP with a BrURA residue in replicating DNA leading to AT to GC transitions. Further analyses involving different cell lines has led to an hypothesis describing the role of mismatch repair in the induction of mutations and SCEs.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.     

Reactions of deoxyribonucleotide synthesis system to irradiation and their modification by radioprotectors

The paper covers the problem on reactions of deoxyribonucleotide (dNTP) synthesis system in blood-forming organs of animals induced by irradiation. The synthesis of dNTP is a rate-limiting stage for DNA synthesis. Cellular requirements for dNTP pools during DNA synthesis are related with ensuring of the accuracy of DNA copying during replication and repair. It has been shown that organism defence mechanisms against irradiation include the following stages: 1. The prompt SOS-activation of dNTP synthesis 30 min later after irradiation, playing the important role in protecting of cell's genetic apparatus from damage. 2. The inhibition of dNTP synthesis within 3-24 h after irradiation resulting to the imbalance of four dNTP and the decrease of their pools. As result of that, the abnormal repair is observed due to depurinations, errors of base incorporations and "misrepair". 3. The restore of dNTP synthesis occurred 2 days later after irradiation. The increase of dNTP pools promotes the increase of DNA synthesis rate as well as proliferative activity of cells. Confirming the fact that the alterations in dNTP pools play essential role in the production of DNA lesions became an important step in understanding of the multistage process leading to radioprotection. To get high and balanced pools of dNTP needed for the increase in the volume of repair of DNA lesions the radioprotectors with high efficiency relative to the survival test were used in experiments. They induced the elevated dNTP synthesis in bone marrow and spleen during the time when the irradiation alone caused the essential prolonged suppression of dNTP synthesis as well as DNA and protein synthesis in organs of nonprotected animals. It has been shown that substances with antioxidant and antiradical activity induced the dNTP synthesis, too. In vivo regulatory factors of dNTP synthesis have been studied to elucidate the mechanisms of getting of high and balanced dNTP pools by using of different substances.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.     

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-2′-deoxyuridine (BrdU) and 5-ethynyl-2′-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.     

Consequences of the depletion of cellular deoxynucleoside triphosphate pools on the excision-repair process in cultured human fibroblasts

DNA excision repair requires the insertion of bases into gaps in the DNA which arise during the removal of damaged sites from the chromatin. The number of bases required is dependent on the amount of damage and the patch size of repair in response to the particular type of damage. In cells in which the ability to synthesize deoxynucleoside triphosphates (dNTPs) has been compromised, repair cannot proceed to completion following doses of DNA-damaging agents which induce repair that requires greater than the steady-state level of dNTPs. Repair is thus not equally sensitive to depletion of dNTPs when measured in rapidly cycling cells with relatively high dNTP pools or in non-cycling cells with significantly smaller pools. Critical depletion of dNTPs results in the production of long-lived DNA strand breaks at repairing sites and reduction in the number of sites initiating repair. On the other hand, elevation of dNTP pools to 10–50-fold normal levels did not inhibit repair. This indicates that dNTP pool depletion but not general pool-imbalance affects DNA excision repair.