Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells

The effect of exposure to elevated temperatures (41-45 degrees C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45 degrees C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45 degrees C for 15 min slowed the rate of DNA repair to 0.17 of the control repair rate. The To for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45 degrees C, respectively. Stepdown-heating (45 degrees C for 15 min followed by repair at 41 degrees C) resulted in greater inhibition of DNA repair (0.11 of the control rate) than was observed after acute heating alone. Repair at 41 degrees C was observed to proceed in unheated cells at a faster rate than at 37 degrees C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45 degrees C indicated an activation energy of 140 kcal mol-1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 degrees C and 45 degrees C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 degrees C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells (45 degrees C for 15 min) were incubated at 37 degrees C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 degrees C (step-down heating; SDH) a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks.     

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.     

Nature of DNA lesions induced in human hepatoma cells, human colonic cells and human embryonic lung fibroblasts by the antiretroviral drug 3'-azido-3'-deoxythymidine

This study tried to clarify the question if nuclear genotoxicity played a role in 3'-azido-3'-deoxythymidine (AZT) toxicity. We investigated cytotoxic and DNA-damaging effects of AZT on human hepatoma HepG2 and human colonic CaCo-2 cells as well as on human diploid lung fibroblasts HEL. The amount of induced DNA damage was measured by standard alkaline single cell gel electrophoresis (SCGE). The nature of induced DNA lesions was evaluated (1) by modified SCGE, which includes treatment of lysed cells with DNA repair enzymes Endo III and Fpg and enables to recognize oxidized bases of DNA, and (2) by SCGE processed in parallel at pH 13.0 (standard technique) and pH 12.1, which enables to recognize alkali labile DNA lesions and direct DNA strand breaks. Cytotoxicity of AZT was evaluated by the trypan blue exclusion technique. Our findings showed that 3-h treatment of cells with AZT decreased the viability of all cell lines studied. SCGE performed in the presence of DNA repair enzymes proved that AZT induced oxidative lesions to DNA in all cell types. In hepatoma HepG2 cells and embryonic lung fibroblasts HEL the majority of AZT-induced DNA strand breaks were pH-independent, i.e. they were identified at both pH values (12.1 and 13.0). These DNA lesions represented direct DNA breaks. In colonic Caco-2 cells DNA lesions were converted to DNA strand breaks particularly under strong alkaline conditions (pH>13.0), which is characteristic for alkali-labile sites of DNA. DNA strand break rejoining was investigated by the standard comet assay technique during 48 h of post-AZT-treatment in HepG2 and Caco-2 cells. The kinetics of DNA rejoining, considered an indicator of DNA repair, revealed that AZT-induced DNA breaks were repaired in both cell types slowly, though HepG2 cells seemed to be more repair proficient with respect to AZT-induced DNA lesions.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Characterization of intracellular DNA strand breaks induced by neocarzinostatin in Escherichia coli cells.

DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.     

Specific DNA alkylation damage and its repair in carcinogen-treated rat liver and brain

The in vivo formation and repair of specific DNA lesions produced by alkylating agents of contrasting carcinogenic potencies were investigated. Male Sprague-Dawley rats were treated with direct-acting alkylating agents methylmethane sulfonate (MMS) or methylnitrosourea (MNU). The amounts of N-3-methyladenine (3-meA), N-7-methylguanine (7-meG), and methylphosphotriesters (mePTE) in the DNA of liver and brain were determined following selective removal of the methylated bases by enzyme 3-meA N-glycosylase from Micrococcus luteus and thermal depurination at neutral pH. Both enzyme- and heat-induced alkali-labile apurinic sites were converted to single-strand breaks on incubation with 0.1 M NaOH. The number of such sites was quantitated following centrifugation of the DNA in alkaline sucrose gradients, fluorescent detection of unlabeled DNA, and estimation of number-average molecular weight. The results show a carcinogen dose-dependent initial linear increase in the number of enzyme- and heat-induced DNA strand breakage in both liver and brain DNA. With a half-life of approximately 3 h, 3-meA was removed from the tissues, whereas 45 to 55% of 7-meG remained unrepaired at 48 h. The study of the alkylation damage induced by MNU treatment of rats showed that the kinetics of repair for 3-meA and 7-meG was similar to the MMS-treated tissues and that mePTE persisted over a 7-day period. The technique developed does not require the use of radiolabeled reagents of DNA and allows for the selective quantitation of DNA alkylation lesions like 3-meA and 7-meG in the presence of nitrosourea-induced phosphotriesters.     

Assessment of radiation-induced DNA strand breaks and their repair in unlabeled cells.

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.     

Effect of inhibitors of poly(ADP-ribose) polymerase on the heat response of HeLa S3 cells

The purpose of this study was to investigate a possible involvement of poly(ADP-ribosyl)ation reactions in hyperthermic cell killing and hyperthermic DNA strand-break induction and repair in HeLa S3 cells. The inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (3AB) and 4-aminobenzamide (4AB), were used as tools in this study. Both inhibitors could sensitize the cells for hyperthermic cell killing equally well, although 3AB is known to be a more effective enzyme inhibitor. The heat sensitization at the level of cell killing could be reversed when the compounds were still present during a 4-h postincubation at 37 degrees C. More heat-induced DNA strand breaks were formed in the presence of 3AB and 4AB. Repair of strand breaks was inhibited during the postincubation at 37 degrees C. Thus the effect of 3AB and 4AB on DNA strand-break repair was different from the cited effect on cell survival. It is concluded that the sensitizing effect of 3AB and 4AB on hyperthermic cell killing is not caused by inhibition of poly(ADP-ribose) polymerase and is also not related to repair of DNA strand breaks.     

DNA damage production in CHO cells at elevated temperatures

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.     

Effect of dose rate on cell killing and DNA strand break repair in CHO cells exposed to internal beta-rays from incorporated [3H]thymidine

Survival as well as repair of DNA strand breaks were studied in CHO cells after exposure to internal beta-rays from incorporated [3H]thymidine at 4 degrees C (equivalent to an exposure at 'infinitely high' dose rate) and at 37 degrees C (low dose rate). DNA strand breaks were determined by the alkaline unwinding technique. In cells exposed at 4 degrees C cell killing was five times higher (Do = 250 decays per cell) than in cells exposed at 37 degrees C (Do = 1280 decays per cell). Strand breaks induced by 3H decay at 37 degrees C were repaired with the same kinetics as those generated at 4 degrees C. Therefore the different degrees of cell killing at 4 degrees C and 37 degrees C cannot be attributed to a difference in the repair kinetics for DNA strand breaks.     

Eumelanin causes DNA strand breaks and kills cells

Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitation of chemically induced DNA strand breaks in human cells via an alkaline unwinding assay

DNA strand breaks induced in human CCRF-CEM cells by electrophilic chemicals (carcinogens/mutagens) can be readily quantitated via a facile alkaline unwinding assay. This procedure estimates the number of chemically induced DNA strand breaks on the basis of the percentage DNA converted from double-stranded to single-stranded form during an exposure to the alkaline unwinding conditions. The assay is based on the assumption that each strand break serves as a strand unwinding point during the alkaline denaturation. The extent of strand separation can be standardized with respect to the initial level of induced strand breaks by the use of X-rays, which produce known levels of DNA strand breaks per rad in mammalian cells. Subsequent to the alkaline exposure, the single- and double-stranded DNA were separated by use of thermostated hydroxylapatite columns (60 degrees C), and the DNA was quantitated via a fluorescence assay (Hoechst 33258 compound). A correlation was shown between mammalian DNA strand-breaking potential (as measured in this procedure) and the propensity of these chemicals to revert Salmonella typhimurium TA100.     

Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.     

The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation

Strand breaks were detected in the DNA of Ehrlich ascites cells as well as in HeLa S3 cells directly after 1-5 hr at 43-45 degrees C by the use of the unwinding in high salt/hydroxylapatite method. The strand breaks found could not be attributed to the decay of incorporated tritiated thymidine. When the cells were incubated at 37 degrees C after the hyperthermic treatments, the amount of strand breaks formed remained at a constant level. Hyperthermia inhibited the repair of "radiation-induced" strand breaks. The repair curves obtained this way show a heat-dose-dependent decrease of the relative weight of the fast component of repair. Similar repair curves of "radiation-induced" strand breaks could be obtained by mixing heat inactivated and vital control cells prior to irradiation. In the latter case, however, the DNA repair was inhibited to a greater extent for identical levels of cell survival. The possible underlying molecular mechanisms are discussed.     

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) (cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), L-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37 degrees C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and L-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.     

Heat-induced DNA cleavage by esperamicin antitumor antibiotics

Esperamicin A1 effectively breaks DNA strands upon heating at 50 degrees C. The preferential DNA cutting sites of heat-activated esperamicin A1 are random and clearly differ from those of thiol- or UV-light-mediated DNA breakage with esperamicin A1. The absence of heat-induced DNA cleavage by esperamicin Z and the induction of the DNA breakage by esperamicin A1 disulfide indicate that (1) the enediyne core plays a significant role in this DNA strand scission and (2) the DNA cutting with the heat-activated esperamicin antibiotics does not necessarily require a trisulfide trigger in the aglycon portion. On the basis of the present results, a probable mechanism for the heat-induced DNA cleavage of esperamicin A1 has been proposed.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.