Relationship between the recovery from sublethal X-ray damage and the rejoining of chromosome breaks in normal human fibroblasts

Using plateau-phase cultures of AG1522 normal human fibroblasts, we examined relationships between the breakage and rejoining of chromosomes and the induction and repair of sublethal damage (SLD) following fractionated doses of X rays. The rate constant for the rejoining of breaks in prematurely condensed interphase chromosomes, measured previously, accurately predicts both the rate of change in survival due to potentially lethal damage (PLD) repair and the rate of change in survival for dose fractionation due to SLD repair. Further, changes in the frequency of chromosome-type deletions and asymmetrical exchange aberrations measured in the first postirradiation mitosis corresponded closely with changes in cell killing when doses were fractionated, and a dose-fractionation- or dose-rate-independent alpha component of damage was similar for aberration and cell killing end points. These results substantiate the hypothesis that sublethal damage repair results from the rejoining of breaks in interphase chromatin produced by a first dose so they no longer are capable of interacting with those produced by a second dose. The fact that the repair of potentially lethal damage is also readily explained on the basis of chromosome break rejoining (M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)) strongly suggests that PLD and SLD repair are different manifestations of the same basic process operating on the same basic lesions.     

A quantitative comparison of potentially lethal damage repair and the rejoining of interphase chromosome breaks in low passage normal human fibroblasts

After long postirradiation incubation periods, the residual frequency of prematurely condensed chromosome fragments following X-ray exposure of noncycling diploid human fibroblasts was found to be correlated with the frequency of chromosome aberrations observed under identical treatment conditions when the cells were subcultured and scored after they reached mitosis. Over a wide range of doses, the proportion of such cells without aberrations at their first metaphase was not significantly different from the proportion able to form macroscopic colonies. Further, the rate of rejoining of interphase chromosome breaks was the same as the rate of increase in survival due to the repair of potentially lethal damage (PLD). These results suggest that there is a one-to-one correspondence between the initial breakage and rejoining of G0 chromosomes and the induction and repair of PLD measured by delayed plating from plateau-phase cultures of these cells.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on cell killing and interphase chromosome breakage in radiation-sensitive cells

The effect of BrdU incorporation on cell radiosensitivity as well as on the induction of chromosome damage by radiation was studied in plateau-phase xrs-5 cells using the premature chromosome condensation (PCC) method. It is well known that xrs-5 cells are sensitive to ionizing radiation and defective in the repair of radiation-induced DNA double-strand breaks, chromosome damage, and potentially lethal damage (PLD). Compared to repair-proficient CHO 10B cells, a reduction was observed in the overall BrdU-mediated radiosensitization in plateau-phase xrs-5 cells for the same degree of thymidine replacement. This finding is interpreted with a model for BrdU-induced radiosensitization advanced previously, in which two distinct components act to produce the overall radiosensitization observed. One component involves processes associated with the increase in initial damage (DNA and chromosome) production per unit absorbed dose and causes an increase in the slope of the survival curve, while the second component involves enhanced fixation of radiation-induced damage (PLD) and causes a reduction in the width of the shoulder of the survival curve. It is suggested that in plateau-phase xrs-5 cells, the deficiency in the repair of radiation-induced damage compromises BrdU-mediated radiosensitization by leaving active only the radiosensitization component that is associated with an increase in damage induction. Enhancement of cell killing by BrdU in plateau-phase xrs-5 cells resulted in a decrease in D0, the relative value of which was similar to the relative increase in the production of chromosome damage as measured by the PCC method. The relative values for the change in D0 and the production of chromosome aberrations were similar in plateau-phase CHO 10B and xrs-5 cells, suggesting that the physicochemical and/or biochemical processes associated with this phenomenon are the same in the two cell lines. Radiosensitization of a magnitude similar to that observed in exponentially growing CHO 10B cells was induced by BrdU in exponentially growing xrs-5 cells. This effect is attributed to a partial expression of the repair gene (transiently during S phase in all cells, or throughout the cycle in a fraction of cells) that permits some repair of radiation-induced damage and which is compromised by BrdU.     

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

The relationship between molecular and cellular repair from potentially lethal damage induced by N-methyl-N'-nitro-N-nitrosoguanidine in Chinese hamster V79 cells

The relationship between molecular and cellular repair from potentially lethal damage (PLD) induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in exponentially growing V79 Chinese hamster cells. We compared the repair processes by an alkaline sucrose sedimentation analysis and a colony formation assay. MNNG-treated cells were exposed to the conditioned medium (CM) from density-inhibited plateau-phase V79 cell cultures, as a post-treatment for the induction of PLD repair. When MNNG-treated cells were postincubated in CM, cell survival continuously increased for 18 h, and during this period, DNA replication was substantially suppressed. CM did not inhibit the rejoining of the single-strand breaks of parental DNA. Rather, parental DNA fragments sedimented more rapidly when postincubated in CM than in fresh medium. These data indicate that cellular recovery from MNNG-induced PLD increases in proportion to the resealing of MNNG-induced single-strand breaks of DNA during the suppression of DNA replication, suggesting that excision repair is involved in the PLD repair process.     

Radioprotective action of WR-1065 on radiation-induced DNA strand breaks in cultured Chinese hamster ovary cells

We have examined the radioprotective effect of WR-1065 on cultured Chinese hamster ovary cells. The effects of the drug on the induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) were measured using alkaline (pH 12.1) and neutral (pH 7.0) elution, respectively. Molecular protection factors (PFs) calculated from these data allowed us to determine whether the degree of modification of strand breakage accurately predicted the PFs measured using the biological end point of cell survival. The drug did protect against the induction of both SSBs and DSBs, although to an extent that did not appear to fully account for the degree of radioprotection in terms of cell killing measured under identical conditions. It is therefore unlikely that radioprotection by WR-1065 occurs simply as a consequence of a general lowering of all types of gamma-ray-induced DNA lesions, and it is possible that the drug could differentially protect against the induction of subsets of these DNA lesions. The rate of SSB rejoining was retarded following preirradiation treatment of cells with WR-1065, but there was no effect on DSB rejoining. Postirradiation treatment with WR-1065 also appeared to retard SSB rejoining but without an accompanying effect on either DSB rejoining or cell survival; however, this effect was largely reversed by the addition of catalase and was therefore probably a result of H2O2 generated by autoxidation of the drug. Based on these observations, it would appear that the molecular actions of aminothiol radioprotective compounds that lead to reduced cell killing are much more complex than previously thought.     

Different oxygen enhancement ratios for induced and unrejoined DNA double-strand breaks in eukaryotic cells

DNA double-strand breaks (DSBs) are 2.9 times more frequently induced in yeast cells exposed to sparsely ionizing 30-MeV electrons under oxic compared to anoxic conditions. The rejoining of DSBs induced under anoxic conditions was investigated under conditions allowing repair of potentially lethal damage and compared to the rejoining of DSBs induced in oxic cells. In contrast to the biphasic rejoining kinetics of DSBs induced in oxic cells, the rejoining kinetics of DSBs induced in anoxic cells is complicated by the formation of secondary DSBs. These arise during postirradiation incubation of cells, presumably as a consequence of repair processes acting on radiation-induced lesions other than DSBs. These secondary DSBs may at least partially explain the finding that a greater fraction of unrejoinable DSBs is present in cells irradiated under anoxic compared to oxic conditions. As a consequence, the oxygen enhancement ratio of the yield of the remaining DSBs is decreasing in the course of DSB rejoining.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polβDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polβDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polβDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H₂O₂ treatment, polβDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H₂O₂, these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polβDN induced increase in residual γH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual γH2AX foci, indicating an escape from γH2AX-mediated DSB repair. In addition, we show that in the polβDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.     

Rapid rejoining of X-ray-induced DNA single-strand breaks in tuberous sclerosis fibroblasts

The formation and rejoining rates of X-ray-induced DNA single-strand breaks (SSBs) were examined in radiosensitive and non-radiosensitive fibroblast lines from patients with tuberous sclerosis (TS), and fibroblasts from normal individuals, using the sensitive and quantitative alkaline elution method. No difference was found between these cell lines in the frequency of DNA SSBs directly produced by X-irradiation at any dose up to 750 rad. Kinetic analysis of the rate of rejoining of DNA SSBs after X-irradiation at 500 rad indicated that the rate of rejoining involved at least two components, an initial fast component and a slower component. TS fibroblast lines, either radiosensitive or nonsensitive, were proficient as to DNA SSB repair, but they showed an increased rate of rejoining in the initial fast repair process, when compared to normal fibroblast lines. Although the molecular basis for the accelerated rejoining of DNA SSBs remains unknown, it is possible that the abnormality may be related to a basic defect in TS.     

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.     

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

The effect of 125I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G1-phase CHO-cells. For this purpose a population of mitotic cells was allowed to divide and progress through S-phase in the presence of 125IdUrd. Cells were subsequently transferred to conditioned medium (C-med) obtained from plateau-phase cultures that allowed cells to divide and accumulate in G1-phase in a non-cycling state. To accumulate 125I-induced damage, cells were kept frozen at -80 degrees C. Freezing was carried out using a new method that optimally preserves cell integrity. After various times of cold storage, cells were thawed and assayed for survival, DNA and chromosome damage, either immediately or after various times in C-med. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragment was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation.     

A comparison of chromosome repair kinetics in G(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts for increased potentially lethal damage repair

Potentially lethal damage (PLD) and its repair were studied in confluent human fibroblasts by analyzing the kinetics of chromosome break rejoining and misrejoining in irradiated cells that were either held in noncycling G(0) phase or allowed to enter G(1) phase of the cell cycle immediately after 6 Gy irradiation. Virally mediated premature chromosome condensation (PCC) methods were combined with fluorescence in situ hybridization (FISH) to study chromosomal aberrations in interphase. Flow cytometry revealed that the vast majority of cells had not yet entered S phase 15 h after release from G(0). By this time some 95% of initially produced prematurely condensed chromosome breaks had rejoined, indicating that most repair processes occurred during G(1). The rejoining kinetics of prematurely condensed chromosome breaks was similar for each culture condition. However, under noncycling conditions misrepair peaked at 0.55 exchanges per cell, while under cycling conditions (G(1)) it peaked at 1.1 exchanges per cell. At 12 h postirradiation, complex-type exchanges were sevenfold more abundant for cycling cells (G(1)) than for noncycling cells (G(0)). Since most repair in G(0)/G(1) occurs via the non-homologous end-joining (NHEJ) process, increased PLD repair may result from improved cell cycle-specific rejoining fidelity of the NHEJ pathway.