Pathways of DNA double-strand break repair during the mammalian cell cycle

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.     

Cell cycle-dependent effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells

Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the Fanconi anemia pathway. Cells were irradiated with IR doses that killed approximately 90% of each asynchronous population, separated into synchronous fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells (xrcc3 mutation) were most resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires HRR. Cells defective in NHEJ (dna-pk(cs) mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes modestly to survival and in a manner that is independent of cell cycle position.     

Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in γ-irradiated CHO cells

The repair of DNA double-strand breaks (DSBs) by homologous recombinational repair (HRR) underlies the high radioresistance and low mutability observed in S-phase mammalian cells. To evaluate the contributions of HRR and non-homologous end-joining (NHEJ) to overall DSB repair capacity throughout the cell cycle after γ-irradiation, we compared HRR-deficient RAD51D-knockout 51D1 to CgRAD51D-complemented 51D1 (51D1.3) CHO cells for survival and chromosomal aberrations (CAs). Asynchronous cultures were irradiated with 150 or 300 cGy and separated by cell size using centrifugal elutriation. Cell survival of each synchronous fraction (～20 fractions total from early G1 to late G2/M) was measured by colony formation. 51D1.3 cells were most resistant in S, while 51D1 cells were most resistant in early G1 (with survival and chromosome-type CA levels similar to 51D1.3) and became progressively more sensitive throughout S and G2. Both cell lines experienced significantly reduced survival from late S into G2. Metaphases were collected from every third elutriation fraction at the first post-irradiation mitosis and scored for CAs. 51D1 cells irradiated in S and G2 had ～2-fold higher chromatid-type CAs and a remarkable ～25-fold higher level of complex chromatid-type exchanges compared to 51D1.3 cells. Complex exchanges in 51D1.3 cells were only observed in G2. These results show an essential role for HRR in preventing gross chromosomal rearrangements in proliferating cells and, with our previous report of reduced survival of G2-phase NHEJ-deficient prkdc CHO cells [Hinz et al., DNA Repair 4, 782–792, 2005], imply reduced activity/efficiency of both HRR and NHEJ as cells transition from S to G2.     

The relevance of the nuclear division cycle to radiosensitivity in yeast

Summary To investigate whether the nuclear division cycle could be related to cycle-specific changes in repair of ionizing radiation damage, we have determined the survival curves after -irradiation of samples taken frequently from synchronously dividing cultures of Saccharomyces cerevisiae cells. Survival was low in G1 and increased during S, reaching a maximum around the end of the S phase, which was maintained in G2. The shape of the survival curves for samples taken from later stages revealed a rapid cycle-specific drop in the radioresistance of individual cells. A simple model was formulated on the assumption that survival is greatly enhanced by the action of an enzymatic repair mechanism which requires duplicated but unsegregated DNA as a substrate. By taking into account the measurable age heterogeneity of samples taken from the synchronous cultures, this model was shown to fit the survival data closely. For an individual cell, the increasing survival during the S phase is thus attributable to a rising fraction of duplicated genome, whereas the rapid decrease in radioresistance at a later stage in the cell cycle may be interpreted as due to the final physical separation of sister chromatids. The start of the latter event was timed to the stage in mitosis when the nucleus begins to move towards the neck of the bud. The data are consistent with the hypothesis that the high radioresistance of cells in late S and G2 is due to the repair of double-stranded DNA breaks by a process involving recombination between sister chromatids.     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.     

The effect of inhibitors of ADP ribosylation on the restoration of the mitotic cycle and on DNA repair after the action of ionizing radiation in Chinese hamster cells

Specific inhibitors of poly(ADP-ribose)polymerase-3-aminobenzamide and 3-metoxybenzamide (6, 12 mM) have been shown to: 1) reduce survival of X-irradiated CHO K1 cells to a slight degree; 2) increase S- and particularly G2-delays in X-irradiated cells, while progressing through the cell cycle as analysed by the DNA flow cytofluorimetry; 3) reduce effectiveness of DNA single-strand breaks repair. The above data suggest a definite role of ADP ribosylation in the cell repair activity.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

Effects of mercuric chloride on synchronized Chinese hamster ovary cells: survival and DNA replication

Chinese hamster ovary (CHO) cells in vitro were treated with HgCl2 at various stages in the cell cycle and the effects of this chemical on cell survival, DNA replication, and cell division were observed. In terms of survival the early G1 cells were the most sensitive to treatment, followed by late G1 and early S, while mid S and late S-G2 treated cells were the least sensitive. Treatment with HgCl2 also resulted in reduced rates of DNA replication and delays in cell division. The early G1 treated cells showed substantially reduced rates of DNA replication followed by 4--5 h division delay. The early S and late S-G2 treated cells had some reduction in their rates of DNA replication followed by corresponding division delay of 2.5 h in the early S treated cells and 1 h in the late S-G2 treated cells.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.     

Human chromosome 5 complements the DNA double-strand break-repair deficiency and gamma-ray sensitivity of the XR-1 hamster variant.

XR-1 is a Chinese hamster ovary (CHO) cell mutant which is unusually sensitive to killing by gamma rays in the G1 portion of the cell cycle but has nearly normal resistance to gamma-ray damage in late S phase. The cell-cycle sensitivity correlates with the mutant's inability to repair DNA double-strand breaks (DSBs) produced by ionizing radiation and restriction enzymes. We have previously shown in somatic cell hybrids of XR-1 cells and human fibroblasts that the XR-1 mutation is a recessive mutation. In this study, using somatic cell hybrids formed between XR-1 and human fibroblasts, we map the human complementing gene to chromosome 5 by chromosome-segregation analysis. This gene biochemically restores the hamster defect to wild-type levels of gamma-ray and bleomycin resistance as well as restoring its proficiency to repair DNA DSBs, suggesting that a single gene is responsible for the XR-1 phenotype. We have tentatively assigned the name XRCC4 (X-ray-complementing Chinese hamster gene 4) to this human gene until its biochemical function in repair is discovered.     

Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair

Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G2. The sensitivity of Chinese hamster ovary cells to G2-arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G2. This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G2 arrest and/or by changes in capability for postirradiation recovery from G2-arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G2-arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G2 arrest, while inhibiting repair of G2-arrest-causing damage makes such an analysis possible. CHO cell monolayers were irradiated (1.5 Gy), then exposed to 5 mM caffeine for periods of 0-10 hr. Cell progression was monitored by the mitotic cell selection procedure. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G2 arrest was expressed. The duration of G2 arrest was independent of the length of the prior caffeine exposure and, since cells of all ages were ultimately examined, the duration of arrest was also independent of cell cycle age at the time of irradiation. This finding indicates that the target for G2-arrest induction is present throughout the cell cycle and that the level of G2-arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G2 arrest.     

Ataxia Telangiectasia Mutated and Rad3 Related (ATR) Protein Kinase Inhibition Is Synthetically Lethal in XRCC1 Deficient Ovarian Cancer Cells

Introduction

Ataxia telangiectasia mutated and Rad3 Related (ATR) protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response.

Methods

In the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed.

Results

ATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells.

Conclusions

Our data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.     

Oxygen enhancement ratio as a function of dose and cell cycle phase for radiation-resistant and sensitive CHO cells

There is still controversy over whether the oxygen enhancement ratio (OER) varies as a function of dose and cell cycle phase. In the present study, the OER has been measured as a function of survival level and cell cycle phase using volume flow cell sorting. This method allows both the separation of cells in different stages of the cycle from an asynchronously growing population, and the precise plating of cells for accurate measurements at high survival levels. We have developed a cell suspension gassing and sampling system which maintained an oxygen tension less than 20 ppm throughout a series of sequential radiation doses. For both radiation-resistant cells (CHO-K1) and a radiation-sensitive clone (CHO-xrs6), we could separate relatively pure populations of G1-phase, G1/S-boundary, S-, and G2-phase cells. Each cell line showed a typical age response, with cells at the G1/S-phase boundary being 4 (CHO-K1) to 12 (CHO-xrs6) times more sensitive than cells in the late S phase. For both cell lines, G1-phase cells had an OER of 2.3-2.4, compared to an OER of 2.8-2.9 for S-phase and 2.6-2.7 for G2-phase cells. None of these age fractions showed a dependence of OER on survival level. Asynchronously growing cells or cells at the G1/S-phase boundary had an OER similar to that of G1-phase cells at high survival levels, but the OER increased with decreasing survival level to a value near that of S-phase cells. These results suggest that the decrease in OER at high survival levels for asynchronous cells may be due to differences in the OERs of the inherent cell age subpopulations. For cells in one cell cycle stage, oxygen appears to have a purely dose-modifying effect.     

Tyrosyltubulin ligase and colchicine binding activity in synchronized Chinese hamster cells

Tyrosyltubulin ligase (TTL) was found to be present in CHO and V79 Chinese hamster cells grown in tissue culture. The enzyme is soluble and requires potassium, magnesium, and ATP for maximum activity and requires tubulin as a substrate. TTL was analyzed through the cell cycle of V79 and CHO Chinese hamster cells. The enzyme showed two peaks of activity in V79 cells at 4 h and 7 h after mitotic selection, corresponding to the early S and mid to late S phases of the cell cycle. In CHO cells the enzyme displayed a major peak of activity at mid S and a minor peak or plateau during early S. Tubulin, as measured by (3H)colchicine binding, was shown to increase through S phase and reach a maximum late in the cycle during G2 approx. 3 h after maximum TTL activity.     

E2F1 is involved in DNA single-strand break repair through cell-cycle-dependent upregulation of XRCC1 expression

The X-ray repair cross complementing group 1 (XRCC1) protein is involved in DNA base excision repair and its expression varies during the cell cycle. Although studies have demonstrated that rapid XRCC1-dependent single-strand break repair (SSBR) takes place specifically during S/G(2) phases, it remains unclear how it is regulated during the cell cycle. We found that XRCC1 is a direct regulatory target of E2F1 and further investigated the role of XRCC1 in DNA repair during the cell cycle. Saos2 primary osteosarcoma cells stably transfected with inducible E2F1-wt or mutant E2F1-132E were treated with hydroxurea (HU) for 36h and were subsequently withdrawn HU for 2-24h to test whether cell-cycle-dependent DNA SSBR requires E2F1-mediated upregulation of XRCC1. We found that SSBR activity, as determined using a qPCR-base method, was correlated with E2F1 levels at different phases of the cell cycle. XRCC1-positive (AA8) and negative (EM9) CHO cells were used to demonstrate that the alterations in SSBR were mediated by XRCC1. The results indicate that E2F1-mediated regulation of XRCC1 is required for cell-cycle-dependent SSBR predominantly in G(1)/S phases. Our observations have provided new mechanistic insight for understanding the role of E2F1 in the maintenance of genomic stability and cell survival during the cell cycle. The regulation of XRCC1 by E2F1 during cell-cycle-dependent SSBR might be an important aspect for practical consideration for resolving the problem of drug resistance in tumor chemotherapies.     

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: 1) cell volume; 2) cell cycle position; and 3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings 1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and 2) demonstrate EC subpopulations in culture.     

Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [(3)H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension.     

Wortmannin增强细胞辐射敏感性的分子机制

 Ｗ ｏｒｔｍ ａｎｎｉｎ（ Ｗ Ｏ Ｒ Ｔ）是 Ｐ Ｉ３ Ｋ 家族激酶特异抑制剂,对 ｐ５３ 野生型及突变型细胞的辐射敏感性均有提高．为了阐明 Ｗ Ｏ Ｒ Ｔ 的辐射增敏机制,通过免疫沉淀及免疫印迹法分析了 Ｗ Ｏ Ｒ Ｔ对辐射引起的细胞 Ｇ２／ Ｍ 转换中关键分子 ｃｄｃ２ 酪氨酸脱磷酸化延迟的影响;通过流式细胞术分析了 Ｗ Ｏ Ｒ Ｔ 对辐射引起的细胞 Ｇ２ 期延迟、细胞凋亡的影响;经报告基因转染的方法分析了 Ｗ Ｏ Ｒ Ｔ对宿主细胞对辐射损伤报告基因修复的影响;发现 Ｗ Ｏ Ｒ Ｔ 可促进受照细胞 ｃｄｃ２ 酪氨酸脱磷酸化、减弱辐射引起的细胞 Ｇ２ 期延迟、增强细胞凋亡并抑制损伤 Ｄ Ｎ Ａ 修复．提示 Ｗ Ｏ Ｒ Ｔ 辐射增敏是通过干扰细胞 Ｇ２ 期检查点调控、抑制损伤 Ｄ Ｎ Ａ 修复和促进细胞凋亡等多种途径实现的．