Responses of Huntington's disease and ataxia telangiectasia lymphoblastoid cells to bleomycin

Ionizing radiation sensitive, mutant human lymphoblastoid cell lines derived from patients with Huntington's disease (HD), or ataxia telangiectasia (AT) both showed cross sensitivity to bleomycin, as assayed by reduced cell viability and increased frequency of chromosome aberrations compared to normal controls. In contrast to AT cells which failed to show inhibition of DNA synthesis after exposure to ionizing radiation, or bleomycin treatment, the sensitive cells from HD patients had depressed rates of DNA synthesis after damage with these agents, similar to that seen in normal cells. In terms of progression through the cell cycle bleomycin damaged AT cells moved from G1 into S and from S to G2 + M at almost the same rate as untreated cells. Bleomycin treated HD cells showed a large proportion of cells blocked in G1, cells were slowed down in S, the rate of entry to G2 + M was reduced and only 5% of cycling cells reached G2. Progress through the cell cycle in normal cells exposed to bleomycin showed a partial block in G1 and the rate of entry to G2 + M was reduced. These differences in response of normal, AT and HD cells to ionizing radiation and bleomycin treatment indicates that the defect underlying the sensitivity is different in HD cells from that in AT cells.     

Cyclic x-ray responses in Mammalian cells in vitro

Various radiation responses in mammalian cells depend on the position of the cell within its generation cycle (that is, its age) at the time of irradiation. Studies have most often been made by irradiating synchronized populations of cells in vitro. Results in different cell lines are not easy to compare, but an attempt has been made here to point out similarities and differences with regard to cell killing and division delay. In general, survival data obtained so far show that, in cells with a short G(1), cells are most sensitive in mitosis and in G(2), less sensitive in G(1), and least sensitive during the latter part of the S period. In cells with a long G(1), in addition to the above, there is usually a resistant phase early in G(1) followed by a sensitive stage near its end. (The latter may be as sensitive as mitosis.) Exceptions to the above, especially in some L cell sublines, have been noted, and a possible explanation is given. In Chinese hamster cells, maximum survival after irradiation occurs during S, but it does not coincide with the time of the maximum rate of DNA synthesis or with the time of the maximum number of cells in DNA synthesis, and changes in survival also occur in cells inhibited from synthesizing DNA. Rather, survival depends on the position the cell has reached in the cycle at that time, which involves not only DNA synthesis but other processes as well. Survival is not completely correlated with DNA synthesis, since halting DNA synthesis just before or just after irradiation only slightly affects survival at its maximum. Division delay exhibits a pattern of response which is similar in most cell lines. Delay is considerable for cells irradiated in mitosis, is small for cells in G(1), increases to a maximum for cells during S, and declines for cells in G(2). L cells or human kidney cells may have a longer delay for cells irradiated in G(2) than for those irradiated in S. The results can be explained in terms of a two-component model of division delay. One component results from the prolongation of the S period due to the reduced rate of DNA synthesis, and the other, a block in G(2), is independent of DNA synthesis. The proportion of the two components may vary in different cell lines.     

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.     

Cell progression after selective irradiation of DNA during the cell cycle

Chinese hamster ovary cells were labeled with [125I]iododeoxyuridine (125IUdR, 0.1184 MBq/ml for 20 min) and the labeled mitotic cells were collected by selective detachment ("mitotic shake off"). The cells were pooled, plated into replicate flasks, and allowed to progress through the cell cycle. At several times after plating, corresponding to G1, S, late S, and G2 plus M, cells were cooled to stop cell cycle progression and to facilitate accumulation of 125I decays. Evaluation of cell progression into the subsequent mitosis indicated that accumulation of additional 125I decays during G1 or S phase was eight to nine times less effective in inducing progression delay than decays accumulated during G2. The results support our previous hypothesis that DNA damage per se is not responsible for radiation-induced progression delay. Instead, 125I-labeled DNA appears to act as a source of radiation that associates during the G2 phase of the cell cycle with another radiosensitive structure in the cell nucleus, and damage to the latter structure by overlap irradiation is responsible for progression delay (M. H. Schneiderman and K. G. Hofer, Radiat. Res. 84, 462-476 (1980].     

The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.     

Initial chromosome damage but not DNA damage is greater in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) exhibit increased sensitivity to ionizing radiation and certain drugs (e.g., bleomycin, neocarzinostatin, and etoposide) as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. To understand better the basis of this sensitivity, three AT and two normal lymphoblastoid cell lines were fractionated into cell cycle phase-enriched populations by centrifugal elutriation and then examined for their survival and their relative initial levels of DNA damage (neutral DNA filter elution) and chromosome damage (premature chromosome condensation). AT cells exhibited decreased levels of survival in all phases of the cell cycle; however, AT cells in early G1 phase were especially sensitive compared with normal cells in G1 phase. While AT and normal cells exhibited similar levels of initial DNA double-strand breaks in exponential populations as well as throughout the cell cycle, AT cells showed nearly twofold higher initial levels of chromosome damage than normal control cells in G1 and G2 phase. These results suggest that there is a higher rate of conversion of DNA double-strand breaks into chromosome breaks in AT cells, perhaps due to a difference in chromatin organization or stability. Thus one determining component of cellular radiosensitivity might include chromatin structure.     

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.     

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

Patients with an inherited syndrome characterized by immunodeficiency, microcephaly, and chromosomal instability: genetic relationship to ataxia telangiectasia.

Fibroblast cultures from six unrelated patients having a familial type of immunodeficiency combined with microcephaly, developmental delay, and chromosomal instability were studied with respect to their response to ionizing radiation. The cells from five of them resembled those from individuals with ataxia telangiectasia (AT) in that they were two to three times more radiosensitive on the basis of clonogenic cell survival. In addition, after exposure to either X-rays or bleomycin, they showed an inhibition of DNA replication that was less pronounced than that in normal cells and characteristic of AT fibroblasts. However, the patients are clinically very different from AT patients, not showing any signs of neurocutaneous symptoms. Genetic complementation studies in fused cells, with the radioresistant DNA synthesis used as a marker, showed that the patients' cells could complement representatives of all presently known AT complementation groups. Furthermore, they were shown to constitute a genetically heterogeneous group as well. It is concluded that these patients are similar to AT patients with respect to cytological parameters. The clinical differences between these patients and AT patients are a reflection of genetic heterogeneity. The data indicate that the patients suffer from a chromosome-instability syndrome that is distinct from AT.     

Degradation of ATM-Independent Decatenation Checkpoint Function in Human Cells is Secondary to Inactivation of p53 and Correlated with Chromosomal Destabilization

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Key Words:

Checkpoints, DNA damage, Decatenation, Topoisomerase II, ICRF-193, Radiation     

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me₂SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me₂SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me₂SO-resistant MELC variant (DR10), cultured with Me₂SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.     

The labelled mitosis curve for a population consisting of fast and slowly cycling cells

In studies of tumour growth, and particularly of tumour treatment with phase-specific chemotherapeutic agents, the fraction labelled mitosis technique is frequently used to estimate the kinetic properties of the cell population making up the tumour. We show here that the FLM technique is in principle very insensitive to the behaviour of slowly cycling cells, even if these constitute a large proportion of the total cell population. Furthermore, since the rate of DNA synthesis is frequently lower in slowly growing cells than in those growing rapidly, there is a higher probability of labelling error associated with the former cells. In view of these theoretical and experimental considerations, it is suggested that considerable caution be used when applying the FLM technique to heterogeneous cell populations such as those of solid tumours.     

Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radiosensitivity from radioresistant DNA synthesis.     

Effects of ionizing radiation on cell cycle progression

Irradiation of normal eukaryotic cells results in delayed progression through the G1, S, and G2 phases of the cell cycle. The G1 arrest is regulated by the p53 tumor suppressor gene product. Irradiation results in increased expression of p53, which in turn induces a 21 kDa protein, WAF 1/Cip 1, that inhibits cyclin CDK kinases. S-phase delay is observed after relatively high doses of radiation. This delay has both radiosensitive and radioresistant components, corresponding to inhibition of DNA replicon initiation and DNA chain elongation, respectively. The mechanism for this delay is as yet undefined, but the extent of the delay appears to be under genetic control and is sensitive to the kinase inhibitor staurosporine. A delay in G2 has been demonstrated in virtually all eukaryotic cells examined in response to irradiation. Our studies have focused on the mechanisms responsible for this delay. Cyclin B1 and p34^cdc2 are cell cycle control proteins that together form a kinase complex required for passage through G2 and mitosis [22]. Control of radiation-induced G2 delay is likely therefore to involve modulation of cyclin B1/p34^cdc2 activity. We have shown in HeLa cells that cyclin B1 expression is decreased in a dose-dependent manner following irradiation. This decrease is controlled at both the level of mRNA and protein accumulation. We have also shown that radiation-sensitive rat embryo fibroblast lines (REF) immortalized with v- or c-myc display a minimal G2 delay when compared to radiation resistant cells transformed with v-myc + H-ras. These REF lines respond to irradiation with a decrease in cyclin B mRNA, which parallels the extent of their respective G2 delays. The duration of the G2 delay in radiation-resistant REF can be shortened by treatment with low doses of the kinase inhibitor staurosporine. We have also been able to markedly reduce the radiation-induced G2 delay in HeLa cells using either staurosporine or caffeine. Attenuation of the G2 delay is accompanied by reversal of the radiation-induced inhibition of cyclin B mRNA accumulation. The results of these studies are consistent with the hypothesis that reduced expression of cyclin B in response to radiation is in part responsible for the G2 delay. The duration of the G2 delay may also be influenced by the activation state of the cyclin B/p34_cdc2 complex.Invited paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Cell cycle progression after cleavage failure: mammalian somatic cells do not possess a "tetraploidy checkpoint"

Failure of cells to cleave at the end of mitosis is dangerous to the organism because it immediately produces tetraploidy and centrosome amplification, which is thought to produce genetic imbalances. Using normal human and rat cells, we reexamined the basis for the attractive and increasingly accepted proposal that normal mammalian cells have a "tetraploidy checkpoint" that arrests binucleate cells in G1, thereby preventing their propagation. Using 10 microM cytochalasin to block cleavage, we confirm that most binucleate cells arrest in G1. However, when we use lower concentrations of cytochalasin, we find that binucleate cells undergo DNA synthesis and later proceed through mitosis in >80% of the cases for the hTERT-RPE1 human cell line, primary human fibroblasts, and the REF52 cell line. These observations provide a functional demonstration that the tetraploidy checkpoint does not exist in normal mammalian somatic cells.     

X-ray-induced G2 arrest in ataxia telangiectasia lymphoblastoid cells

Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiectasia (AT) lymphoblastoid cells and normal human cells. Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in AT cells than in normal cells as previously reported by other investigators. However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistinguishable from that in normal cells. Moreover, no significant difference in cell viability was noted between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar. These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to more stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there. The possible reasons for this greater accumulation are discussed.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects

The conserved protein kinase Chk1 is believed to play an important role in checkpoint responses to aberrant DNA structures; however, genetic analysis of Chk1 functions in metazoans is complicated by lethality of Chk1-deficient embryonic cells. We have used gene targeting to eliminate Chk1 function in somatic DT40 B-lymphoma cells. We find that Chk1-deficient DT40 cells are viable, but fail to arrest in G(2)/M in response to and are hypersensitive to killing by ionizing radiation. Chk1-deficient cells also fail to maintain viable replication forks or suppress futile origin firing when DNA polymerase is inhibited, leading to incomplete genome duplication and diminished cell survival after release from replication arrest. In contrast to embryonic cells, however, Chk1 is not required to delay mitosis when DNA synthesis is inhibited. Thus, Chk1 is dispensable for normal cell division in somatic DT40 cells but is essential for DNA damage-induced G(2)/M arrest and a subset of replication checkpoint responses. Furthermore, Chk1-dependent processes promote tumour cell survival after perturbations of DNA structure or metabolism.