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The tumor suppressor gene p53 is thought to be a key factor in the onset of G1 cell-cycle arrest following DNA damage. However, here we describe cells derived from a patient with Bloom's syndrome, lacking any detectable p53 protein, that still shows a functional G1 cell-cycle checkpoint after irradiation with UV-C. Comparison with cells from other Bloom's patients showed that the absence of p53 protein is not a specific characteristic of Bloom's syndrome.

The demonstrations of frequent allelic deletions in lung and colon cancers have reemphasized the importance of clastogenesis in carcinogenesis. We have investigated the mechanisms of induction of chromosome aberrations in ultraviolet-irradiated diploid human fibroblasts. Cells were irradiated with UV at various times during a parasynchronous wave of cell proliferation and then harvested during the first mitosis that followed irradiation. Metaphase spreads were stained with Geimsa and the yields of chromosome aberrations were quantified. Ultraviolet irradiation induced primarily chromatid-type chromosome aberrations which included chromatid breaks and exchanges. Frequencies of aberrations displayed significant differences according to the phase of the cell cycle in which irradiation occurred and the time after irradiation when metaphases were harvested. Fibroblasts that were irradiated when in G0 and then immediately replated to stimulate cell division and cells that were at the S/G2 border when irradiated displayed the fewest numbers of aberrations. For G0-irradiated cells, the first entering mitosis carried a higher frequency of aberrations than those collected 2-4 h later. In contrast, for S/G2-irradiated cells the first into mitosis displayed fewer aberrations than subsequent fractions. Cells that were irradiated when at the G1/S border displayed the greatest numbers of aberrations with the frequencies of chromatic exchanges being significantly increased over all other times of irradiation. These studies confirm that UV is an S-phase-dependent clastogen and point to the G1/S border as a time of maximal sensitivity to clastogenesis. Irradiation of G1 cells was shown to produce a fluence-dependent reduction in the rate of entry of cells into the S-phase. There appeared to be a point late in G1 beyond which cells were resistant to irradiation and experienced less delay in S phase entry. Ataxia telangiectasia fibroblasts failed to delay entry to S phase following UV-irradiation in G1 and displayed hypersensitivity to UV-induced chromosomal aberrations. The delay in entry of damaged cells into the S phase may have the beneficial effect of providing more time for repair of potentially clastogenic DNA damage before the onset of DNA replication.

The effects of ultraviolet light on cellular DNA replication were evaluated in an asynchronous Chinese hamster ovary cell population. BrdUrd incorporation was measured as a function of cell-cycle position, using an antibody against bromodeoxyuridine (BrdUrd) and dual parameter flow cytometric analysis. After exposure to UV light, there was an immediate reduction (approximately 50%) of BrdUrd incorporation in S phase cells, with most of the cells of the population being affected to a similar degree. At 5 h after UV, a population of cells with increased BrdUrd appeared as cells that were in G1 phase at the time of irradiation entered S phase with apparently increased rates of DNA synthesis. For 8 h after UV exposure, incorporation of BrdUrd by the original S phase cells remained constant, whereas a significant portion of original G1 cells possessed rates of BrdUrd incorporation surpassing even those of control cells. Maturation rates of DNA synthesized immediately before or after exposure to UV light, measured by alkaline elution, were similar. Therefore, DNA synthesis measured in the short pulse by anti-BrdUrd fluorescence after exposure to UV light was representative of genomic replication. Anti-BrdUrd measurements after DNA damage provide quantitative and qualitative information of cellular rates of DNA synthesis especially in instances where perturbation of cell-cycle progression is a dominant feature of the damage. In this study, striking differences of subsequent DNA synthesis rates between cells in G1 or S phase at the time of exposure were revealed.

The temporal relationship between DNA damage and DNA replication may be critical in determining whether the genetic changes necessary for cellular transformation occur after DNA damage. Recent characterization of the mechanisms responsible for alterations in cell-cycle progression after DNA damage in our laboratory have implicated the p53 (tumor suppressor) protein in the G1 arrest that occurs after certain types of DNA damage. In particular, we found that levels of p53 protein increased rapidly and transiently after nonlethal doses of gamma irradiation (XRT) in hematopoietic cells with wild-type, but not mutant, p53 genes. These changes in p53 protein levels were temporally linked to a transient G1 arrest in these cells. Hematopoietic cells with mutant or absent p53 genes did not exhibit this G1 arrest, through they continued to demonstrate a G2 arrest. We recently extended these observations of a tight correlation between the status of the endogenous p53 genes and this G1 arrest after XRT and this cell-cycle alteration after XRT was then established by transfecting cells lacking endogenous p53 genes with a wild-type gene and observing acquisition of the G1 arrest and by transfecting cells processing endogenous wild-type p53 genes with a mutant p53 gene and observing loss of the G1 arrest after XRT. These observations and their significance for our understanding of the mechanisms of DNA damage-induced cellular transformation are discussed.

We have assessed the role of the p53 tumor suppressor gene in cell cycle arrest and cytotoxicity of ionizing radiation in 17 Burkitt's lymphoma and lymphoblastoid cell lines. Cell cycle arrest was assessed by flow cytometry of cells 16 h following irradiation. In addition to the usual G2 arrest, the cell lines exhibited three types of responses in G1: Class I, strong arrest in G1 following radiation; Class II, minimal arrest; and Class III, an intermediate response. All Class I cells contained normal p53 genes. Of the ten lines that showed minimal G1 arrest, eight had mutant p53 alleles, and two lines were heterozygous for p53 mutations. Both of the lines showing an intermediate response contained wild-type p53. Our results are consistent with the view that mutations abrogate the ability of p53 to induce G1 arrest following radiation. Studies with the heterozygotes showed that the mutant protein can have a dominant negative influence upon wild-type p53, and the reduced ability of two normal p53 lines to arrest in G1 indicated that p53 function can be impaired by other mechanisms. The radiosensitivity of most of the lines appeared to depend on the ability of p53 to induce a G1 arrest. The mean radiation dose that inhibited proliferation of the Class I lines by 50% was 0.98 Gy. Of the eight p53 mutant cell lines tested, five lines required approximately 2.9 Gy to cause a 50% inhibition of cell proliferation. The two heterozygotes were also more resistant to radiation than the Class I cells (50% inhibitory dose, 2.1 and 2.9 Gy). Our results suggest that radioresistance is afforded by a loss of function of wild-type p53, which would normally induce a G1 arrest and promote cell death in the presence of DNA damage.

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

We show here that the arrests of cells in G2 phase of the cell cycle induced by either staurosporine or ionizing radiation are closely related phenomena governed by a common kinase signaling pathway. The protein kinase inhibitor staurosporine induces a complete G2-phase arrest in exponentially growing TK6 human lymphoblastoid and V79 Chinese hamster fibroblast cells. Both cell types are equally sensitive to the kinase inhibitor and the arrest is dependent on its continued presence. Caffeine completely abrogates this arrest at concentrations comparable to those which abrogate radiation-induced G2-phase arrest. The kinetics of caffeine-induced release of both kinds of arrest are essentially identical. The activity of p34cdc2 kinase was also found to increase in a parallel fashion after caffeine-induced release of both kinds of arrest. As opposed to those transformed cell types which arrest only in G2 phase in response to staurosporine, immortalized C3H 10T1/2 fibroblasts and Muntjak skin fibroblasts display both G1- and G2-phase arrests. The results suggest that staurosporine and radiation interact with regulatory pathways in the cell cycle, and specifically with a caffeine-sensitive signal transduction pathway which recognizes DNA damage, regulates the G2/M-phase transition, and attenuates the biological consequences of radiation exposure.

The distribution of the DNA content of radiation-induced micronuclei was analysed in several cell lines (Chinese hamster, Syrian hamster and mouse NIH-3T3 cells) by flow cytometry. Frequency and DNA content of micronuclei were measured simultaneously using fluorescence and forward scatter signals of micronuclei and nuclei in suspension stained with ethidium bromide. Computerized random breakage of chromosomes and random combination of fragments was performed to compare the measured micronucleus distributions in synchronized cells irradiated during G1-phase with calculated distributions. The measured DNA distribution of radiation-induced micronuclei was found to be influenced by several factors: (1) the DNA distribution and the centromeric index of the chromosomes in the various cell lines; (2) the cell cycle phase at time of micronucleus measurement due to DNA synthesis in micronuclei; (3) the presence of chromosome fragments in micronuclei; and (4) the presence of whole chromosomes in micronuclei. These factors were shown to be responsible for the previously found large radiation-induced micronuclei which could not be explained by the classic assumption only that radiation-induced micronuclei are mainly produced by single acentric fragments.

Two methods involving labelling cells with bromodeoxyuridine (BrdUrd) have been used to study by flow cytometry the effect of hyperthermia (43 degrees C for up to 1 h) on Chinese hamster V79 cells. One method involved the use of an antibody to BrdUrd after pulse-labelling the cells either before or at time intervals after treatment. In the second method, the cells were incubated continuously in BrdUrd after heat treatment, and the components of the cell cycle were then visualized by staining with a combination of a bis-benzimidazole and ethidium bromide. All three methods showed that heating at 43 degrees C stopped DNA synthesis which, at 37 degrees C, subsequently recovered reaching the normal rate 8-12 h later. The cells in S phase at the time of treatment then progressed to G2 where they were further delayed. Cells heated in G1. after the recommencement of synthesis, progressed around the cycle, albeit slower than in unheated cells. The difference between the cells in G1 and S phases at the time of treatment may account for the greater sensitivity of S phase cells to hyperthermia.

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.

A study was made of the dose dependence of the chromosome aberration frequency in human lymphocytes exposed to 60Co-gamma radiation and neutrons (mean energy of 0.85 MeV) at the G0 stage and in different periods of the G1 and G1/S stages of the cycle. With gamma irradiation the dose dependence for cells at the G1 and G1/S stages was at a higher level than that for cells at the G0 stage, whereas the opposite picture was observed for cells exposed to neutron radiation. The difference was also noted in the time-response curves where gamma radiation increased and neutrons, on the contrary, decreased the aberration yield in the cells that passed from G0 to G1 stage. The experimental data obtained are attributed to activation of repair system at the G1 stage which is mainly conditioned by chromatin decondensation; the activating, that is, the functional factor influences the aberration induction with gamma irradiation, while the decondensation, that is, the structural factor, with neutron irradiation.

Cell cycle effects of very high LET particles on synchronous V79 Chinese Hamster cells have been studied in a track segment experiment by means of flow cytometric methods. Cells were irradiated with 10 MeV/u Pb-ions (LET = 13500 keV/micrometers) at an average fluence of 2 particles per cell nucleus, corresponding to a survival level of about 25%. Instantaneous drastic reductions of cell proliferation in all cycle phases have been observed, which affect the cell cycle for at least 50 hours after exposure to heavy ions. These findings are in clear contrast to the results from low LET radiation experiments, where significant delays can only be observed in S-phase and G2M-phase and for comparatively short time intervals of a few hours. Additionally, high LET radiation gives rise to prolonged DNA synthesis bypassing cell division, which leads to cells with DNA content greater than that of G2M-cells.

We have been studying variations of radiosensitivity and endogenous cellular factors during the course of progression through the human and hamster cell cycle. After exposure to low-LET radiations, the most radiosensitive cell stages are mitosis and the G1/S interface. The increased activity of a specific antioxidant enzyme such as superoxide dismutase in G1-phase, and the variations of endogenous thiols during cell division are thought to be intracellular factors of importance to the radiation survival response. These factors may contribute to modifying the age-dependent yield of lesions or more likely, to the efficiency of the repair processes. These molecular factors have been implicated in our cellular measurements of the larger values for the radiobiological oxygen effect late in the cycle compared to earlier cell ages. Low-LET radiation also delays progression through S phase which may allow more time for repair and hence contribute to radioresistance in late-S-phase. The cytoplasmic and intranuclear milieu of the cell appears to have less significant effects on lesions produced by high-LET radiation compared to those made by low-LET radiation. High-LET radiation fails to slow progression through S phase, and there is much less repair of lesions evident at all cell ages; however, high-LET particles cause a more profound block in G2 phase than that observed after low-LET radiation. Hazards posed by the interaction of damage from sequential doses of radiations of different qualities have been evaluated and are shown to lead to a cell-cycle-dependent enhancement of radiobiological effects. A summary comparison of various cell-cycle-dependent endpoints measured with low- or high-LET radiations is given and includes a discussion of the possible additional effects introduced by microgravity.

The biological effects of vibrational and linear acceleration on the alga Chlorella vulgaris were studied. Periodic vibration in the frequency range of 4-4000 Hz with vibrational acceleration up to 16 g did not affect the survival and mutability of Chlorella cells and did not modify the effects of acute gamma-radiation. However, random vibration similar to that occurring during launch of spaceships, combined with linear acceleration increased the radiation damage to algae produced by acute gamma-radiation at a dose of 10000 r. This effect is seen only in cells at the beginning of the G1 stage, which precedes DNA synthesis.