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Purpose: To assess the cell cycle-dependent influence of gemcitabine on ionizing radiation-induced DNA double-strand breakage (DSB) and rejoining measured by pulsed-field gel electrophoresis (PFGE)., Materials and Methods: WIDR cells (human colon carcinoma) were synchronized by serum starvation/stimulation providing populations with 7% (G1) or 50% S-phase cells, respectively. Following drug treatment (0.5 microg/ml for 2 hours) cells were irradiated (up to 90 Gy) or incubated for repair (up to 6 h after 40 Gy). Cell cycle changes were monitored by flow cytometry, DNA fragmentation was assessed by PFGE as fraction of electrophoretically mobile DNA., Results: Without drug treatment, irradiated S-phase cells exhibited lower PFGE signals than the G1 cells due to the well known electrophoretic immobility of replicative DNA fragments, but DSB rejoining was not different. Gemcitabine pretreatment increased the apparent initial radiation-induced DNA fragmentation specifically for S-phase cells. This effect was rapidly reversed (1 h) during incubation for repair., Conclusions: The data indicate that gemcitabine causes the formation of additional radiation-induced DSB in S-phase cells or destabilizes the replicative structures that otherwise prevent DNA fragment migration during PFGE. The latter would be rapidly restituted superimposing DSB rejoining. This is discussed in relation to the recently proposed role of mismatch repair in gemcitabine radiosensitization.

Purpose: The roles of DNA-dependent protein kinase (DNA-PK) and ATM in the cell-cycle-dependent radiosensitivity in human cells were investigated., Methods and Materials: A DNA-PK activity-deficient human glioblastoma cell line M059J, ataxia telangiectasia cell lines AT3BISV and AT5BIVA, and control cell lines were used. Wortmannin inhibited DNA-PK and ATM activities. Cells were synchronized by hydroxyurea. Progression through the cell cycle was analysed by flow cytometry., Results: M059J exhibited hyper-radiosensitivity throughout the cell cycle, with extreme hyper-radiosensitivity in G to early S-phase compared with the control cell line M059K. AT3BISV and AT5BIVA exhibited hyper-radiosensitivity throughout the cell cycle but showed a similar pattern of cell-cycle-dependent radiosensitivity to that observed in LM217 or HeLa cells. In AT3BISV and AT5BIVA, radiosensitization by wortmannin was observed throughout the cell cycle and was most prominent in G1 to early S-phase. Wortmannin did not sensitize M059J to ionizing radiation in any cell-cycle phase. DNA-PK activities were not different throughout the cell cycle., Conclusion: The results suggest that (1) non-homologous endjoining plays a dominant role in G1 to early S-phase and a minor role in late S to G2-phase in repairing DNA double-strand breaks, (2) the role of ATM in repairing double-strand breaks may be almost cell-cycle-independent and (3) the dominant role of non-homologous end-joining during G1 to early S-phase is not due to cell-cycle-dependent fluctuations in DNA-PK activity.

Purpose: Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertheless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control., Materials and Methods: Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously., Results: The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Bell, 4197 and EA14 are p53 wild-types and show a G1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so., Conclusions: The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G1 block or with p53 status.

Purpose: To investigate whether in Werner's syndrome cells the G2 phase of the cell cycle has some abnormal response to post-treatment with agents such as caffeine and hydroxyurea known to interfere with cellular response to DNA damage., Materials and Methods: Two Werner's syndrome lymphoblastoid cell lines (KO375 and DJG) and the normal cell line SNW646 were exposed to 50 cGy of X-rays or mitomycin-C and posttreated with caffeine or hydroxyurea in the G2 phase of the cell cycle., Results: Hydroxyurea post-treatment potentiated the X-ray-induced aberration levels both in the normal and Werner's syndrome (KO375 and DJG) cell lines; in contrast caffeine was only effective in the normal cell line. Similar results were observed when Werner's syndrome cells were treated in the G1 phase with the S-dependent agent mitomycin-C and post-treated with caffeine in G2, extending the observation that Werner's syndrome cells are unaffected by caffeine G2 post-treatment., Conclusions: These results show a lack of caffeine effect in Werner's syndrome cells, suggesting an involvement of the Werner's syndrome protein in the signal transduction pathway by which caffeine could override the DNA damage induced G2 checkpoint.

Purpose: To study the mechanisms underlying the loss of G2/M checkpoint control which leads to mitotic catastrophe in human tumour cells following exposure to ionizing radiation., Materials and Methods: Asynchronous HeLa S3 cells were irradiated with doses of 5, 10 and 20 Gy X-rays. Cell-cycle progression and cyclin B1 levels were measured using bivariate flow-cytometric techniques as a function of time after irradiation. As indicators of mitotic catastrophe, the appearance of spontaneous premature chromosome condensation (SPCC) and cells presenting nuclear fragmentation were analysed using microscopy. Cyclin B1-dependent kinase activity was determined in immunoprecipitates and analysed using gel electrophoresis., Results: After X-irradiation of HeLa cells, delays in late S and G2 phases of the cell cycle were followed by SPCC and nuclear fragmentation, both indicative of mitotic catastrophe. The kinetics of appearance of cells that had apparently undergone mitotic catastrophe (i.e. the fraction of cells exhibiting nuclear fragmentation) was independent of the dose-dependent radiation-induced division delay, while the extent of fragmentation (expressed as the number of nuclear fragments per fragmented cell) did increase with dose. Also observed was a 5-fold elevation of cyclin B1 levels in late S/G2 cells, which correlated temporally with the observed delays late in the cell cycle. Following the appearance of elevated cyclin levels, cyclin B1-associated histone H1 kinase activity showed similar increases; these increases in kinase activity occurred prior to increases in the fraction of cells exhibiting nuclear fragmentation., Conclusions: In human cells, cyclin B1 gene expression occurs in late S and G2 phases, and thus the increase observed in this protein may be due to the increased time spent by cells in these phases as a result of cell-cycle delays caused by the radiation exposure. It is possible that, under these conditions, over accumulation of cyclin B1 dilutes the mitosis-inhibitory action of the weel or other inhibitory pathways. Thus, this study presents a possible mechanism for G2/M checkpoint abrogation following ionizing radiation which may depend solely on effects associated with perturbed cell-cycle progression.

To consider the therapeutic potential of radiation effect modifiers it is necessary to balance the modification of the injury in tumours with that in different types of normal tissue. It is especially important to ensure that the effects that have been demonstrated in preclinical experiments are both qualitatively and quantitatively relevant for the radiation doses that will be used in clinical schedules. Most radiobiology studies are initially performed with large single doses or a few large fractions, and from those results predictions have sometimes been made of the potential clinical benefit from a radiation modifier. In the clinic they will be used with many repeated small fractions of about 2 Gy over a period of several weeks. The effects will be quantitatively different in these two dose ranges for a variety of reasons. No modifiers of radiation effect are truly dose-modifying over the whole dose spectrum. They all have a differential effect on the type of damage inflicted at high and low dose levels, i.e. those described by the linear and quadratic terms in the LQ model. This means that every modifier has a dose or dose per fraction dependence on the magnitude of the sensitization or protection. The details of that dose dependence will vary with the alpha/beta ratio of the tissue under consideration. Furthermore all tissues and tumours contain a mixture of cells, with different proliferative, redox and other characteristics that influence their sensitivity to radiation and their susceptibility to the radiomodifier. The influence of different subsets of cells changes as a fractionated treatment progresses and the sensitive cells are eradicated, leaving more resistant survivors. The overall response to a fractionated schedule then depends critically on whether there is re-assortment of cells from the resistant phase into more sensitive or modifiable phases before the next fraction in the series. In addition, the magnitude of dose modification depends totally on the standard curve against which the comparison is made. The reference standard is different in preclinical laboratory studies and in conventional clinical experience. Those differences must be considered when moving from the laboratory to the clinic and back again. The effect of these different factors is considered using the linear quadratic model to dissect the components. Examples are provided to demonstrate the clinical relevance.

Adenine prevents the down-regulation of DNA synthesis in gamma-irradiated EAT and HL60 cells. This protective effect is related to the initiation of DNA replication. The effectiveness of this protection increases with the concentration of adenine in both cell lines. The maximum protective effect of adenine induced in EAT cells occurs at about 10 times lower concentration as compared with HL60 cells; however, the level of this effect is almost the same in both cell lines. The results indicate that the prevention of the down-regulation of DNA synthesis in gamma-irradiated EAT and HL60 cells is conditioned mainly by the arrest of the cells in the early S-phase. It is evident that DNA synthesis in cells beyond G1/S boundary is insensitive to gamma-radiation.

UV-B-induced perturbations of cell cycle progression in asynchronous human keratinocytes were analysed during two cell cycles with respect to their cell cycle stage at the time of irradiation using BrdUrd/Hoechst flow cytometry. Exponentially growing SCL-2-keratinocytes exposed to UV-B radiation showed a short delay in G1-phase exit and were blocked in the S and G2/M phases of the first cell cycle. UV-A wavelengths did not show any detectable effect on cell cycle progression. In contrast, 137Cs-irradiation of these cells induced a temporary G2 block only. Micronucleus frequency increased in gamma-irradiated cells as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine starting immediately after 137Cs gamma-irradiation prevented accumulation of cells in G2 phase, but did not influence the frequency of micronuclei. In UV-B-irradiated keratinocytes, however, the damage-induced cell cycle perturbations were merely reduced by caffeine, but not eliminated. Compared with gamma-irradiation a moderate induction of micronuclei was observed in UV-B-irradiated cells. Caffeine, however, potentiated the induction of micronuclei by UV-B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV-B- and gamma-irradiation that may be repaired through different pathways.

The potentiation effects of arabinofuranosyl cytosine (ara-C), hydroxyurea, 3-aminobenzamide, and caffeine on the yield of chromosome aberrations were examined at the first-cleavage metaphase in mouse zygotes irradiated with X-rays in S and G2 phases (7.5 and 12 h after insemination respectively) in vitro. Potentiation effects were found in both S and G2 for all chemicals except ara-C. Caffeine completely released G2 arrest, which was not overcome by any other chemicals, and enhanced chromosome aberrations most effectively. Caffeine enhanced chromosome aberrations more in G2 irradiation than in S irradiation, and the frequency of chromatid-type aberrations was remarkably enhanced in G2 irradiation. The results in this study demonstrated an existence of two types of caffeine effects in the mouse zygotes irradiated in S and G2; a cancellation of G2 arrest and direct inhibition of DNA repair pathways. The chromosomal damage induced in G2 was effectively repaired by a repair pathway, which is affected by caffeine, on the other hand, the progression of cell cycle, not repair inhibition, was a major factor of caffeine effect on the yield of chromosome aberrations in S irradiation.

Recent evidence casts doubt on the hypotheses that fluoropyrimidine-mediated radiosensitization is related to cytotoxicity or to cell cycle redistribution into the G1/S boundary. We hypothesized that cells that are capable of progressing into S phase in the presence of fluorodeoxyuridine may also be more susceptible to radiation-induced damage. To test this hypothesis, fluorodeoxyuridine (FdUrd)-treated HT29 human colon cancer cells were separated by centrifugal elutriation into four fractions (1-4) containing a range of cells from those at the G1/S boundary (fraction 1) to those which had progressed approximately 11% into S phase (fraction 4). We found that fraction 4 cells showed significantly greater radiosensitization than fraction 1 cells. We also compared the effects of fluorodeoxyuridine on HT29 and SW620 human colon cancer cells. We found that, in contrast with HT29 cells, SW620 cells arrested at the G1/S boundary and were minimally radiosensitized. Finally, we found that an increase in sensitivity was correlated with a decrease in the rate of repair of DNA double-strand and single-strand breaks (assessed by asymmetric field inversion gel electrophoresis and alkaline elution respectively). These findings are consistent with the hypothesis that fluorodeoxyuridine-mediated radiosensitization depends on S phase progression and a decreased ability to repair radiation-induced DNA damage.

The proliferation response and changes in cellularity of mouse tongue epithelium were studied after single doses of X-rays and during 3 weeks of daily irradiation. A single dose of 13 Gy resulted in minimum cellularity (70% of control values) on days 3-5 and complete restoration on day 7. Mitotic activity ceased for 1 day followed by normal-to-supranormal values until day 15. A wave of abnormal mitoses was observed with a peak at days 4-7. Daily irradiation with 3 or 4 Gy induced neither major structural nor visible cellular damage. Cellularity decreased to approximately 60% during week 1 and subsequently remained at 60-70%. The proliferation activity was reduced to approximately 8% by day 2. Mitotic activity during weeks 2 and 3 was subnormal-to-normal, with a dose-dependent increase to normal counts during the first weekend and a distinct overshoot over the second weekend respectively. A proliferation model is presented to explain the present findings and previous functional measurements of changes in tissue tolerance. Its major features are accelerated symmetrical stem cell divisions and abortive divisions of sterilized cells.

The comparative induction of chromatid aberrations by 238Pu alpha-particles, or by 250 kVp X-rays was investigated in V79 Chinese hamster cells. Metaphases were sampled at hourly intervals postirradiation up to 8 h and BrdU/FPG staining methods were used to distinguish G2, S and G1 phase cells. Two experiments were performed. In the first, an alpha-particle dose of 0.41 Gy was compared with an X-ray dose of 1.5 Gy used in a previously published study. In the second, an X-ray dose of 1.2 Gy was used in parallel with 0.41 Gy of alpha-particles to produce a similar overall frequency of interchanges, and allow comparative ratios to be derived for other aberration types. At these isoexchange doses, alpha-particles produce relatively less gaps and breaks, particularly in late G2, and significantly more isochromatid deletions. A very high proportion of the isochromatid deletions were incomplete after alpha-particles compared with X-rays, but no difference in incompleteness was found for interchanges. With X-rays, about 6% of interchanges are complex intra-interchange forms. At similar exchange frequencies this increases to 26.7% for alpha-irradiation, suggesting increased multiple lesion interaction. Differences in dose distribution between alpha-particles and X-rays are discussed and mitotic delay is examined after separation of the analysed cells into damaged and undamaged classes.

Chinese hamster ovary cells were labelled with 125I-iododeoxyuridine (1.15 x 10(3) Bq/ml) for 12 h, then synchronized by mitotic selection, plated for cell cycle traverse, and harvested during successive stages of the cell cycle for freezing and accumulation of 125I decays. Cell viability was evaluated by the colony-forming assay. Cells subjected to 125I decays during the G1 phase exhibited exponential survival curves with an N = 1 and a D0 = 38-41 decays/cell. A continuous increase in 125I resistance was observed as cells progressed through the S phase and cells in late-S/G2 yielded shouldered survival curves with a N = 2 and a D0 = 78-84 decays/cell. After mitosis, the radiation resistance of cells returned to G1 values. These findings suggest that the primary target for radiation-induced cell death is duplicated during S phase, with G1 cells containing one target and G2 cells two targets. Dual targets, although located within a single cell, act as independent entities as if already distributed between two separate daughter cells. Therefore, the colony-forming assay provides survival values representative of single cells/single targets only for cells irradiated during the G1 phase of the cell cycle. For cells irradiated in S or G2 phases, when intracellular target multiplicity > 1, the colony-forming assay systematically gives higher values of cell survival by up to 100% due to the target multiplicity. Experiments with external X-rays confirm these conclusions.

DNA synthetic activity and DNA content of individual cells can be determined simultaneously by means of two-parameter flow cytometry. We used this method to study the effects of irradiation and/or hyperthermia on the proliferation of human melanoma cells in vitro. In untreated cultures, most of the cells with an S-phase DNA content showed incorporation of BrdU, but a small percentage did not. The fraction of these quiescent S-phase cells increased after irradiation (up to 8 Gy X-rays) and/or hyperthermia (up to 6 h at 42 degrees C or up to 2 h at 43 degrees C). Four days after the treatment up to 50% of the S-phase cells did not incorporate BrdU. There was a clear dose dependence for irradiation and hyperthermia alone or in combination. Generally, the combined effects seemed to be additive. Possible pitfalls of the technique used were taken into consideration. Our main practical conclusion is that single-parameter measurements of DNA content are insufficient to characterize the proliferative status of cell populations, especially after irradiation and/or hyperthermia, because a large part of those cells identified as being in S-phase may be quiescent.

Quiescent S-phase cells, i.e. cells with an S-phase DNA content that do not show BrdU incorporation, can be induced in a dose-dependent manner by irradiation and/or hyperthermia (Zölzer et al. 1992). As they begin to appear only 48-72 h after treatment, they do not seem to be related to early cell cycle disturbances, but rather to late events involved with cell death. We therefore determined colony forming ability under the same conditions, and tried to correlate the two parameters. Although, in general, higher frequencies of unlabelled S-phase cells were associated with lower survival, there were interesting differences. At the same level of survival, for instance, quiescent cells were induced more efficiently by hyperthermia than by irradiation. Additional experiments with split dose protocols showed that while cell killing was reduced by fractionation, the frequency of unlabelled S-phase cells increased. This further corroborates our conclusion that there is no simple relationship between the two parameters. Quiescence in S-phase is not just an expression of cell death irrespective of the treatment by which it may have been caused.