Martin H. Schneiderman

Radiation-Induced Division Delay in Synchronized Chinese Hamster Ovary Cells in Monolayer Culture

Chinese hamster ovary cells in monolayer culture, synchronized by mechanically selecting mitotic cells in the absence of drugs, were x-irradiated with 150, 300, or 600 rads at frequent intervals du...

Radiation-induced cycle delay in synchronized Chinese hamster cells: comparison between DNA synthesis and division

Chinese hamster ovary fibroblasts (MI > 90%) were synchronized by mitotic selection and x-irradiated at two-hour intervals during the cell cycle with 150, 300, or 600 rad. Cell progression was monitored by pulse labeling with [3 H] TdR at one-hour intervals, followed by autoradiography and liquid scintillation counting. Irradiated mitotic and G1 cells were delayed in initiating DNA synthesis (0.4-2.7 hr), but the rate of entry into and out of S, the duration of S, and the rate of DNA synthesis remained unchanged. Cells irradiated during S incorporated [3 H] TdR at a reduced rate and were delayed in entering G2. The fraction of cells capable of entering S was not reduced by any time-dose combination. The delay in entering and leaving the S phase which resulted from a dose of 150 or 600 rad was about 40% of that from 300 rad. The division delay, however, increased linearly with dose and exponentially with cell age. The ratio of division delay to S delay for G1 cells irradiated with 150 and 300 rad was appro...

The Target for Radiation-Induced Division Delay

In an effort to elucidate the subcellular target responsible for radiation-induced division delay, Chinese hamster ovary (CHO) cells growing in monolayer cultures were pulse-labeled with /sup 125/IUdR and the cell kinetics monitored by counting the mitotic cells selected every 10 min. Our results showed that /sup 125/I had to be incorporated into DNA to cause a perturbation of cell progression; unlabeled G/sub 2/ cells were unperturbed. To evaluate the mechanism of /sup 125/I-induced division delay, /sup 125/IUdR-labeled cells were permitted to accumulate /sup 125/I decays either during the S phase or during both S and G/sub 2/ phases. The results indicated that cells which accumulated /sup 125/I decays only during the S phase did not experience enhanced delay. In contrast, the yield of mitotic cells was reduced in cells which accumulated /sup 125/I decays during S plus G/sub 2/. Analysis of the data suggests that the target for radiation-induced mitotic delay is not the DNA, but a cell structure which comes in contact with the DNA during G/sub 2/ or early M phase.

The paradoxical nature of DNA damage and cell death induced by 125I decay

Chinese hamster ovary cells were synchronized at the G1/S-phase boundary of the cell cycle and pulse-labeled for 10 min with 125I-iododeoxyuridine 30 min after entering the S phase. Cell samples were harvested for freezing and 125I-decay accumulation at intervals ranging from 15 to 480 min after termination of labeling. The survival data showed a marked shift from cell killing characteristic of low-LET radiation to that more characteristic of killing by high-LET radiation with increasing intervals between DNA pulse-labeling and decay accumulation. Cells harvested and frozen within 1 h after pulse-labeling yielded a low-LET radiation survival response with a pronounced shoulder and a large D0 of up to 0.9 Gy. With longer chase periods the shoulder and the D0 decreased progressively, and cells harvested 5 h after pulse-labeling or later exhibited a high-LET survival response (D0: 0.13 Gy). Two interpretations for these findings are discussed. (1) If DNA is the sole target for radiation death, the results indicate that DNA maturation increases radiation damage to DNA or reduces damage repair. (2) If radiation cell death involves damage to higher-order structures in the cell nucleus, the findings suggest that newly replicated DNA is not attached to these structures during the initial low-LET period, but 125I starts to induce high-LET radiation effects as labeled DNA segments become associated with the target structure(s). On balance, or data favor the latter interpretation.

Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 micrograms/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 micrograms/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 micrograms/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 micrograms/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.

Radiation-induced division delay in Chinese hamster ovary fibroblast and carcinoma cells: dose effect and ploidy.

The mitotic selection procedure for cell cycle analysis was utilized to investigate the G/sub 2/ transition point for and the duration of radiation-induced division delay in diploid and tetraploid Chinese hamster ovary (CHO) fibroblasts and in Chinese hamster ovarian carcinoma cells. The location of the radiation-induced division delay transition point was dose independent at high doses and located approximately 42 min before division. At lower doses only an estimate of the point of blockade was possible; but the G/sub 2/ transition point appeared to be earlier in the cell cycle. The duration of radiation-induced division delay was dose dependent. This response is consistent with a sensitive population of cells in late G/sub 2/ that define the location of the transition point and the length of division delay. There was no difference observed in the dose response for radiation-induced division delay between the pseudotetraploid cell line of CHO and the pseudodiploid parent strain. However, in the cell line derived from a spontaneous Chinese hamster ovarian carcinoma the division delay was 39 +- 4 min/Gy. Therefore, radiation-induced division delay is independent of chromosome ploidy, but can show intraspecies cell line specificity.

G2 Cells: Progression Delay and Survival

The progression of Chinese hamster ovary (CHO) G2 cells into mitosis and their survival was measured after X-ray doses up to 4.0 Gy. S-phase cells were prevented from reaching mitosis by labeling with 125IUdR for 10 min prior to irradiation of the exponentially growing monolayer of cells. Mitotic cells, located past the radiation-induced division delay transition point, did not suffer a delay and were selected separately prior to the recovery of the G2 cells. The results show that (1) up to 400 min after radiation only 55% of the G2 cells recovered after about 2.5 Gy; (2) the progression delay of the G2 cells that recovered was 52.5 min/Gy; and (3) the survival curve D0 for these cells, 2.45 Gy, indicated a radioresistant population.

Targets for Radiation-induced Cell Death: Target Replication During the Cell Cycle Evaluated in Cells Exposed to X-rays or 125I Decays

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.

125I-UDR induced division delay.

Mitotic selection for cell cycle analysis was used to investigate the effects of (3)H and (125)I, incorporated into DNA, on the cell cycle progression of Chinese hamster ovary (CHO) cells. The results indicate that S-phase cells were delayed and G(2) cells were not.

Association between the division delay target and DNA late in the cell cycle.

The precise cell cycle time of association between labeled DNA (the radiation source) and the non-DNA cell structure whose damage is responsible for radiation-induced division delay was measured. Mitotic cells were selected from a monolayer of Chinese hamster ovary cells for 80 min (nine shakes) to establish the rate of cell progression into mitosis. The cell monolayers were then exposed to 0.1295 MBq/ml 125IUdR for 10 min to label the cells in S phase. After pulse labeling, mitotic cell selection was continued for various times (between 0 and 120 min) before 125I decays were accumulated at 4 degrees C. After 2 h in the cold, the cells were rewarmed and the selection of mitotic cells was continued. (Cooling had a small, transient affect on subsequent cell progression.) As the time between labeling and cooling was increased, the fraction of cells selected in mitosis decreased, indicating that an increasing proportion of 125I-labeled cells had entered a sensitive phase of the cell cycle where 125I decays are particularly effective in producing radiation-induced division delay. It is hypothesized that during this sensitive period (from -25 to +90 min of the S/G2 boundary), the labeled DNA comes into sufficiently close contact with a non-DNA structure to facilitate damage to this structure by overlap irradiation from 125I decays in the DNA.