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In recent years, the use of gold-based nanoparticles in radiotherapy has been extensively studied, and the associated radiosensitization mechanism has been evaluated in a variety of in vitro studies. Given that mitotic catastrophe is widely involved in radiation-induced cell death, we evaluated the effect of gold nanoparticles on this key event. Most of the methods currently used to visualize and quantify morphological changes and multinucleation are manual. To circumvent this time-consuming step, we developed and optimized an image processing workflow (based on freely accessible software and plugins) for the automated quantification of mitotic catastrophes. We validated this approach in three cell lines by comparing the number of radiation-induced mitotic catastrophes detected using the automated and manual methods in the presence and absence of nanoparticles. With the Bland-Altman analysis, the automated and manual counting methods were found to be fully interchangeable. The ultimate goal of this work was to determine whether mitotic catastrophe was critically involved in radiationinduced cell death after prior exposure to gold nanoparticles. In the radioresistant U87 cell line, exposure to gold nanoparticles was associated with a shorter time course for the events related to mitotic catastrophe, which peaked at 96 h postirradiation. Mitotic catastrophe was dose-dependent in both the presence and absence of gold nanoparticles. These results demonstrate that cell exposure to gold nanoparticles led to an increase in mitotic catastrophe events, and confirm the marked radiosensitizing effect observed in clonogenic assays.

The synthetic peptide TAT-RasGAP 317-326 has been shown to potentiate the efficacy of anti-cancer drugs. In this study, we explored the action of TAT-RasGAP 317-326 when combined with radiation by investigating its radiosensitizing activity in vitro and in vivo. To investigate the modulation of intrinsic radiosensitivity induced by TAT-RasGAP 317-326 , clonogenic assays were performed using four human cancer cell lines, HCT116 p53 +/+ (ATCC: CCL-247), HCT116 p53 -/- , PANC-1 (ATCC: CRL-1469) and HeLa (ATCC: CCL-2), as well as one nontumor cell line, HaCaT (CLS: 300493). Next, to investigate tumor growth delay after irradiation, HCT116 cell lines were selected and xenografted onto nude mice that were then treated with TAT-RasGAP 317-326 alone or in combination with radiation or cisplatin. Afterwards, cell cycle and death modulation were investigated by quantification of micronuclei and apoptosis-related protein array. TAT-RasGAP 317-326 radiosensitized all four human carcinoma cell lines tested but displayed no effect on normal cells. It also displayed no effect when administered as monotherapy. This radiosensitizing effect was confirmed in vivo in both p53-positive and p53-negative HCT116 xenografts. TAT-RasGAP 317-326 combined with radiation enhanced the number of cells in S phase and subsequently delayed cell death, but had almost no effect on major apoptosis-related proteins. TAT-RasGAP 317-326 is a radiosensitizing agent that acts on carcinoma cells and its radiosensitizing effect might be mediated, at least in part, by the enhancement of mitotic cell death.

We examined the effect of epidermal growth factor (EGF) treatment in mice that received bone marrow transplantation (BMT) after 11 Gy whole-body irradiation. C57Bl/6 mice were divided into three treatment groups: 0 Gy; 11 Gy ((60)Co, single dose, 0.51 Gy/min) with BMT (5 × 10(6) bone marrow cells isolated from green fluorescent protein syngeneic mice, 3-4 h postirradiation); and 11 Gy with BMT and EGF (2 mg/kg applied subcutaneously 1, 3 and 5 days postirradiation). Survival data were collected. Bone marrow, peripheral blood count and cytokines, gastrointestine and liver parameters and migration of green fluorescent protein-positive cells were evaluated at 63 days postirradiation. Epidermal growth factor increased survival of irradiated animals that received BMT from 10.7 to 85.7% at 180 days postirradiation. In the BMT group, we found changes in differential bone marrow and blood count, plasma cytokine levels, gastrointestinal tissues and liver at 63 days postirradiation. These alterations were completely or in some parameters at least partially restored by epidermal growth factor. These findings indicate that epidermal growth factor, administered 1, 3 and 5 days postirradiation in combination with bone marrow transplantation, significantly improves long-term prognosis.

Epidemiological data reveals the gastrointestinal (GI) tract as one of the main sites for low-LET radiation-induced cancers. Importantly, the use of particle therapy is increasing, but cancer risk by high-LET particles is still poorly understood. This gap in our knowledge also remains a major limiting factor in planning long-term space missions. Therefore, assessing risks and identifying predisposing factors for carcinogenesis induced by particle radiation is crucial for both astronauts and cancer survivors. We have previously shown that exposure to relatively high doses of high-energy (56)Fe ions induced higher intestinal tumor frequency and grade in the small intestine of Apc(Min/+) mice than γ rays. However, due to the high number of spontaneous lesions (∼30) that develop in Apc(Min/+) animals, this Apc mutant model is not suitable to investigate effects of cumulative doses <1 Gy, which are relevant for risk assessment in astronauts and particle radiotherapy patients. However, Apc(1638N/+) mice develop a relatively small number of spontaneous lesions (∼3 per animal) in both small intestine and colon, and thus we propose a better model for studies on radiation-induced carcinogenesis. Here, we investigated model particle radiation increases tumor frequency and grade in the entire gastrointestinal tract (stomach and more distal intestine) after high- and low-radiation doses whether in the Apc(1638N/+). We have previously reported that an increase in small intestinal tumor multiplicity after exposure to γ rays was dependent on gender in Apc(1638N/+) mice, and here we investigated responses to particle radiation in the same model. Phenotypical and histopathological observations were accompanied by late changes in number and position of mitotic cells in intestinal crypts from animals exposed to different radiation types.

The cellular response to chemoradiotherapy was investigated in cells of the HCT116 (wild-type TP53) and HT29 (mutated TP53) human colorectal cancer cell lines to better understand how the chemotherapeutic agent 5-fluorouracil (5-FU) acts as a radiosensitizer in vitro and how it contributes to the well-documented greater efficacy of chemoradiotherapy compared to radiotherapy (or chemotherapy) alone. A bolus 5-FU treatment protocol that simulated actual clinical clearance kinetics was used with a radiation dose given within 90 min after drug addition. The involvements of key signaling pathways (DNA damage response, cell cycle progression, cell proliferation, cell death) in cell responses were investigated concurrently, allowing for direct correlations of numerous treatment response phenotypes. Early DNA damage response, substantial cell death, loss of clonogenicity, and senescence characterized both radiotherapy- and chemoradiotherapy-treated cultures but not chemotherapy-treated cultures. The largest G(2)/M arrests and strongest correlation of senescence with non-clonogenicity were seen in radiotherapy- and chemoradiotherapy-treated HCT116 cell cultures, suggesting that functional TP53 could play a role in maintaining/inducing these cellular phenotypes. Overall, chemoradiotherapy proved to be the most effective treatment modality since it resulted in the strongest growth inhibitions, largest G(2)/M arrests, largest fractions of senescent cells, and complete loss of clonogenicity in both cell lines.

Human gliomas are among the most aggressive tumors, and they respond poorly to treatment. The efficacy of surgical, radiation and chemotherapy treatment of these tumors is limited by the development of resistance. Interventions aimed at altering the response of these tumors to radiation or chemotherapy treatments are needed to improve survival rate and prognosis. Glioblastomas are generally p53 (TP53) functional tumors; however, DNA repair pathways are activated in these tumors instead of the pathways to apoptosis. Thus resistance to treatment is seen in the ability of these tumors to overcome cell death. We present data that demonstrate that U87MG glioblastoma cells transduced with a dominant-negative p53 adenovirus construct become sensitized to radiation-induced mitotic catastrophe through abrogation of G(2)/M checkpoint control and overaccumulation of cyclin B1. These findings suggest that interventions abrogating the G(2)/M checkpoint sensitize these cells to radiation-induced mitotic catastrophe and may represent a novel mechanism to increase the efficacy of radiation in wild-type p53 gliomas that are resistant to apoptosis.

Histone H2AX is phosphorylated and forms foci in response to exposure to ionizing radiation. It has been thought that phosphorylated histone H2AX foci reflect unrepaired DNA double-strand breaks; however, we report here the localization of phosphorylated histone H2AX foci at the site of rejoined DNA double-strand breaks. We observed that phosphorylated histone H2AX foci remained even 96 h after exposure to X rays in interphase cells. To clarify the localization of residual phosphorylated histone H2AX foci, we examined localization of focus formation on mitotic chromosomes irradiated with X rays. We found that phosphorylated histone H2AX foci were located not only on chromosomal fragments but also on intact metaphase chromosomes without fragments. In anaphase cells, chromosomal bridges, which resulted from illegitimate rejoining of DNA broken ends, had phosphorylated histone H2AX foci. These foci were detected as individual small spots 30 min after X irradiation, but foci detected 20 or 96 h after X irradiation were clustered along the chromosomal bridges. These results indicate that phosphorylated histone H2AX foci persist if DNA breaks are rejoined. It is suggested that "residual" foci indicate an aberrant chromatin structure by illegitimate rejoining but not a DNA double-strand break itself.

Cells respond to genotoxic insults such as ionizing radiation by halting in the G2 phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G2, and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G2 chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G2 chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G2 chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G2 assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G2 compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G2 checkpoint control in the prostate patients. There was no significant correlation of G2 radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G2 radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G2 aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3sigma increased the amount of cell death at 96 h, from 52% to approximately 70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3sigma expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3sigma has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05-1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3sigma resulted in an increase in apoptosis and a decrease in survival.

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.

Calculations based on plausible parameters taken from the existing experimental database, and new measurements on the cell cycle dependence of low-dose hyper-radiosensitivity (HRS) of non-tumorigenic HeLa x skin fibroblast human hybrid cells, provide the first experimental evidence that the selective killing of a transformation-sensitive G(2)/M-phase subpopulation as a consequence of low-dose HRS could account in part for the observed reduction of induced transformation frequencies at low doses to values below that observed spontaneously. However, it is clear that other mechanisms associated with classical adaptive response, such as induced DNA repair, are also likely to be involved.

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6-11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9-12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G(2)/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

It has been known for many years that caffeine reduces or eliminates the G2-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or gamma rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G2-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G2-phase delays after exposure to gamma rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G2-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G2-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the gamma-ray-induced G2-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca(++) from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G2-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G2-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G2-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G2-phase delay through a Ca(++)-independent mechanism, such as the inhibition of a cell cycle-regulating kinase.

Similar to what has been observed after irradiation, the fraction of G(2)-phase cells increases as a consequence of heat treatment. On the basis of cell cycle distributions alone, however, it is difficult to say whether the two results are related. In particular, comparison is complicated by the fact that the accompanying changes in the S-phase transition are different. These changes play a minor role after irradiation but constitute by far the most important cell cycle effect after heat treatment. Two-parameter flow cytometry was used here to study the proliferation of human melanoma cells in vitro. Cultures were pulse-labeled with BrdU after irradiation and/or heat treatment and were fixed either immediately or after a delay of up to 36 h. DNA-synthesizing cells were identified with the help of an FITC-conjugated antibody against BrdU; DNA was quantified after staining with propidium iodide. In this way, the cell cycle distribution could be determined and the progression through the cell cycle could be analyzed. From the movement of labeled cells through the cycle, in particular the appearance of labeled cells in the G(1) compartment (after they had gone through mitosis), the delay in G(2) phase could be determined. The duration of the G(2)/M phase in control cells was about 6 h. This was increased to 12, 13 and 16 h after irradiation (4 Gy X rays), heat treatment (1 h at 43 degrees C), and a combination of the two, respectively. In all these cases, the G(2)-phase block was completely overcome within 48 h after treatment, whereas changes in the S phase were still observable at this time. As expected, the radiation-induced G(2)-phase block was almost completely removed by incubating the cells with 5 or 10 mM caffeine. In the case of hyperthermia alone or in combination with radiation, however, caffeine was somewhat less effective. This does not mean, however, that the mechanisms involved are necessarily different. It can also be seen as a result of the differences in the time course of events. The long delay in S phase after heat treatment may lead to a loss of susceptibility to caffeine by the time the cells move into the G(2) phase.

We have used the method of differential display of mRNAs to search for changes in gene expression associated with radioadaptation triggered by low doses of ionizing radiation in human lymphoblasts. We isolated a cDNA designated PB13 that was down-regulated as early as 1 h after irradiation with 4 Gy in cells adapted by a pre-exposure to a dose of 2 cGy, compared to 3 h in nonadapted cells (4 Gy alone). Northern analysis confirmed the differential expression of a 2.4-kb transcript that was repressed for at least 10 h after irradiation. The major part of the PB13 cDNA was identical to the human CDC16 mRNA. When using either PB13 or CDC16 cDNA as probes, similar radiation-induced alterations in gene expression were observed. Expression of the CDC16 gene was also repressed after oxidative stress with H(2)O(2). The CDC16 protein belongs to the anaphase-promoting complex (APC) that controls progression through mitosis. The repression of expression of the CDC16 gene by ionizing radiation could result in delayed progression of damaged cells through mitosis. This cycle delay would occur earlier in adapted cells and would allow a more rapid and efficient repair that could contribute to the tolerance to subsequent irradiation.

Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40-60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G(2)-phase block, where the fraction of cells in G(2) phase reaches a maximum after 18-20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G(1)- and G(2)-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G(2) phase has reached a maximum shortens the normal recovery from G(2)-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3-2.8, 8.6-85 and 52-74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3-1.4, 2.3-3.0 and 1.2-2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G(2)-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.

2-Methoxyestradiol. 2-Methoxyestradiol (2-ME) is an endogenous estradiol metabolite that disrupts microtubule function, suppresses murine tumors, and inhibits angiogenesis. Since some microtubule inhibitors have been shown to alter radiosensitivity, we have evaluated 2-ME as a radiation enhancer in vitro. H460 human lung cancer cells were plated, treated with 2-ME for 24 h, and irradiated; then colony-forming ability was assessed. The radiation dose enhancement ratios (DERs) using this protocol were 1.3, 1.8 and 2.1 for 1, 1.5 and 2 microM 2-ME, respectively. Using a single-cell plating protocol, the respective DERs were 1.2, 1.5 and 1.8. The parent compound of 2-ME, beta-estradiol, did not enhance radiation effects at equally cytotoxic doses. Isobologram analysis showed that 1 microM 2-ME was additive with radiation, but that 1.5 and 2 microM were synergistic. Cell cycle analysis showed a dose-dependent increase in the percentage of cells in the radiosensitive G(2)/M phase after a 24-h treatment with 2-ME; a threefold increase in the percentage of cells in G(2)/M phase was observed using 2 microM 2-ME. Treatment with 2 microM 2-ME almost completely inhibited repair of sublethal damage (SLD) as shown using split-dose recovery. Radiosensitive, repair-deficient murine SCID (severe combined immunodeficient) cells did not show enhancement of radiation effects with 2 microM 2-ME, but enhancement was observed in the wild-type parental cells (CB-17). SCID cells complemented with human DNA-dependent protein kinase restored radioenhancement by 2-ME. In addition, MCF-7 breast cancer cells were also radiosensitized by 2 microM 2-ME (DER = 2.1). These data suggest that 2-ME is a potential radiation sensitizer, in addition to its previously reported antitumor and antiangiogenic properties. We have verified the antiangiogenic activity of 2-ME in vitro using human endothelial cells. Based on these results, we hypothesize that the mechanism of radiation enhancement may involve redistribution of cells into G(2)/M phase by 2-ME, and that the resulting population of cells is repair-deficient and thus radiosensitive.

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.

The purpose of this paper is to determine the relationship between the response to radiation and the appearance of apoptosis and micronuclei with Trp53 protein in murine tumors after irradiation. Two murine tumors, EL4, which was derived from a mouse lymphoma, and FM3A, which was derived from a mouse mammary carcinoma, were locally irradiated with 15 Gy and sections were stained with H&E and an anti-Trp53 antibody. The response to radiation was greater in EL4 tumors than in FM3A tumors. The frequency of apoptotic cells in EL4 tumors was 6.1 +/- 1.2% at time zero, reached a peak of 36.3 +/- 3. 8% at 6 h, and then decreased with time through 72 h to 2.5 +/- 1.5% after 15 Gy irradiation. In FM3A tumors, no apoptotic cells were detected at 0, 1, 3, 6 or 24 h after exposure. At 48 and 72 h, the frequency was only 3.0 +/- 0.6% and 1.3 +/- 0.3%. Apoptotic cells increased significantly at 3, 6 and 24 h after irradiation in EL4 tumors (P < 0.008) and at 48 and 72 h in FM3A tumors (P < 0.006). The frequency of Trp53-positive cells was 17.9 +/- 2.2 and 15.2 +/- 2.3% at time zero in EL4 and FM3A tumors, respectively, increased to 74.5 +/- 4.5% in EL4 cells (P = 0.001), and increased to 33.9 +/- 1. 1% in FM3A cells (P = 0.005) 1 h after irradiation. Trp53-positive micronuclei appeared in cells in both tumors from 24 to 72 h after irradiation. The frequency of Trp53-positive micronuclei was 3.8 +/- 0.5 and 13.5 +/- 1.3% at 24 h in EL4 and FM3A tumors, respectively, and gradually decreased by 72 h. After exposure to 15 Gy, Trp53-positive micronuclei increased significantly in FM3A tumors compared to EL4 tumors at both 24 and 48 h (P < 0.02). The frequency of these micronuclei increased with increasing dose in FM3A tumors, and the difference between these percentages after 3 Gy and after 5, 10 and 15 Gy was significant (P < 0.02). Many apoptotic cells were observed in the radiosensitive EL4 tumor after irradiation. Death by apoptosis may be related to an early response to radiation in these tumors. The appearance of micronuclei may be an important mechanism of cell death in FM3A tumors in which no apoptosis was induced.

Cells of the TP53-deficient human leukemia cell line HL60 continue to progress throughout the cell cycle and arrest in the G2/M phase during protracted exposure to exponentially decreasing low-dose-rate radiation. We have hypothesized that G2/M-phase arrest contributes to the extent of radiation-induced cell death by apoptosis as well as to overall cell killing. To test this hypothesis, we used caffeine and nocodazole to alter the duration of G2/M-phase arrest of HL60 cells exposed to exponentially decreasing low-dose-rate irradiation and measured the activity of G2/M-phase checkpoint proteins, redistribution of cells in the phases of the cell cycle, cell death by apoptosis, and overall survival after irradiation. The results from these experiments demonstrate that concomitant exposure of HL60 cells to caffeine (2 mM) during irradiation inhibited radiation-induced tyrosine 15 phosphorylation of the G2/M-phase transition checkpoint protein CDC2/p34 kinase and reduced G2/M-phase arrest by 40-46% compared to cells irradiated without caffeine. Radiation-induced apoptosis also decreased by 36-50% in cells treated with caffeine and radiation compared to cells treated with radiation alone. Radiation survival was significantly increased by exposure to caffeine. In contrast, prolongation of G2/M-phase arrest by pre-incubation with nocodazole enhanced radiation-induced apoptosis and overall radiation-induced cell killing. To further study the role of cell death by apoptosis in the response to exponentially decreasing low-dose-rate irradiation, HL60 cells were transfected with the BCL2 proto-oncogene. The extent of G2/M-phase arrest was similar for parental, neomycin-transfected control and BCL2-transfected cells during and after exponentially decreasing low-dose-rate irradiation. However, there were significant differences (P < 0.01) in the extent of radiation-induced apoptosis of parental and neomycin- and BCL2-transfected cells after irradiation, with significantly less radiation-induced apoptosis and higher overall survival in BCL2-transfected cells than similarly irradiated control cells. These data demonstrate that radiation-induced G2/M-phase arrest and subsequent induction of apoptosis play an important role in the response of HL60 cells to low-dose-rate irradiation and suggest that it may be possible to increase radiation-induced apoptosis by altering the extent of G2/M-phase arrest. These findings are clinically relevant and suggest a novel therapeutic strategy for increasing the efficacy of brachytherapy and radioimmunotherapy.

Experiments were performed to determine whether diurnal variations in apoptosis in the mouse small intestine after irradiation with 2.5 Gy gamma rays depended on the time of day that the mice were irradiated, the time of day that the mice were sacrificed or the interval between irradiation and sacrifice. Experiments were performed with a 12-h light:dark regimen with the light period from 6:00 to 18:00 h. With fixed intervals of 6 h and 24 h between irradiation and sacrifice, a peak in induced apoptosis (16%) was observed in mice sacrificed at 8:00 h, two times higher than the nadir of response at 23:00 h (8%). When variable intervals were used between irradiation and measurement of apoptosis, i.e. sacrifice, at 8:00 h or 23:00 h, the induced apoptosis was dependent on the interval, with a peak for 18-h intervals. However, the level of apoptosis was always about twofold higher when measured at 8:00 h than at 23:00 h. No correlation was observed between diurnal variations in apoptosis and survival of mouse intestinal crypts. The diurnal variations in apoptosis after irradiation can be interpreted either in terms of expression of apoptosis during the G2/M phase of the cell cycle in partially synchronized cells, or in terms of a systemic mechanism such as diurnal variation in the neurohormone melatonin.

It has been shown that p53- human colorectal cancer cells arrest after DNA damage in a G2-like state and may then undergo DNA synthesis without intervening mitosis (Waldman et al., Nature 381, 713-716, 1996). To further clarify the role of p53 in the regulation of the G2/M-phase checkpoint, we have studied cells of three closely related human lymphoblastoid cell lines (TK6, WTK1 and TK6E6, an HPV16 E6-transfected TK6 line) with differing p53 status. The cells were irradiated with 1.5-12 Gy gamma rays with or without 2 mM caffeine. There was no evidence of uncoupling of DNA synthesis and mitosis after irradiation in the p53- cell lines, WTK1 and TK6E6, suggesting that this uncoupling may not be a universal phenomenon. The apparent formation of tetraploid cells after irradiation of cells of the p53- WTK1 line was due to the occurrence of a G2-phase block in a pre-existing tetraploid population. These results support the conclusion that control of the G2/M-phase checkpoint after irradiation may differ among different cell types.

The goal of the present study was to assess changes in proliferation in the mouse jejunum after irradiation and the role of the growth factors EGF, TGF-alpha and TGF-beta 1-3 in the proliferative response. Our working hypothesis was that feedback signals from the villus to cells in the crypt regulate proliferation, and that the growth factors EGF and TGF-alpha with their common receptor EGF-R are involved in stimulation of proliferation, while the growth factors TGF-beta 1-3 with their receptors TGF-beta RI and TGF-beta RII are involved in inhibition of proliferation during this regulation. Immunohistochemical detection methods and automated image analysis were used for objective quantification of growth factor expression. The data indicate that, after 5 Gy irradiation, growth stimulation in the crypts takes place before major changes in the villi are observed. However, the combination of the reduction in the cell number, the number of cells expressing TGF-beta 1-3 and the reduction in the level of expression of TGF-beta 1-3 in the villi may cause the release of crypt cells from regulatory growth inhibition and initiate a proliferation-stimulating signal by an increase in the production of TGF-alpha and EGF. Regulation of proliferation after initiation of a proliferative response seems to be related more to the growth factors EGF, TGF-alpha and TGF-beta 3 in the crypts than to villus cellularity or growth factor expression, supporting the concept of stem cell autoregulation as a mechanism of cell regeneration in the intestinal crypt.

An analytical model is presented that describes radiation-induced cellular inactivation in the presence of sublethal damage repair, cellular repopulation and redistribution in the mitotic cycle (the 3 Rs). The parameters of the model are measurable experimentally. Also taken into account are the initial age distribution of the cell population, the fact that subgroups of cells progress through the cycle at different speeds, the effects of a dose of radiation on the duration of the four phases of the cycle (G1, S, G2, M), the possibility that a certain fraction of the cells are quiescent, and cell loss and/or cell removal from the proliferating population. Survival probabilities are expressed as linear-quadratic functions of dose where the coefficient alpha and beta as well as the recovery constant (t0) are taken to depend on the position of the cell in the mitotic cycle. Explicit analytical expressions for inactivation probability are given for clonogenic cells exposed to continuous or fractionated radiation. Two model calculations are used to illustrate the formalism: in one, the redistribution of cells during fractionated therapy is examined. In the other calculation, it is shown that it is sufficient to take into account differences in proliferation rates and the change in the ratio alpha/beta within the generation cycle for cells that may have otherwise equal response to acute exposures to explain that in a fractionated treatment protocol late-responding cells are more sensitive to the dose per fraction than early-responding cells. It is not necessary to invoke differences in radiosensitivity between these two classes of cells.

Exposure of synchronized populations of mouse C3H 10T1/2 cells to a single dose (0.6 Gy) of 5.9 MeV neutrons at intervals after mitotic shake-off results in a distinctive variation in the oncogenic transformation frequency through the cell cycle. Previous findings show a sensitive window for X-ray-induced oncogenic transformants at late times after mitotic shake-off (14-16 h). Optimal sensitivity to neutrons was observed for cell populations irradiated soon after mitotic shake-off (4-6 h), where the majority of cells would be in the G1 phase of the cell cycle. Additionally, enhanced sensitivity was also found for that period after shake-off (14-16 h) which was maximally sensitive to X rays corresponding to cell populations with a high proportion of G2-phase cells. That is, low-LET radiation (250 kVp X rays) largely appears to produce oncogenic transformants in G2-phase cells, while intermediate-LET radiation (5.9 MeV neutrons) is effective principally on G1- and, to a somewhat lesser extent, G2-phase cells. Cells irradiated with neutrons showed less variation for lethality through the cell cycle than those irradiated with X rays, in agreement with previous findings. The mechanistic basis for the difference in the response of cells in the different phases of the cell cycle to radiations of different quality is unknown but is suggestive of distinct ("signature") molecular changes leading to the observed oncogenic transformation response.

Oncogenic transformation of synchronized C3H 10T1/2 cells was determined after exposure to 4.3 MeV alpha particles (LET = 101 keV/microns). Two synchronization techniques were tested using basic and modified protocols: one based on the release of cells from contact inhibition and the second on the mitotic shake-off method. Progression of cells through the cycle was followed as a function of time by flow cytometric analysis, DNA labeling for passage through S phase, the growth curve for the cell number and mitotic index measurements. The conclusion is that, although the release of cells from confluence provides higher yields of synchronized cells, mitotic shake-off proved to be the best way of collecting a synchronized population of minimally perturbed cells. Cells synchronized by mitotic shake-off were irradiated with 0.30 Gy in the interval between 2 and 10 h corresponding to G1 and early S phases. For comparison asynchronous populations were irradiated in parallel. Oncogenic transformation frequency, corrected for background, in mid-G1 phase was (18 +/- 4) x 10(-5) (average values of frequencies at 4 and 6 h) compared with the value of (8 +/- 4) x 10(-5) for the asynchronous population. While these data are suggestive of a trend toward a slightly increased sensitivity in mid-G1 phase, it is not statistically significant. The surviving fraction is constant in G1 phase.

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137Cs gamma irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of gamma radiation. We studied the effect of several pharmacological agents on the radiation-induced G2/M block and DNA fragmentation. The agents which reduced the radiation-induced G2/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G2/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G2/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G2/M block, and to use this effect in the treatment of patients with malignant disease.

A two- to threefold increase in the rate of neoplastic transformation in cells irradiated at a dose rate of 0.22 cGy/min with fission-spectrum neutrons compared to that at 10.7 cGy/min has been confirmed with the use of alkaline phosphatase chromogenic substrate Western Blue staining to detect foci of neoplastically transformed cells through their expression of a tumor-associated antigen, the end point of the HeLa x skin fibroblast human hybrid cell transformation assay. To investigate whether the inverse dose-rate effect is due to the existence of a period in the cell cycle in which cells are significantly more sensitive to neoplastic transformation than in the rest of the cell cycle, as has been postulated previously (Rossi and Kellerer, Int. J. Radiat. Biol. 50, 353-361, 1986; Brenner and Hall, Int. J. Radiat. Biol. 58, 745-758, 1990; Elkind, Int. J. Radiat. Biol. 59, 1467-1475, 1991), we compared the sensitivity of late mitotic/early G1-phase and mid-G1-phase cells with that of asynchronous cells. The rationale for examining these particular cell cycle phases was based on the fact that mitosis has been hypothesized to be a candidate for the extremely sensitive period, and on a preliminary report that mid-G1-phase C3H 10T1/2 cells may exhibit enhanced sensitivity for neutron-induced transformation. A nominal dose of 45 cGy of fission-spectrum neutrons was delivered at approximately 10 cGy/min. The data indicate that neither late mitotic/early G1-phase nor mid-G1-phase cells are significantly more sensitive than asynchronous cells. Further, the dependence on the phase of the cell cycle for neoplastic transformation of CGL1 cells induced by fission-spectrum neutrons is different from that previously demonstrated for gamma radiation, where late-mitotic cells were approximately five times more sensitive than mid-G1-phase and asynchronous cells (Redpath and Sun, Radiat. Res. 121, 206-211, 1990).

Upon exposure of cells to radiation delivered at a continuous low dose rate, cell proliferation may be sustained with the cells exhibiting a constant doubling time that is independent of the total dose. The doubling time or mitotic delay under these conditions has been shown to depend on the dose rate in HeLa, V79 and P388F cells (Mitchell et al., Radiat. Res. 79, 520-536, 1979; Fox and Gilbert, Int. J. Radiat. Biol. 11, 339-347, 1966). Reanalysis of the data for these particular cell lines shows that there is a threshold dose rate for mitotic delay, and that above the threshold there is a linear relationship between the length of mitotic delay and the logarithm of the dose rate which is referred to as the dose-rate response. We have observed the same relationships for L5178Y (LY)-R and LY-S cells exposed to low-dose-rate radiation. The threshold dose rates for LY-R, LY-S and P388F cells are similar (0.01-0.02 Gy/h) and are much lower than for V79 and HeLa cells. The slope of the dose-rate response curve is the greatest for HeLa cells, followed in order by LY-S, V79 and P388F cells, and finally by LY-R cells. The slopes for HeLa and LY-R cells differ by a factor of 35.

Cells from patients with ataxia telangiectasia (AT) are abnormal in their response to irradiation as judged by clonogenic survival and accumulation in G2 phase. The relationship of the results of these two assays, however, is still a matter of controversy. Flow cytometry was used to measure the distribution of cells in the phases of the cell cycle after 2 Gy irradiation in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) and SV40-transformed fibroblasts. AT cells showed increased and prolonged accumulation in G2/M phase regardless of the cell type (lymphoblastoid or fibroblast) or complementation group (A, C or D). To test the hypothesis that prolonged accumulation of AT cells in G2 phase after irradiation was not simply a reflection of their radiosensitivity, we gave iso-survival radiation doses to SV40-transformed fibroblasts of two AT and one control cell lines. The two AT cell lines exited from the G2/M-phase block more slowly than control cells after each dose tested. This implies that prolonged accumulation in G2/M phase in AT cells is not directly related to radiosensitivity as measured by clonogenic survival, but that factors involved in the exit from G2 phase after irradiation may be abnormally regulated. We found that G2-phase arrest of AT cells did not necessarily result in a fatal consequence in the first cell cycle after irradiation. Furthermore, G2-phase arrest did not lead to detectable DNA fragmentation characteristic of apoptosis as judged by gel electrophoresis.

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD). Previously it was found that PLD repair could be partially inhibited by increasing the extracellular pH (pHe) of the spent medium from its normal value of 6.7-6.8 to 7.6 during postirradiation holding. The present study shows that PLD repair is also inhibited by reducing the pHe of the spent medium to 6.0. The effects of altering pHe on rejoining of DNA double-strand breaks (DSBs) as measured by neutral filter elution and on mitotic delay and chromosome aberrations seen after releasing cells from the plateau phase were investigated. Neither increasing nor decreasing the pHe of the spent medium had an effect on radiation-induced mitotic delay. Rejoining of DSBs was significantly inhibited by holding at pHe 6.0 but not affected by holding at pHe 7.6. At 2 h after irradiation about 51% of unrejoined breaks remained at pHe 6.0, compared to about 15% at pHe 6.7 or 7.6. However, holding at pHe 7.6 appeared to cause a marginal change in the kinetics of rejoining of DSBs. Repair of lesions leading to dicentric and acentric chromosome aberrations did not occur when cells were held at pHe 6.0, since less than 10% of these aberrations disappeared from cells held for 24 h before subculture. In contrast, holding plateau-phase cells at pHe 7.6 vs 6.7 caused a small but significant reduction in the disappearance of dicentrics but had no effect on the rate or extent of the disappearance of acentrics. These data have led us to hypothesize that inhibition of PLD repair by holding at pHe 6.0 is related both to inhibition of pH-dependent DNA repair enzymes and to induction of changes in DNA which lead to misrepair when the cells are released from plateau phase. Inhibition of PLD repair by holding at pHe 7.6, on the other hand, is related primarily to changes in DNA structure which promote misrepair.

The killing effects of ionizing radiation on MOLT-4 cells were studied using colony formation assays, dye exclusion tests, and cell growth analysis. When MOLT-4 cells were exposed to X rays, the fraction of cells stained with erythrosin B increased markedly during the incubation after irradiation at 37 degrees C and reached a maximum within 24 h. In contrast, no such increase was observed for L5178Y cells or their derivative, radiosensitive mutant M10 cells, at a dose resulting in the same level of cell survival as measured by the colony formation assay. Detailed analysis of cell survival as measured by the dye exclusion test in comparison with that measured by the colony formation assay in MOLT-4 cells indicated that MOLT-4 cells are subject to interphase death as well as reproductive death. Analysis of subclones showed a wide variety of differences in the level of stained cells using the dye exclusion test. The results confirmed the population heterogeneity of MOLT-4 cells. Moreover, none of the subclones showed exactly the same cell survival when this was measured with the two assays. The present results strongly suggest that both modes of cell death may occur in single MOLT-4 cells. Possibly apoptosis is the mechanism accounting for both modes of death in these cells.

When cells are subjected to irradiation, their progression through the cell cycle can be arrested. If the arrested cells are subjected to additional damage, their period of arrest is prolonged. Under continuous low-dose-rate irradiation, the cumulative nature of arrest time leads to a geometric increase in the arrest time as a function of the dose rate. Above a certain cell-line-specific dose rate, the arrest duration becomes infinite and cell proliferation ceases. We find that the lowest dose rate (critical dose rate) required to stop cell proliferation during continuous irradiation is the reciprocal of the mitotic delay per gray of high-dose-rate irradiation. The calculated critical dose rates for X rays agree with those measured by Mitchell et al. (Radiat. Res. 79, 537-551, 1979), provided that the critical dose rate is identified with the minimum dose rate which allows no more than one population doubling. This specific identification of the critical dose rate is explained on the basis of cell cycle kinetics. A kinetic mechanism for cell cycle arrest and recovery leads to the critical dose rate as the reciprocal sensitivity for G2 arrest.

A mathematical model of the progression of cells through the mitotic cycle under continuous low-dose-rate irradiation is described. The model considers explicitly two special cases: (a) when a fraction of cells disintegrate and disappear after mitosis and (b) when a fraction of cells which have reached mitosis do not progress further but do not disintegrate either. We have established a relationship between the parameters of the model and dose and/or the age of the cell at exposure. This formalism is applied to studies of the effects of dose rate on HeLa cells (Mitchell, Bedford, and Bailey, Radiat. Res. 79, 520-536, 1979; 80, 186-197, 1979). Detailed information on the fraction of cells of a certain biological age at a given chronological time is needed because of the variation in the radioresponse of the cells as a function of age.

An in vitro microscopic assay for mitosis-inducing activity in mitotic HeLa cells was developed and used to demonstrate that cells irradiated and arrested in G2 phase of the cell cycle contain an inhibitor of mitosis. This assay system has a number of advantages over other assays including the use of autologous components (HeLa nuclei and mitotic cell extracts) in contrast to the microinjection method with Xenopus oocytes and without the requirements for microinjection expertise and Xenopus oocytes. The radiation-inducible inhibitor was detected at the lowest radiation dose tested (2 Gy) with maximal activity achieved within 30 min after radiation. Inhibitor activity decayed with time after radiation (2 Gy) with no activity detected at 6 h even though the cells remained in G2 phase, suggesting that either synthesis or activation of additional components is necessary for recovery from G2 arrest. The inhibitor activity was not detected in irradiated cells treated with caffeine to induce premature recovery from G2 arrest.

Radiation-induced mitotic delay is under investigation in the fission yeast, Schizosaccharomyces pombe. A large range of cell cycle- and radiation-sensitive mutants of this yeast is available to facilitate this effort. Through an examination of such mutants it has been shown that the X-ray transition point and the p34cdc2 execution point are coincident; wee1- strains are not delayed by irradiation; and the radiation-sensitive mutants rad1-1, rad3-136, rad9-192, and rad17-W are not delayed by radiation or by inhibitors of DNA synthesis, including hydroxyurea. A model is proposed: Damaged DNA generates a signal to delay mitosis which is carried by the products of the rad genes to activate the tyrosine kinase p110wee1. This in turn inactivates the serine/threonine kinase p34cdc2, thereby blocking entry to mitosis. Unreplicated DNA also initiates a signal to delay mitosis which is carried by these same rad genes but, as indicated in the literature, transmission to p34cdc2 does not require p110wee1. The delay-deficient rad mutants may possess some properties of tumor suppressor genes, with implications for mutagenesis and oncogenesis.

A model for damage, repair, killing, and repopulation of myelopoietic marrow is presented. Evaluation produces time and dose-rate profiles during and following any complex irradiation. Equations model variable dose rates, multiple exposures, different sources, and arbitrary intervals between treatments. If factors which dominate the control of biological processes can be demonstrated, an option is to set biological rate constants to experimentally determined values. Previously, knowledge did not permit identification of dominating biological processes and their temporal rates. But a unique feature of this study is that unspecified lesions for killing and injury of cells are evaluated from mortality data on the animal species of choice. "Unspecified" is used to indicate a condition of assumption-free modeling of molecular processes, whereby rate constants for cellular effects are simply computed directly from animal mortality data. Coefficients (estimated by maximum-likelihood methods for nonspecific processes) are compared with experimental values for specific processes. The model has many uses, including modeling of the myelopoietic potential as a function of time. Another option is to calculate the whole-body survival curve for cells that control myelopoiesis as a result of the treatment schedule. Also through simple extensions of the model, an extremely complex protocol can be identified with an equivalent prompt dose value--even for partial-body, fractionated exposures.

The eyes of Sprague-Dawley rats were irradiated with doses of 2.5-10 Gy 250-kVp X rays, 1.25-2.25 Gy fission-spectrum neutrons (approximately 0.85 MeV), or 0.1-2.0 Gy 600-MeV/A 56Fe particles. Lens opacifications were evaluated for 51-61 weeks following X and neutron irradiations and for 87 weeks following X and 56Fe-particle irradiations. Average stage of opacification was determined relative to time after irradiation, and the time required for 50% of the irradiated lenses to achieve various stages (T50) was determined as a function of radiation dose. Data from two experiments were combined in dose-effect curves as T50 experimental values taken as percentages of the respective T50 control values (T50-% control). Simple exponential curves best describe dose responsiveness for both high-LET radiations. For X rays, a shallow dose-effect relationship (shoulder) up to 4.5 Gy was followed at higher doses by a steeper exponential dose-effect relationship. As a consequence, RBE values for the high-LET radiations are dose dependent. Dose-effect curves for cataracts were compared to those for mitotic abnormalities observed when quiescent lens epithelial cells were stimulated mechanically to proliferate at various intervals after irradiation. Neutrons were about 1.6-1.8 times more effective than 56Fe particles for inducing both cataracts and mitotic abnormalities. For stage 1 and 2 cataracts, the X-ray Dq was 10-fold greater and the D0 was similar to those for mitotic abnormalities initially expressed after irradiation.

The hair follicle or its differentiated product, the hair, which represents the linear historical record of the follicular proliferative activity, could provide a biological dosimeter of value for dose distribution determinations after accidental exposure. Here we present some further studies on irradiated mouse hair follicles and hair, and discuss the difficulties in obtaining similar data for humans. The incidence of cell death in the follicles has been shown elsewhere to be maximum 12 h after irradiation, and it increases with dose. Here we confirm that doses of 0.2-0.4 Gy can be readily detected. We show here that there is only a little more cell death in the larger follicles even though they contain many more cells and mitotic figures. About one-third of all the dead cell fragments in a follicle can be seen in a good longitudinal follicle section. Mitotic activity declines progressively with dose in the large follicles, which start with more mitotic cells, showing the dose-dependent changes most readily. The dead cells are morphologically identical to apoptotic cells at the level of the light microscope, and they fragment into several bodies, the number of which increases with dose. The total number of apoptotic bodies or fragments in whole large follicles increases almost 100-fold over a range of 1.3 Gy (from 0.2 to 1.5 Gy) and about tenfold over the range 0.2-0.5 Gy. The estimated number of dead (apoptotic) cells increases about sevenfold over the same 1.3-Gy range. The width of the middle portion of the broadest, awl, hairs measured 12 days after irradiation decreases with increasing dose. About 80% of the hairs show an obvious reduction in width after 2 Gy and the effects of a dose of about 1 Gy can be detected. The width of the hair is reduced by 10-14% per Gy. A comparison has been made between BDF1 (black) and BALB-c (albino) mice. The large follicles contain similar numbers of mitotic cells, but the BALB-c mice are more sensitive both in terms of the radiation-induced apoptosis and in terms of a reduction in awl hair width.

To investigate the effect of irradiation on lymphocyte mitosis we utilized a T-lymphocyte colony assay; this sensitive technique avoided the methodological problems associated with previous techniques such as tritiated thymidine uptake. Irradiation doses in the range of 0.5 to 4 Gy had the effect of decreasing the lymphocytes' capacity to divide as shown by their reduced T-colony-forming ability. This radiosensitivity, however, was found to vary according to the timing of irradiation in relation to the stage of colony growth. Through survival curve analysis it was shown that the colony-forming cells could withstand higher irradiation doses without losing their replicative ability when they were irradiated prior to or during early stages of PHA stimulation. A similar "protection" by PHA stimulation has been well documented for nondividing T-lymphocyte functions but, due to the lack of a suitable methodology, had hitherto not been described for dividing T-lymphocytes. These results provide evidence that the early events associated with PHA stimulation might lead to an improved ability of T-lymphocytes to withstand higher doses of irradiation before mitotic death is registered.

The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2 mM) and Ro-20-1724 (18 microM) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by gamma radiation. These results (1) emphasize the organism and agent specificity of cyclic nucleotide responses, (2) indicate that the kinetics and apparent refractory response are compatible with the function of previously identified membrane receptor systems, i.e., a blue light receptor and a choleragen receptor mediating cyclic AMP responses, and (3) suggest a relationship between the agent-specific cyclic nucleotide responses and the modification of gamma-radiation-induced delay of mitosis by the same agents, i.e., a decrease in the delay by those which elevate cyclic GMP levels and an increase in the delay by those which elevate only cyclic AMP levels.