Teratogenic effects of mild heat stress during mouse embryogenesis: effect of Trp53

Hyperthermia can be teratogenic in fetal mice exposed during organogenesis, an effect considered to be due to heat-induced apoptosis of cells in the developing organs. We exposed pregnant mice carrying Trp53(+/+), Trp53(+/-) and Trp53(-/-) fetuses to mild whole-body hyperthermia that raised their core temperature to 40.5 degrees C for 60 min on either day 10 or 11 of gestation. On day 18 of gestation, the fetuses were removed from control and hyperthermia-treated mice and genotyped, and tail length was measured. Limb digits were examined for abnormalities. Tail length in unheated control fetuses was influenced by Trp53 status. A complete lack of functional Trp53 (Trp53(-/-)) but not partial lack of function (Trp53(+/-)) resulted in shorter tails compared to Trp53(+/+) fetuses, indicating a role for Trp53 in the regulation of tail lengthening in mouse fetuses. In all three genotypes, hyperthermia on gestation day 10 resulted in tails shorter than unheated controls, and hyperthermia on day 11 resulted in tails longer than controls. There was no effect on limb digit abnormalities. The data suggest that Trp53-dependent or independent apoptosis may not be directly involved in heat-induced teratogenesis, but that the primary teratogenic effect of heat results from the disruption of another tail length-regulating process that is independent of Trp53. However, the nature of the teratogenic outcome of that disruption depends on the gestation time. The ability of Trp53 to additionally regulate the tail lengthening process was also sensitive to the effects of heat, but that sensitivity again depended on the time of the heat stress during gestation.     

Influence of prior exposure to low-dose adapting radiation on radiation-induced teratogenic effects in fetal mice with varying Trp53 function

Teratogenesis in tails and limb digits of fetal mice with varying Trp53 status was examined after exposure of pregnant females to 4 Gy gamma radiation with and without a prior 30-cGy exposure. Prior low-dose exposure modified the teratogenic effects of radiation in a manner dependent upon Trp53 status and gestation time. A 4-Gy exposure on gestation day 11 resulted in tail shortening and digit abnormalities. A 30-cGy exposure 24 h prior to a 4-Gy radiation exposure on day 11 reduced the extent of both digit abnormalities and the tail-shortening effects in Trp53(+/+) fetuses and also reduced tail shortening in Trp53(+/-) fetuses, but to a lesser extent. However, the pre-exposure enhanced the tail-shortening effects of 4 Gy in Trp53(-/-) fetuses. In contrast, a 30-cGy exposure given 24 h prior to a 4-Gy exposure on gestation day 12 had no effect on the reduced tail length resulting from the 4-Gy exposure of Trp53(+/+) or Trp53(+/-) fetuses, but it partly protected Trp53(-/-) fetuses against reduced tail length. A 4-Gy exposure alone on day 12 did not result in any increase in the frequency of digit abnormalities in Trp53(-/-) fetuses so any protective effect of the preirradiation could not be detected. However, the preirradiation did result in protection against in digit abnormalities in Trp53(+/-) fetuses. We conclude that radiation-induced teratogenesis reflects both Trp53-dependent and independent processes that lead to apoptosis, and these respond differently to prior adapting doses.     

Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress

Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0-24 h) and chronic (3-33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3-4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.     

Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status

We reported previously that a radiation-induced adaptive response existed in the late period of embryogenesis, and that radiation-induced apoptosis in the predigital regions was responsible for digital defects in embryonic ICR mice. To investigate the possible involvement of the Trp53 gene and radiation-induced apoptosis in radiation-induced adaptive responses in embryogenesis, the present study was conducted using Trp53 wild-type (Trp53(+/+)) and Trp53 heterozygous (Trp53(+/-)) embryonic mice of the C57BL/6 strain. The existence of a radioadaptive response in the Trp53(+/+) embryonic mice was demonstrated by irradiating the embryos with 5 or 30 cGy on embryonic day 11 prior to a challenging irradiation at 3 Gy on embryonic day 12. The two conditioning doses at 5 and 30 cGy significantly suppressed the induction of apoptosis by the challenging dose in the predigital regions of limb buds in the Trp53(+/+) embryonic mice, while no such effect was found in the Trp53(+/-) embryonic mice. These findings indicate that induction of a radioadaptive response in embryogenesis is related to Trp53 gene status and the occurrence of radiation-induced apoptosis.     

Effect of low-level radiation on the death of male germ cells

Hormetic and adaptive responses induced by low-level radiation in hematopoietic and immune systems have been observed, as shown by stimulatory effects on cell growth and resistance to subsequent radiation-induced cytogenetic damage. However, in terms of cell death by apoptosis, the effects of low-level radiation are controversial: Some studies showed decreased apoptosis in response to low-level radiation while others showed increased apoptosis. This controversy may be related to the radiation doses or dose rates and also, more importantly, to the cell types. Testes are one of the most radiosensitive organs. The loss of male germ cells after exposure to ionizing radiation has been attributed to apoptosis. In the present study, the effects of low-level radiation at doses up to 200 mGy on mouse male germ cells in terms of apoptosis and the expression of apoptosis-related proteins were examined at different times after whole-body exposure of mice to low-level radiation. In addition, the effect of pre-exposure to low-level radiation on subsequent cell death induced by high doses of radiation was examined to explore the possibility of low-level radiation-induced adaptive response. The results showed that low-level radiation in the dose range of 25-200 mGy induced significant increases in apoptosis in both spermatogonia and spermatocytes, with the maximal effect at 75 mGy. The increased apoptosis is most likely associated with Trp53 protein expression. Furthermore, 75 mGy low-level radiation given pre-irradiation led to an adaptive response of seminiferous germ cells to subsequent high-level radiation-induced apoptosis. These results suggest that low-level radiation induces increased apoptosis in male germ cells but also induces a significant adaptive response that decreases cell death after a subsequent high-dose irradiation.     

Prenatal radiation-induced limb defects mediated by Trp53-dependent apoptosis in mice

We reported previously that in utero radiation-induced apoptosis in the predigital regions of embryonic limb buds was responsible for digital defects in mice. To investigate the possible involvement of the Trp53 gene, the present study was conducted using embryonic C57BL/6J mice with different Trp53 status. Susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both Trp53 status and the radiation dose; i.e., Trp53 wild-type (Trp53(+/+)) mice appeared to be the most sensitive, Trp53 heterozygous (Trp53(+/-)) mice were intermediate, and Trp53 knockout (Trp53(-/-)) mice were the most resistant. These results indicate that induction of apoptosis and digital defects by prenatal irradiation in the later period of organogenesis are mediated by the Trp53 gene. These findings suggest that the wild-type Trp53 gene may be an intrinsic genetic susceptibility factor that is responsible for certain congenital defects induced by prenatal irradiation.     

Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae

BACKGROUND: Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. RESULTS: After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. CONCLUSIONS: DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. We propose that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes.     

Increased resistance of the radiosensitive M10 mutant cells of the L5178Y mouse lymphoma cell line to heat-induced apoptosis.

M10 cells, which are deficient in the repair of DNA DSBs and are therefore radiosensitive, are about twofold more thermoresistant than their parental L5178Y cells. We found that, after heat shock at 43 degrees C under conditions resulting in 10% survival (D(10)), M10 cells did not undergo apoptosis, whereas L5178Y cells did undergo apoptosis. M10 cells, but not L5178Y cells, constitutively expressed Hsp72 protein. Moreover, the M10 cells accumulated higher amounts of the heat-inducible form of Hsp72. The patterns of activation of the DNA-binding activity of HSF (heat-shock factor) differed in M10 and L5178Y cells. In response to heat shock, M10 cells accumulated greater amounts of Trp53 protein (formerly known as p53) than the parental cells. Cdkn1a (formerly known as p21, Waf1) was constitutively expressed and further accumulated after heat shock only in M10 cells. We suggest that heat-inducible Hsp72 to a larger extent, and constitutive Hsp72 to a lesser extent, together with Cdkn1a may be involved in the protection of M10 cells against heat-induced apoptosis. Apoptosis in these cells is likely to occur in Trp53-dependent manner.     

Geldanamycin analog 17-DMAG limits apoptosis in human peripheral blood cells by inhibition of p53 activation and its interaction with heat-shock protein 90 kDa after exposure to ionizing radiation

Exposure to ionizing radiation induces p53, and its inhibition improves mouse survival. We tested the effect of 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) on p53 expression and function after radiation exposure. 17-DMAG, a heat-shock protein 90 (Hsp90) inhibitor, protects human T cells from ionizing radiation-induced apoptosis by inhibiting inducible nitric oxide synthase (iNOS) and subsequent caspase-3 activation. Using ex vivo human peripheral blood mononuclear cells, we found that ionizing radiation increased p53 accumulation, acute p53 phosphorylation, Bax expression and caspase-3/7 activation in a radiation dose- and time postirradiation-dependent manner. 17-DMAG inhibited these increases in a concentration-dependent manner (IC(50) = 0.93 ± 0.01?μM). Using in vitro models, we determined that inhibition of p53 by genetic knockout resulted in lower levels of caspase-3/7 activity 1?day after irradiation and enhanced survival at 10?days. Analysis of p53-Hsp90 interaction in ex vivo cell lysates indicated that the binding between the two molecules occurred after irradiation but 17-DMAG prevented the binding. Taken together, these results suggest the presence of p53 phosphorylation and Hsp90-dependent p53 stabilization after acute irradiation. Hsp90 inhibitors such as 17-DMAG may prove useful with radiation-based cancer therapy as well as for general radioprotection.     

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.     

Radiation-induced apoptosis and its relationship to loss of clonogenic survival

Ionizing radiation can be an effective inducer of apoptosis and studies of many aspects of the pathways and mechanisms involved in this apoptosis induction have been published. This review stresses two aspects: the relationship between apoptosis and loss of clonogenic ability in irradiated cells and the time course for the appearance of apoptosis after radiation exposure. Although it was initially assumed that apoptosis occurred relatively quickly (within hours) after irradiation, evidence is presented and discussed here showing that apoptosis can occur at long times after irradiation (out to 20 days) in some cell types. This late, or delayed, apoptosis occurs after the cells have divided once or several times. The impact of delayed apoptosis on loss of clonogenicity after irradiation remains unclear. It seems likely that in some cell types, e.g., fibroblasts, the occurrence of late apoptosis is minimal and may have little impact on long term cell survival of the population, but in at least one instance, with a cell line of hematopoietic origin, it appears that late apoptosis can account for all the loss of clonogenicity in irradiated cells. The role of p53 in radiation-induced apoptosis is also discussed, with data presented showing that both p53-dependent and independent pathways for radiation-induced apoptosis exist, depending on the cell type.     

Uncoupling p53 functions in radiation-induced intestinal damage via PUMA and p21

The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.     

DNA double-strand breaks in human lymphocytes after single irradiation by low doses of pulsed X-rays: non-linear dose-response relationship

Effects of ionizing radiation registered in cells after low dose irradiation are still poorly understood. A pulsed mode of irradiation is even more problematic in terms of predicting the radiation-induced response in cells. Thus, the aim of this paper was to study and analyze the effects of dose and frequency of pulsed X-rays on the frequency of radiation-induced DNA double-strand breaks and their repair kinetics in human peripheral blood lymphocytes in vitro. Analysis of radiation-induced gammaH2AX and 53BP1 repair foci was used to assess the DNA damage in these cells. The dose-response curve of radiation-induced foci of both proteins has shown deviations from linearity to a higher effect in the 12-32 mGy dose range and a lower effect at 72 mGy. The dose-response curve was linear at doses higher than 100 mGy. The number of radiation-induced gammaH2AX and 53BP1 foci depended on the frequency of X-ray pulses: the highest effect was registered at 13 pulses per second. Moreover, slower repair kinetics was observed for those foci induced by very low doses with a nonlinear dose-response relationship.     

Biology of marrow stromal cell lines derived from long-term bone marrow cultures of Trp53-deficient mice.

To investigate the effect of Trp53 (formerly known as p53) on stromal cells of the hematopoietic microenvironment, long-term bone marrow cultures were established from mice in which the Trp53 gene had been inactivated by homologous recombination (Trp53(-/-)) or their wild-type littermates (Trp53(+/+)). Long-term bone marrow cultures from Trp53(-/-) mice continued to produce nonadherent cells for 22 weeks, while Trp53(+/+) cultures ceased production after 15 weeks. There was a significant increase in the number of nonadherent cells produced in Trp53(-/-) long-term bone marrow cultures beginning at week 9 and continuing to week 22 (P < 0.02). The Trp53(-/-) cultures also showed significantly increased cobblestone island formation indicative of early hematopoietic stem cell-containing colonies beginning at week 10 (P < 0.01). Cobblestone islands persisted until weeks 15 and 22 in Trp53(+/+) and Trp53(-/-) cultures, respectively. Co-cultivation experiments in which Trp53(+/+) Sca1(+)lin- enriched hematopoietic stem cells were plated on Trp53(-/-) stromal cells showed increased cobblestone island formation compared to Trp53(-/-) Scal+lin- cells plated on Trp53(+/+) or Trp53(-/-) stromal cells. Radiation survival curves for clonal bone marrow stromal cells revealed a similar D0 for the Trp53(+/+) and Trp53(-/-) cell lines (1.62 +/- 0.16 and 1.49 +/- 0. 08 Gy, respectively; P = 0.408), and similar n (8.60 +/- 3.23 and 10.71 +/- 0.78, respectively) (P = 0.491). Cell cycle analysis demonstrated a G2/M-phase arrest that occurred 6 h after irradiation for both Trp53(+/+) and Trp53(-/-) stromal cell lines. After 10 Gy irradiation, there was no significant increase in the frequency of apoptosis detected in Trp53(+/+) compared to Trp53(-/-) marrow stromal cell lines. In the stromal cell lines, ICAM-1 was constitutively expressed on Trp53(+/+) but not Trp53(-/-) cells; however, a 24-h exposure to TNF-alpha induced detectable ICAM-1 on Trp53(-/-) cells and increased expression on Trp53(+/+) cells. To test the effect of Trp53 on the radiation biology of hematopoietic progenitor cells, the 32D cl 3 cell line was compared with a subclone in which expression of an E6 inserted transgene accelerates ubiquitin-dependent degradation of Trp53, thus preventing accumulation of Trp53 after genotoxic stress. The radiation survival curves were similar with no significant difference in the D0 or n, or in the percentage of cells undergoing apoptosis after 10 Gy irradiation between the two cell lines. Cells of the 32D-E6 cell line displayed a G2/M-phase arrest 6 h after 10 Gy, while cells of the parent line exhibited both a G2/M-phase arrest and a G1-phase arrest at 24 and 48 h. The results suggest a complex mechanism of action of Trp53 on the interactions between stromal and hematopoietic cells in long-term bone marrow cultures.     

Modification of rat thymocyte membrane properties by hyperthermia and ionizing radiation.

Thymocytes are one the most widely used cell models for the study of radiation-induced interphase death. This cell-type was chosen for the study of hyperthermic and radiation effects on two membrane-related processes implicated in the interphase death of cells: Na+-dependent 2-aminoisobutyric acid (AIB) transport and cyclic 3'-5' adenosine monophsophate formation. The response of AIB transport to heat is dose-dependent, but the biphasic thermal response curve (AIB uptake versus time) differs fom the sigmoidal radiation response curve. Heating thymocytes for 20-30 min at 43 degrees C stimulates AIB uptake. Additional heating at 43 degrees C, however, markedly reduces AIB uptake. Despite the immediate stimulating effect of heat (30 min at 43 degrees C), the thymocyte has already developed irrepairable impairments, as demonstrated by the fractionated heating experiments. The heat-induced impairment of AIB uptake is mainly on the Na+-dependent component of neutral amino-acid transport, affecting primarily the maximal rate of uptake, i.e. Vmax. Additional evidence for heat-induced plasma membrane damage is the alteration in cAMP levels. Heating thymocytes for 30 min or longer at 43 degrees C causes a massive rise in cAMP level within the cell. This differs from thymocytes exposed to radiation where no rise in cAMP is observed.     

Organ-specific gene expressions in C57BL/6 mice after exposure to low-dose radiation

The possibility that radiation-induced alterations in gene expression are tissue specific and are related to apoptosis was examined using samples from brain, heart, lung, spleen and intestine from female C57BL6 mice after exposure to 0.2 Gy radiation. Apoptosis was the highest in spleen and intestine, moderate in lung, and absent in brain and heart. However, the mRNA expression of Trp53 and Cdkn1a (p21) after irradiation was not different among the organ types, and immunohistochemistry revealed that all the organs expressed these two proteins after irradiation. When expression patterns of 23 genes in the organs were examined by RT-PCR, neogenine, Apo1, nuclease sensitive element binding protein 1, syntaxin, cyclin G1, hNOP56, paraoxonase and glutathione peroxidase were overexpressed after irradiation in all the organs sampled, suggesting them as universal exposure markers for low-dose radiation. Sialyltransferase may be a candidate for radiation detection in spleen and intestine, which are radiosensitive organs. Because Sod1 (Cu/ZnSOD) and alphaB crystalline were expressed only in spleen, and protein tyrosine kinase and platelet membrane glycoprotein lib were expressed in both spleen and lung, these genes may also be potential markers for detection of radiation exposure, especially low-dose radiation, in these tissues. These data suggested possible tissue-specific markers of low-dose radiation exposure and suggested potential novel genetic modifiers of radiation response.     

Repair of radiation-induced DNA damage in thermotolerant and nonthermotolerant HeLa cells

The effect of heat exposure on the repair of radiation-induced DNA damage which inhibits the ability of nuclear DNA to undergo supercoiling changes was studied using the fluorescent halo assay in thermotolerant and nonthermotolerant (normal) cells. The assay utilizes an intercalating, fluorescent dye to unwind and rewind endogenous DNA supercoils. When HeLa cells are exposed to 17.3 Gy radiation the ability of DNA to be rewound into supercoils is completely inhibited. However, the ability of DNA to rewind is 70% restored by 30 min after irradiation. Both thermotolerant and normal cells exposed to 45 degrees C for 30 min prior to irradiation had a rewinding ability intermediate between control and unheated cells, but there was no restoration of rewinding ability up to 3 h postirradiation. Thus, when irradiation immediately followed heating, there was no difference between thermotolerant and normal cells. However, when various time intervals were imposed between heating and irradiation, a difference in the ability of the cells to recover from heat-induced alterations became apparent. In normal cells after 6 h of postheat incubation the cells' ability to restore DNA supercoiling was approximately the same as that of control cells, while in thermotolerant cells only 2 h was required to repair the ability to restore supercoiling at the same rate. The rate of repair of DNA remained correlated with relative nuclear protein content as measured by fluorescein isothiocyanate staining in both thermotolerant and normal cells, indicating a possible relationship between the two.     

G2/M-phase arrest and death by apoptosis of HL60 cells irradiated with exponentially decreasing low-dose-rate gamma radiation.

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.