Gy Exposure Research Articles

INFLAMMATORY CELL RECRUITMENT FOLLOWING THORACIC IRRADIATION

Ionizing radiation leads to a progressive injury in which a monocyte/macrophage-rich pneumonitis is followed by a chronic progressive fibrosis. In the present study, the role of macrophage/monocyte recruitment in the genesis of radiation-induced pulmonary fibrosis was examined. The objectives were threefold: (i) characterize the inflammatory cells recruited into the lung during the development of radiation-induced fibrosis; (ii) investigate changes in lung response following depletion of resident alveolar macrophages in vivo prior to radiation treatment; (iii) assess if inhalation of low levels of endotoxin would potentiate the radiation-initiated injury. One group of fibrosis-sensitive C57BL/6 mice was irradiated with a single dose of 15 Gy to the thorax. In a second group, resident inflammatory cells were depleted using clodronate, encapsulated into liposomes, 48 hours prior to irradiation with a single dose of 15 Gy to the thorax. Control animals were sham irradiated. All groups of animals then were examined 8, 16, or 24 weeks post irradiation. No difference in total cell numbers or cell differentials was observed between irradiated mice or those that were both liposome treated and irradiated at any time point. At 16 weeks, mice that received radiation showed a 5- to 6-fold increase in lymphocytes regardless of treatment as compared to control animals. At 24 weeks post irradiation, select groups were exposed to lipopolysaccharide (LPS) and examined 24 hours post inhalation. Lavageable protein was increased several fold in mice that received both radiation and LPS exposure as compared to 15 Gy or LPS exposure alone. These results demonstrate: (i) macrophages and lymphocytes are the predominately recruited cell types through 24 weeks post irradiation; (ii) recovery of inflammatory cells, regardless of prior macrophage depletion, were similar, suggesting that early responses are primarily driven by parenchymal cell injury; (iii) thoracic irradiation-induced injury can cause sensitization to a secondary stimulus that may result in injuries/responses not predicted by evaluating exposures individually.

Ginseng reduces the micronuclei yield in lymphocytes after irradiation

To assess the effect of Chinese ginseng in modifying the radiation-induced micronuclei (MN) yield in human G o peripheral blood lymphocytes (PBL), we conducted the cytokinesis-blocked (CB) MN assay in blood samples obtained from healthy volunteers ( n=4). Before 137Cs ex vivo irradiation, mononuclear cell cultures from each sample were incubated 24 h with different concentrations (0–2000 μg ml −1) of crude water extract of ginseng dry root. We found that (1) at 0 Gy and without the presence of ginseng, MN yield (mean±S.E.M.) was 11.7±2.7 per 1000 binucleated (BN) cells. Different concentrations of ginseng crude water extract did not affect the MN yields and the proliferative activity of PBL; (2) after 1 and 2 Gy exposure, radiation alone sharply increased the MN yields, respectively, to 119.6±17.4 and 340.5±20.9 per 1000 BN cells. However, treatment with ginseng for 24 h before radiation exposure, resulted in a significant linear decline of MN yields as ginseng concentration increases. Compared to radiation alone, the extent to which ginseng water extract reduced the MN yields induced by 1 Gy exposure was 46.0% at 1500 μg ml −1 and 61.5% at 2000 μg ml −1, and with 2 Gy exposure, it was 38.6% at 1500 μg ml −1 and 46.5% at 2000 μg ml −1; (3) MN data suggested a tendency for overdispersion relative to the Poisson model; and (4) over the different levels of ginseng concentrations, the trend in micronucleated BN index was as similar as that of the MN yields. These results indicated that (1) ginseng crude water extract exerts no apparent cytogentic effect on human PBL at concentrations up to 2000 μg ml −1 as evaluated by the CBMN assay; and (2) the protection of ginseng water extract against 137Cs-induced MN in human PBL is concentration-dependence. Therefore, our findings indicated that ginseng may have therapeutic value as a possible radioprotector for normal tissue during radiotherapy of cancer patients.

Use of the Leksell Gamma Knife for localized small field lens irradiation in rodents.

For most basic radiobiological research applications involving irradiation of small animals, it is difficult to achieve the same high precision dose distribution realized with human radiotherapy. The precision for irradiations performed with standard radiotherapy equipment is +/-2 mm in each dimension, and is adequate for most human treatment applications. For small animals such as rodents, whose organs and tissue structures may be an order of magnitude smaller than those of humans, the corresponding precision required is closer to +/-0.2 mm, if comparisons or extrapolations are to be made to human data. The Leksell Gamma Knife is a high precision radiosurgery irradiator, with precision in each dimension not exceeding 0.5 mm, and overall precision of 0.7 mm. It has recently been utilized to treat ocular melanoma and induce targeted lesions in the brains of small animals. This paper describes the dosimetry and a technique for performing irradiation of a single rat eye and lens with the Gamma Knife while allowing the contralateral eye and lens of the same rat to serve as the "control". The dosimetry was performed with a phantom in vitro utilizing a pinpoint ion chamber and thermoluminescent dosimeters, and verified by Monte Carlo simulations. We found that the contralateral eye received less than 5% of the administered dose for a 15 Gy exposure to the targeted eye. In addition, after 15 Gy irradiation 15 out of 16 animals developed cataracts in the irradiated target eyes, while 0 out of 16 contralateral eyes developed cataracts over a 6-month period of observation. Experiments at 5 and 10 Gy also confirmed the lack of cataractogenesis in the contralateral eye. Our results validate the use of the Gamma Knife for cataract studies in rodents, and confirmed the precision and utility of the instrument as a small animal irradiator for translational radiobiology experiments.

Indoor light on thermoluminescence of CVD diamond film used as a high-energy photon dosimeter

The effect of light on polycrystalline diamond film that was produced by chemical vapor deposition and is used as a thermoluminescent dosimeter should be considered, although some researchers have indicated that such an effect was theoretically unlikely to happen. A 15 min exposure to a normal desk light bulb induces significant thermoluminescence (TL) comparable to a 0.5 Gy exposure to high-energy photons. This light-induced TL will be saturated within 2 h. The saturated TL intensity depends on the frequency of the light and the blue light dominates. The TL peak area at a temperature of 605 K is insensitive to light but is sensitive to high-energy photons. Another peak at about 410 K is caused by light only, because the TL from the ionization radiation at the same location is bleached. The effect of light could be easily distinguished by a numerical or an experimental method. Lamps with a green lampshade or pure red lights are suggested for use as indoor light sources. To reduce the effect of light, pre-heating treatment before readout is also suggested.

Combined modality therapy of NCI-H460 human lung cancer using epidermal growth factor receptor inhibitor, OSI-774, and ionizing radiation in vitro and in vivo

quantitatively inhibits the EGFand radiation-induced activation of the EGFR and its downstream target MAPK, an important mediator of the cytoprotective, pro-proliferative radiation response. Using EGF and HRG, peptide growth factors that interact with either ERBB1 or ERBB3/ERBB4, respectively, we have extended our findings by determining the inhibitory effect of EGFR-CD533 on activation of the entire ERBB RTK network. We demonstrate that EGFR-CD533 not only inhibited the EGF-induced activation of ERBB1 and ERBB2 in MDA-MB-231 and T47D cells, but also completely blocked the HRGdirected stimulation of ERBB2 and ERBB4 in T47D cells. This inhibitory effect is not limited to the receptor level but also leads to MAPK inhibition and cellular radiosensitization. The broad spectrum of inhibitory interactions of the EGFR-CD533 with ERBB receptors is further supported by results from experiments in U-373 cells with EGFRvIII, which is irresponsive to EGF but elicits a 3to 5-times greater response to radiation after a single 2 Gy exposure. Expression of this mutant EGFR confers radioresistance, however, the concomitant expression of both EGFRvIII and EGFR-CD533 eliminates radiation-induced activation of both wild-type EGFR and EGFRvIII (which does not typically engage in dimerizations), completely disrupts the EGFR/MAPK cytoprotective signaling cascade, and thus leads to radiosensitization in vitro and in vivo.

Cellular Responses to Low Dose Heavy-ion Exposure in Human Cell

The human lymphoblastoid cell line TK6 was used to study the cellular responses after low-dose (100, 200, 500 mGy) or high-dose (3 Gy) of X rays, C (22 keV.micron-1) and Fe (1000 keV.micron-1) ion exposures. p53 protein induction in individual cells was determined by indirect immunofluorescence staining. Cell-cycle progression after heavy-ion exposure was determined by using a laser scanning cytometer. A characteristic pattern of cell-cycle progression was observed with 3 Gy exposure of Fe ions but not with 100 mGy. Similarly such a pattern with 100 mGy C ion exposure did not match that with 3 Gy. The proportion of p53-induced cells is proportional to the probability of cell being hit by a primary heavy ion. The observed low-dose effect can be reflected in the probability of a hit, although detailed nature about their energy deposition must be considered for more precise estimation of such an effect. New detection methodology must be developed for identification of heavy-ion specific cellular responses.

Study of the gonocyte cell cycle in irradiated TP53 knockout mouse foetuses and newborns

Purpose : To investigate the role of p53 in gonocyte cell-cycle arrest in rodents with or without radiation exposure. Materials and methods : Pregnant p53 (+/-) mice, mated with p53 (-/-) males, were exposed to 137 Cs γ-rays at day 18 postcoitum (p.c.) at doses ranging from 1.5 Gy to 3 Gy. The gonocyte cell cycle was studied in p53 (-/-) male foetuses and newborns after BrdU incorporation and DAPI staining, and compared with those of p53 wild-type animals. Results : The proliferation of gonocytes in wild-type mice is normally arrested between day 16 p.c. and birth, a period when p53 is strongly expressed in gonocytes; p53 high expression is prolonged in all gonocytes after 3 Gy irradiation. In p53 (-/-) mice, this period of gonocyte cell-cycle arrest is not modified, compared with wild-type mice. It is also prolonged after a 3 Gy exposure. Conclusion : Two hypotheses are proposed. Either p53 is not involved in the control of gonocyte cell-cycle arrest in control and irradiated mice, or its role is redundant in this process.

Dopaminergic neurons are protected from ionizing irradiation and 6-hydroxydopamine-induced apoptosis by overexpression of the Manganese Superoxide Dismutase (MnSOD) transgene

Dopaminergic neurons are protected from ionizing irradiation and 6-hydroxydopamine-induced apoptosis by overexpression of the Manganese Superoxide Dismutase (MnSOD) transgene

Induction of minisatellite mutations in the mouse germline by low-dose chronic exposure to γ-radiation and fission neutrons

Germline mutation induction at mouse minisatellite loci by paternal low-dose (0.125–1 Gy) exposure to chronic (1.66×10 −4 Gy min −1) low-linear energy transfer (low-LET) γ-irradiation and high-LET fission neutrons (0.003 Gy min −1) was studied at pre-meiotic stages of spermatogenesis. Both types of radiation produced linear dose–response curves for mutation of the paternal allele. In contrast to previous results using higher doses, the pattern of induction of minisatellite mutation after chronic γ-irradiation was similar to acute (0.5 Gy min −1) exposure to X-rays, indicating that the elevated mutation rate was independent of the ability of the cell to repair damage induced immediately or over a period of up to100 h. Chronic exposure to fission neutrons was more effective than acute or chronic low-LET exposure (relative biological effectiveness, RBE=3.36). The data also provide strong support for the previous conclusion that increases in minisatellite mutation rate are not caused by radiation-induced DNA damage at minisatellite loci themselves, but rather from damage induced by ionising radiation elsewhere in the genome/cell.

Application of Individually Calibrated Solid LiF,Ti (MTS-N) Detectors in Clinical Dosimetry

The applicability of individually calibrated solid LiF:Mg,Ti detectors (MTS-N, Institute of Nuclear Physics, Krakow, Poland) in clinical dosimetry was investigated. By attending to repeatability and stability of annealing and readout parameters, it was possible to select from a group of about 100 MTS detectors calibrated in external 60 Co or 9 MV photon beams a subgroup of at least 70% of these detectors whose individual response factors (IRFs) did not vary by more than 2.5% (SD) from the mean value over six cycles of annealing 1 Gy exposure and readout. A 3-step readout of detectors was found to be at least as accurate and more convenient than linear readout. MTS-N detectors were applied in in vivo dosemeter assemblies and in intercalibration measurements of 60 Co beams.

Teratogen update: radiation and Chernobyl.

The 1986 nuclear reactor accident at Chernobyl caused nonuniform radiocontamination of air and land, primarily within regions of the former Soviet Union and Western Europe. Major exposure groups included the reactor workers, villagers evacuated from within 30 km of the accident, the "liquidators" who decontaminated the evacuation zone afterward, those in radiocontaminated villages not evacuated, and "others" not in the latter categories. The possibility of being exposed to radiation caused considerable anxiety, especially among pregnant women. Were teratogenic levels of radiation (> or = 0.1 Gy) exposure attained? To date there is no consistent proof that this level of radiation exposure was received. Nevertheless, thousands of induced abortions were performed. Radioiodine (I-131) caused thyroid cancer in young children in portions of Belarus, the Ukraine, and Russia. It is not known but very possible that I-131 fetal thyroid exposure contributed to this observation. The relationship between mental retardation and radiation exposure has not been confirmed. Leukemia and other cancers, while predicted for the liquidators (mainly males), has not been found in the other exposure groups at this time. Investigations of aborted fetuses and newborns in Belarus showed an increase in the frequency of both congenital and fetal abnormalities in high and low Cs-137 contaminated regions. This study is unreliable due to detection and selection biases. Accident and environmental factors unrelated to radiation doses may have contributed to these observations. Occasional positive teratogenic studies in less contaminated regions of Western Europe are suspect because of the low radiation doses received. There is no substantive proof regarding radiation-induced teratogenic effects from the Chernobyl accident.

Effect of neutron-gamma radiation on dopamine and serotonin metabolism in the rat brain: a regional analysis.

The concentrations of dopamine (DA) and its metabolites and the levels of 5-hydroxyindoleacetic acid (5-HIAA), the metabolite of serotonin, were determined in discrete cerebral areas of rats 3 hr after (neutron-gamma) irradiation at 4 and 7 Gy. After the 7 Gy irradiation, no significant effect was observed. After the 4 Gy exposure, the most marked difference between irradiated and control rats was in the levels of DA and its metabolites in the striatum. We observed a decrease of DA, HVA, and DOPAC levels in the striatum and an opposite pattern in the substantia nigra. Whatever the brain area observed, an increase of 5-HIAA levels was noted.

Characterization of the response of human bone marrow endothelial cells to in vitro irradiation.

Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation-induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2. 5 or 10Gy. Our results showed a time- and a dose-dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM-1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM-1 on their surface than non-irradiated cells, whereas no change in VCAM-1, E-selectin and PECAM-1 expression was observed. An increased production of G-CSF, GM-CSF, IL-8, IL-6, IL-1alpha, IL-11, MIP-1alpha and SCF and no production of LIF, TNF-alpha, TPO and IL-3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells.

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells.

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21(Cip-1/WAF1) via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the specific MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased approximately sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24-48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4-8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizing radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro.

Ionizing Radiation Activates c-Jun NH2-Terminal Kinase (JNK/SAPK) via a PKC-Dependent Pathway in Human Thyroid Cells

Thyroid gland is known to be higher sensitive to carcinogenic effects of external ionizing radiation (IR) than other tissues. To clarify the cell-specific response following irradiation, activations of c-Jun NH2-terminal kinases (JNKs), which is one of mitogen-activated protein kinases (MAPKs) family members, and extracellular signal-regulated kinase (ERK) were examined in primary cultured human thyroid cells in comparison with human diploid fibroblast cells, WI-38. Although UV exposure strikingly induced JNK activity in both cells, the dose-response increase following IR exposure was observed in thyroid cells with the maximal JNK activity (3.5 fold induction) obtained at 10 Gy exposure, but no increase in WI-38 cells. The JNK activity was reached a maximum of 2.2 fold induction at 30 min after 5 Gy exposure and then sustained for at least 12 hr. On the other hand, ERK activity was not stimulated in thyroid cells following irradiation. The effects of 12-O-tetradecanoylphorbol β-acetate (TPA) mimicked those of radiation on JNK cascade and 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine 2HCl (H7) and pretreatment with TPA blocked JNK activation following irradiation. Our results demonstrate that IR stimulates JNK activity in cultured human thyroid cells but not in fibroblasts indicating distinct activation and regulation mechanisms of JNK cascade. The JNK activation following IR exposure is mediated at least partially through a PKC-dependent pathway.

Continued expression of a tissue specific activated oncogene in the early steps of radiation-induced human thyroid carcinogenesis.

Ionizing radiation is a well-known risk factor of cancer development, but the mechanism of radiation induced carcinogenesis is not clear. Chromosomal rearrangements induced by radiation most likely are one of the principal genetic alterations resulting in malignant transformation. The chimeric BCR-ABL associated with chronic myelogenous leukemia (CML) and H4-RET oncogenes associated with thyroid papillary carcinoma are the result of a translocation and inversion, respectively. In vitro studies showed these genes were induced by high-doses of X-irradiation in cell lines. Studies also show that therapeutic external X-ray doses as high as 60 Gy for treatment of various childhood cancers including Hodgkin's disease significantly increase the risk of thyroid cancer. Therefore, we examined the induction and persistence of these chimeric genes in human thyroid tissues transplanted in scid mice after 50 Gy exposure as a function of time for 2 months to elucidate the early events of thyroid carcinogenesis. The H4-RET genes were detected on day 2 and throughout the 2 month period. On the other hand, BCR-ABL genes were detected on day 2 and were undetectable subsequently. These results suggest that ionizing radiation causes various oncogene activations, but cells with only specific gene alteration uniquely associated with thyroid carcinogenesis are selectively retained demonstrating one of the early events in the beginnings of radiation carcinogenesis in human thyroid tissues.

Induction of multiple PCR-SSCPE mobility shifts in exons in cultures of normal human urothelium exposed to low-dose gamma radiation

We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma -radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma -radiation from a 60Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene postirradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53 -related genomic instability.

Annexin I in fibrotic rat lung and cultured lung fibroblasts following irradiation

. Radiation-induced lung fibrosis is a result of collagen accumulation in the interstitium, partly due to increased collagen synthesis by fibroblasts. One feature of active collagen synthesis is increased membrane trafficking in the fibroblasts. A group of proteins called annexins is believed to play a regulatory role in membrane fusion and exocytosis. Therefore, increased annexin activity might be expected in the fibrotic lung. We tested this hypothesis by measuring annexin I levels, hydroxyproline content and ultrastructural changes in radiation-induced pulmonary fibrosis in rat. Three months after a single exposure to 30 Gy of X-rays to the right hemithorax, the right lung of the rat was atrophied and fibrotic with a concomitant increase in size of the shielded left lung. Electron micrographs revealed that the irradiated lung was ladened with interstitial collagen fibrils, with increased number of fibroblasts amongst them. Hydroxyproline concentration in the irradiated lung was nearly twice that in the sham-irradiated lung. Annexin I in the irradiated lung, on the other hand, was markedly reduced, and barely detectable on immunoblots. Since increased annexin I might precede enhanced collagen production, we also measured annexin I levels in rat lungs 3 days after 30 Gy irradiation and correlated that with hydroxyproline concentration. We found no appreciable difference in annexin I levels and hydroxyproline content between sham-irradiated and irradiated lungs at 3 days. To determine whether annexin I levels in cultured fibroblasts were altered by irradiation, we assayed annexin I in cultured rat lung fibroblasts 3 days after 0-10 Gy exposure, with concomitant measurement of 14C-proline incorporation. The annexin I level in fibroblasts irradiated with 10 Gy X-rays was 55% higher than in shamirradiated fibroblasts. However, incorporation of 14C-proline into collagenase-sensitive macromolecules in the culture medium and extracellular matrix was not different between these two groups of cells. These data demonstrate a radiation-induced increase in immunoreactive annexin I in cultured lung fibroblasts, but fail to support the hypothesis of a positive correlation between annexin I concentration and fibrosis in irradiated rat lung.

Late and persistent up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression by ionizing radiation in human endothelial cells in vitro

. Adhesion molecules play a key role in cellular traffic through vascular endothelium, in particular during the inflammatory response when leukocytes migrate from blood into tissues. Since inflammation is one of the major consequences of radiation injury, we investigated the effect of ionizing radiation on cellsurface expression of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in cultured human umbilical vein endothelial cells (HUVEC). Flow cytometry performed on irradiated HUVEC revealed both a time- (from 2 to 10 days) and dose- (from 2 to 10 Gy) dependent up-regulation of basal expression of ICAM-1, and no induction of VCAM-1 or E-selectin. The radiationinduced increase in ICAM-1 expression on HUVEC was correlated with augmented adhesion of neutrophils on irradiated endothelial cells. Interleukin-6 (Il-6) or other soluble factors released by irradiation were not involved in the enhanced ICAM-1 expression by irradiation. Northern blot analysis showed an overexpression of ICAM-1 mRNA from 1 to 6 days after a 10 Gy exposure. Our data suggest that ICAM-1 participates in the radiation-induced inflammatory reaction of the endothelium.

Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells

In the present study we investigated the effect of gamma-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = O.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G2M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G2-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B1 expression showed that, following 2 and 5 Gy, the fraction of cyclin B1-expressing cells was superimposable upon that of G2M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B1-expressing cells was half the G2M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B1 protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B1 expression as a possible cause for the persistent G2 block in irradiated M14 cells.