Evidence for induction of DNA double strand breaks in the bystander response to targeted soft X-rays in CHO cells

This study investigated the role of DNA double strand breaks and DNA base damage in radiation-induced bystander responses in Chinese hamster ovary (CHO) cell lines. Two CHO repair-deficient clones, xrs5 (DNA double strand break repair-deficient) and EM9 (DNA base excision repair-deficient) were used in addition to the wild type (CHO). The Gray Cancer Institute ultrasoft X-ray microprobe is a powerful tool for investigating the bystander response, because it permits the irradiation of only a single nucleus of a cell, as reported previously. In order to investigate the bystander effect in each repair-deficient cell line, we irradiated a single cell within a population and scored the formation of micronuclei. When a single nucleus in the population was targeted with 1 Gy, elevated numbers of micronuclei were induced in the neighbouring unirradiated cells in the EM9 and xrs5 cell lines, whereas induction was not observed in CHO. The induction of micronuclei in xrs5 was significantly higher than that in EM9. Under these conditions, the surviving fraction in the neighbouring cells was significantly lower in xrs5 than in the other cell lines, showing a higher cell killing effect in xrs5. To confirm that bystander factors secreted from irradiated cells caused these effects, we carried out medium transfer experiments using conventional X-irradiation. Medium conditioned for 24 h with irradiated cells was transferred to unirradiated cells and elevated induction of micronuclei was observed in xrs5. These results suggest that DNA double strand breaks rather than base damage are caused by factors secreted in the medium from irradiated cells.     

The time dependence of bystander responses induced by iron-ion radiation in normal human skin fibroblasts

Although bystander effects have been shown for some high-LET radiations, few studies have been done on bystander effects induced by heavy-ion radiation. In this study, using a Transwell insert co-culture system, we have demonstrated that irradiation with 1 GeV/nucleon iron ions can induce medium-mediated bystander effects in normal AG01522 human fibroblasts. When irradiated and unirradiated bystander cells were combined in shared medium immediately after irradiation, a two- to threefold increase in the percentage of bystander cells with gamma-H2AX foci occurred as early as 1 h after irradiation and lasted at least 24 h. There was a twofold increase in the formation of micronuclei in bystander cells when they were co-cultured with irradiated cells immediately or 1 or 3 h after irradiation, but there was no bystander effect when the cells were co-cultured 6 h or later after irradiation. In addition, bystander micronucleus formation was observed even when the bystander cells were co-cultured with irradiated cells for only 1 h. This indicates that the crucial signaling to bystander cells from irradiated cells occurs shortly after irradiation. Moreover, both gamma-H2AX focus formation and micronucleus formation in bystander cells were inhibited by the ROS scavengers SOD or catalase or the NO scavenger PTIO. This suggests that ROS and NO play important roles in the initiation of bystander effects. The results with iron ions were similar to those with X rays, suggesting that the bystander responses in this system are independent of LET.     

Effects of heavy ions and energetic protons on normal human fibroblasts

At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirradiated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.     

Effects of diethylstilboestrol-dipropionate on SCEs, micronuclei, cytotoxicity, aneuploidy and cell proliferation in maternal and foetal mouse cells treated in vivo

The effect of diethylstilboestrol-dipropionate on the frequency of SCEs and micronuclei, cytotoxicity, aneuploidy and cell proliferation rates of foetal liver and maternal bone marrow cells following exposure of pregnant mice was measured. An increase in the number of aneuploid and polyploid cells was observed in both tissues. There was no effect on micronuclei frequency, SCE frequency, or cell proliferation rate.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

Bystander effects in unicellular organisms

Radiation-induced bystander effects have been seen in mammalian cells from diverse origins. These effects can be transmitted through the medium to cells not present at the time of irradiation. We have developed an assay for detecting bystander effects in the unicellular eukaryote, the fission yeast Schizosaccharomyces pombe. This assay allows maximal exposure of unirradiated cells to cells that have received electron beam irradiation. S. pombe cells were irradiated with 16-18 MeV electrons from a pulsed electron LINAC. When survival of the irradiated cells decreased to approximately 50%, forward-mutation to 2-deoxy-d-glucose resistance increased in the unirradiated bystander cells. Further increase in dose had no additional effect on this increase. In order to detect this response, it was necessary for the irradiated cell/unirradiated cell ratio to be high. Other cellular stresses, such as heat treatment, UV irradiation, and bleomycin exposure, also caused a detectable response in untreated cells grown with the treated cells. We discuss evolutionary implications of these results.     

The bystander effect-induced formation of micronucleated cells is inhibited by antioxidants, but the parallel induction of apoptosis and loss of viability are not affected

X-rays induce various DNA damages including strand breaks that lead to formation of micronuclei and chromosomal aberrations as well as increased number of apoptotic cells. Similar effects appear when non-irradiated cells are treated with medium collected from cultures of irradiated cells (irradiation conditioned medium - ICM). This phenomenon was termed "bystander effect". A number of studies suggest that bystander effect appears to be associated with up-regulation of oxidative metabolism. We thus compared the effects of antioxidant Vitamins C and E on the frequency of micronuclei and apoptotic cells in both directly irradiated cell cultures and in cultures exposed to ICM. Addition of Vitamins C or E (1-40 microg/ml) to culture medium after exposure to radiation or ICM reduced the frequency of micronuclei in a concentration-dependent manner. These vitamins had no effect on cell viability, clonogenic survival or the frequency of apoptotic cells under both conditions tested. These results show that the bystander effect causes micronucleation in addition to other known effects and suggest that the factors causing micronucleation by X-irradiation, oxidative DNA damage and incomplete repair, are regulated by apoptosis-independent pathways.     

SURVIVAL OF AN UNIRRADIATED SALMONELLA TYPHIMURIUM MARKER STRAIN INOCULATED IN POULTRY FEEDS AFTER IRRADIATION

The present study was designed to compare unirradiated Salmonella typhimurium survival during storage after inoculation in either irradiated or unirradiated poultry feed. The effects of irradiation (5 kGy) on the indigenous feed microflora and on the survival of marker strain of S. typhimurium contaminated after irradiation treatment were determined during 56 days of storage of either soybean meal (SBM) or meat and bone meal (MBM) based feeds. The initial aerobic bacterial populations were reduced more than 90% in both SBM (4.96 to 4.08 ± 0.03 log₁₀ CPU/g feed) and MBM (5.12 to 3.90 ± 0.03) by irradiation. Irradiation treatment reduced the average fungal counts during 56 days of storage in both SBM (4.24 to 2.74 ± 0.03) and MBM (4.38 to 2.15 ± 0.03) containing feeds. However, unirradiated S. typhimurium populations inoculated after irradiation of the feed were not different in either irradiated or nonirradiated SBM and MBM based feeds. Therefore, the differences in fungal versus bacterial sensitivity among the feed types and storage times suggests that gamma irradiation can alter the makeup of indigenous microbial populations in feed but this does not appear to have a discernible influence on subsequent survival of unirradiated S. typhimurium added as a dry inoculum after irradiation.     

Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.     

The influence of gamma radiation and substrate on mycotoxin production by Fusarium culmorum IMI 309344

Mycotoxin production (deoxynivalenol (DON), acetyl deoxynivalenol (A DON) and zearalenone) by Fusarium culmorum inoculated on to maize (heat sterilized, irradiation sterilized and non-sterile) and irradiated to 1 kGy or 3 kGy, or unirradiated, was investigated over a period of time. Lowest mycotoxin production was observed on non-sterile maize which may be due to the presence of a competitive microflora on non-sterile maize. In general, mycotoxin production was higher on heat-sterilized grain as compared to irradiation-sterilized maize. It was suggested that this pattern of mycotoxin production was possibly caused by changes in the grain brought about by autoclaving, which favoured mycotoxin production and possibly induced changes in irradiation-sterilized maize which inhibited mycotoxin production. On sterile maize, there was no significant difference in DON production by unirradiated, 1 kGy and 3 kGy irradiated cultures up to 56 d of incubation; between days 56 and 77 of incubation, DON production increased rapidly with largest increases occurring in irradiated (1 kGy and 3 kGy) cultures. On non-sterile grain, neither DON nor A DON were detected in unirradiated cultures of F. culmorum but were detected in cultures irradiated to 1 kGy and 3 kGy. In practice grain should be stored under conditions of temperature and moisture content which prevent fungal growth. However, in this study, the grain was stored under conditions that were approaching ideal for growth of the test organism. The results highlight that irradiation disinfestation of grain must be combined with good grain handling practices so that excessive mycotoxin production can be prevented during storage.     

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.     

Bystander-mediated genomic instability after high LET radiation in murine primary haemopoietic stem cells

Communication between irradiated and unirradiated (bystander) cells can result in responses in unirradiated cells that are similar to responses in their irradiated counterparts. The purpose of the current experiment was to test the hypothesis that bystander responses will be similarly induced in primary murine stem cells under different cell culture conditions. The experimental systems used here, co-culture and media transfer, are similar in that they both restrict communication between irradiated and bystander cells to media borne factors, but are distinct in that with the media transfer technique, cells can only communicate after irradiation, and with co-culture, cells can communication before, during and after irradiation. In this set of parallel experiments, cell type, biological endpoint, and radiation quality and dose, were kept constant. In both experimental systems, clonogenic survival was significantly decreased in all groups, whether irradiated or bystander, suggesting a substantial contribution of bystander effects (BE) to cell killing. Genomic instability (GI) was induced under all radiation and bystander conditions in both experiments, including a situation where unirradiated cells were incubated with media that had been conditioned for 24h with irradiated cells. The appearance of delayed aberrations (genomic instability) 10-13 population doublings after irradiation was similar to the level of initial chromosomal damage, suggesting that the bystander factor is able to induce chromosomal alterations soon after irradiation. Whether these early alterations are related to those observed at later timepoints remains unknown. These results suggest that genomic instability may be significantly induced in a bystander cell population whether or not cells communicate during irradiation.     

Changes in micronucleus frequency resulting from preirradiation of cell culture surfaces

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.     

Effect of gamma irradiation on microbial load and sensory characteristics of aniseed (Pimpinella anisum)

Seeds of anise (Pimpinella anisum) were exposed to doses of 0, 5, 10, 15 and 20kGy in a (60)Co package irradiator. Irradiated and unirradiated samples were stored at room temperature. Microbial populations on seeds, total and inorganic soluble solids in water extract and sensory properties of the latter were evaluated after 0, 6 and 12 months of storage. Results indicated that gamma irradiation reduced the aerobic plate counts of aniseed. Immediately after irradiation, the total soluble solids in an extract of irradiated seeds were greater than those of unirradiated ones. The total soluble solids in an extract of irradiated and un-irradiated seeds increased after 6 and 12 months of storage. There were no significant differences (p>0.05) in inorganic soluble solids between the water extract of irradiated and unirradiated aniseeds. Sensory evaluation indicated that gamma irradiation improved sensory characteristics of aniseed water extract tested immediately after irradiation; however, after 12 months of storage, no significant differences (p>0.05) were found in color, taste or flavor between extract of irradiated and unirradiated seeds.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.     

Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells

Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICdR-resistant mutants did not have a growth advantage over wild-type virus in irradiated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis?) became more mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not lead to higher mutagenesis in unirradiated cells. Thus the data did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic.     

Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation.

The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.     

Dependence of the nucleoside effect on linear energy transfer.

L929 cells were irradiated by cyclotron-produced neutrons and by 14.8 MeV monoenergetic neutrons. For comparison cells were also irradiated by 60Co gamma rays. Following irradiation cells were treated by an equimolar solution of deoxyribonucleosides, and the effect on cell survival measured. Results show that nucleoside treatment was efficient after low-LET irradiation: gamma ray survival curves were altered by deoxyribonucleosides in terms of significantly increased extrapolation numbers only, but without Do change. Cells irradiated by neutrons from either of the two sources did not respond to nucleoside treatment, and consequently their survival curves remained unaltered. These results show that the nucleoside effect does occur after low-LET irradiation, but apparently not following high-LET irradiation. Since deoxyribonucleosides as well as other cell breakdown products are released in irradiated and necrotic tumours due to massive cell destruction, such a nucleoside effect could possibly enhance the cell survival and thus effect the result of radiotherapy. Absence of the nucleoside effect in case of high-LET irradiation may therefore be an additional potential gain from neutrons in radiotherapy.     

Potentiation of radiation lethality in HeLa cells by combined mild hyperthermia and chloroquine.

Evidence is presented for the interaction of X irradiation, slightly toxic levels of chloroquine, and mild hyperthermia in the inactivation of colony-forming ability in asynchronous HeLa cells. A three-way interaction was observed which resulted in the potentiation of radiation-induced lethality. There was little evidence of toxicity in unirradiated cells incubated for 3 h with 0.1 mM chloroquine at either 37 or 41 degrees C. The radiopotentiation factor, which is similar to the dose modification factor, was determined from dose-response curves by relating the reciprocal of the slope (D0) of the reference survival curve to that of the survival curve of cells receiving the combined postirradiation treatment with chloroquine and mild hyperthermia. Radiopotentiation factors larger than 1.7 were obtained irrespective of whether the reference D0's were obtained from survival curves for cells irradiated at 37 degrees C without drug or from cells receiving postirradiation treatment with heat or drug only.     

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.