X radiation causes a persistent induction of reactive oxygen species and a delayed reinduction of TP53 in normal human diploid fibroblasts

Multiple genetic changes are required for the development of a malignant cell. The frequency of such changes in cancer cells is higher than can be explained through random mutation, and it was proposed that a subpopulation of cells develop a persistent mutator phenotype. Evidence for such a phenotype has been observed in mammalian cells after treatment with ionizing radiation. The mechanism that promotes this effect has not been defined, but proposed explanations include increased levels of reactive oxygen species (ROS) in irradiated cells and their progeny. The tumor suppressor TP53 is of prime importance in coordinating the cellular response to damage, and it has been suggested to have a role in regulating the cellular redox state. We investigated the persistence of induced levels of ROS in normal diploid human cells for 1 month after X-ray exposure and the role of TP53 in this oxidant response. X radiation induced an oxidant response that persisted for 2 weeks after exposure in cells with normal TP53 function. ROS levels in cells with abrogated TP53 function were decreased in magnitude and duration. X radiation caused a primary transient induction of TP53 followed by a reinduction of TP53 5 days after irradiation. This reinduction persisted for at least 2 days and coincided with the largest induction of apoptosis. The persistently elevated levels of ROS and delayed reinduction of TP53 reported here are further evidence of the delayed effects of ionizing radiation and add to the growing number of such observations.     

Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt

The biological effects of low-dose radiation have been investigated and debated for more than a century, but its cellular effects and regulatory mechanisms remain poorly understood. This study shows the human cellular responses to low-dose radiation in CCD-18 Lu cells, which are derived from normal human lung fibroblasts. We examined a colony-forming assay for cell survival by ionizing radiation. Live cell counting and cell cycle analysis were measured for cell proliferation and cell cycle progression following low-dose irradiation. We examined Raf and Akt phosphorylation to determine the proliferation mechanism resulting from low-dose radiation. We also observed that p53 and p21 were related to cell cycle response. We found that 0.05 Gy of ionizing radiation enhanced cell proliferation and did not change the progression of the cell cycle. In addition, 0.05 Gy of ionizing radiation transiently activated Raf and Akt, but did not change phospho-p53, p53 and p21 in CCD-18 Lu cells. However, 2 Gy of ionizing radiation induced cell cycle arrest, phosphorylation of p53, and expression of p53 and p21. The phosphorylation of Raf and Akt proteins induced by 0.05 Gy of ionizing radiation was abolished by pre-treatment with an EGFR inhibitor, AG1478, or a PI3k inhibitor, LY294002. Cell proliferation stimulated by 0.05 Gy of ionizing radiation was blocked by the suppression of Raf and Akt phosphorylation with these inhibitors. These results suggest that 0.05 Gy of ionizing radiation stimulates cell proliferation through the transient activation of Raf and Akt in CCD-18 Lu cells.     

The current status of the adaptive response to ionizing radiation in mammalian cells

The results of numerous studies indicate that cells can become refractory to the detrimental effect of ionizing radiation when previously exposed to a low, “adapting dose”;. This phenomenon has been termed an “adaptive response”; to ionizing radiation. It has been postulated that the induced radioresistance is due to the induction of DNA repair systems which efficiently protect the adapted cells from the effects of a subsequent, high “challenging dose”;. However, a direct proof of this hypothesis is still lacking. The analyzed endpoints include chromosomal aberrations, survival, mutations, genetic instability and DNA damage repair measured by the comet assay. Frequently contradictory results were published by different authors. For example some authors observed a reduced frequency of apoptosis in adapted cells, whereas others reported the opposite. The source of variablity of the adaptive response in human lymphocytes remains unresolved. While there is no doubt that an adapting dose can trigger some protecting mechanisms within the cell it appears that there is no single, universal mechanism of the adaptive response that is valid for all cell types and irradiation conditions.     

Radiation sensitivity of fibroblast strains from patients with Usher's syndrome, Duchenne muscular dystrophy, and Huntington's disease

The colony-forming ability of 10 normal human fibroblast cell strains and of 10 strains representing 3 degenerative diseases of either nerve or muscle cells was determined after exposure of the cells to X-rays or beta-particles from tritiated water. Both methods of irradiation yielded similar comparative results. The fibroblast strains from the 5 Usher's syndrome patients and from 1 of the 2 Huntington's disease patients were hypersensitive to radiation, while those from the 3 Duchenne muscular dystrophy patients and the second Huntington's disease patient had normal sensitivity to radiation. These results indicate both disease-specific and strain-specific differences in the survival of fibroblasts after exposure to ionizing radiation.     

Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks, as a possible means for identifying individuals who are mildly hypersensitive to ionizing radiation, such as some ATM heterozygotes. We compared levels of gamma-H2AX foci after irradiation in cells from six apparently normal individuals as well as from individuals from two separate AT families including the proband, mother, father and three unaffected siblings in each family. After a 1-Gy single acute (high-dose-rate) gamma-ray dose delivered to noncycling contact-inhibited monolayers of cells, clear differences were seen between samples from normal individuals (ATM(+/+)) and probands (ATM(-/-)) at nearly all sampling times after irradiation, but no clear distinctions were seen for cells from normal compared to obligate heterozygotes (ATM(+/-)). In contrast, after 24 h of continuous irradiation at a dose rate of 10 cGy/h, appreciable differences in numbers of foci per cell were observed for cells from individuals for all the known ATM genotypes compared with controls. Four unaffected siblings had mean numbers of foci per cell similar to that for the obligate heterozygotes, whereas the other two had mean values similar to that for normal controls. We determined independently that those siblings with mean numbers of foci per cell in the range of ATM heterozygotes carried the mutant allele, while both siblings with a normal number of foci per cell after irradiation had normal alleles. A more limited set of experiments using lymphoblastoid cell strains in the low-dose-rate assay also revealed distinct differences for normal compared to ATM heterozygotes from the same families and opens the possibility of using peripheral blood lymphocytes as a more suitable material for an assay to detect mild hypersensitivities to radiation among individuals.     

Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.     

Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that may underlie radiation cataractogenesis, warranting further investigations.     

Fractionated X-radiation treatment can elicit an inducible-like radioprotective response that is not dependent on the intrinsic cellular X-radiation resistance/sensitivity

Inducible responses are well documented to play a role in the radiation response of cells. However, it is not known whether clinically relevant fractionated X-radiation treatment could elicit an inducible-like radioprotective response and whether there is a direct correlation between the inducible radiation response phenomenon and the intrinsic radiation response of the cell. Therefore, the purpose of this study was to determine whether closely related human colorectal tumor (HCT116) clones treated with fractionated X rays could elicit an inducible-like radiation response to a subsequent acute (i.e. single) X-ray challenge, and whether the magnitude of the inducible-like response correlates with the intrinsic X-ray resistance of the responding clones. After fractionated X irradiation, only the radiosensitive clone showed enhanced clonogenic survival with a subsequent acute X-ray exposure. Cell cycle changes or the selection of subclones with increased intrinsic radiation resistance induced by the fractionated X rays were excluded as the basis of this enhanced tolerance, suggesting the presence of an inducible-like radioprotective response. Using the comet assay, we found similar amounts of intrinsic DNA damage among the clones after acute X irradiation. Our findings demonstrate that fractionated X-ray treatment can elicit an inducible-like radioprotective response and represent the first evidence that this response is independent of the intrinsic radiation resistance/sensitivity of the responding cells.     

Differential effects of radical scavengers on X-ray-induced mutation and cytotoxicity in human cells

The cytotoxic and mutagenic effects of X irradiation on a human lymphoblast cell line were examined in the presence of two radioprotective agents which modulate damage to DNA. The cells were treated with X rays alone or in the presence of either dimethyl sulfoxide or cysteamine. Surviving fraction and mutation to trifluorothymidine resistance (tk locus) and to 6-thioguanine resistance (hgprt locus) were measured. Survival was enhanced when the cells were irradiated in the presence of dimethyl sulfoxide; the D0 rose from 58 to 107 rad. However, at both genetic loci the induced mutant fractions were identical in the presence or absence of dimethyl sulfoxide. Survival was enhanced to a greater degree when the cells were irradiated in the presence of cysteamine; the D0 rose from 58 to 200 rad. Cysteamine also protected the cells from X-ray-induced mutation; the frequencies of X-ray-induced mutation at both the tk and hgprt loci were reduced by 50-75%. No protective effects were observed unless dimethyl sulfoxide or cysteamine was present during irradiation. These findings are discussed in terms of the hypothesis that, unlike for cell killing, radiation-induced mutagenesis in human lymphoblast cells is not mediated by the actions of aqueous free radicals, but rather by the direct effects of ionizing radiation.     

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and gamma-rays. Association with a reduced ability to repair potentially lethal damage

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease characterized by mental and physical retardation, microcephaly, dwarfism, retinitis pigmentosa and a hypersensitivity to sunlight. Cells originating from patients also exhibit, in vitro, a hypersensitivity to UV radiation. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains (GM739, BOR, CS697, CS698 and KA) and two normal ones (HF19 and GP) to UV- and gamma-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to gamma-rays has been reported for 4 out of the 5 CS cell strains investigated. However, these CS cell strains are less sensitive to gamma-rays than are ataxia telangiectasia cells. The KA cell strain exhibits a normal response to gamma-irradiation. Repair of potentially lethal damage (PLD) after UV- and gamma-irradiation was investigated by using unfed plateau-cell cultures. Under these conditions, control cells show a great capacity to repair PLD (10- to 30-fold survival increase at 1% survival level). The two CS strains (GM739 and BOR), which are hypersensitive to both UV- and gamma-irradiation, exhibit no or only little PLD repair after treatment. In contrast, the normal response of KA cells to gamma-rays is associated with a normal PLD repair capability. This latter cell strain exhibits an intermediate sensitivity to UV and shows an intermediate PLD repair capacity. The response of CS cell strains after gamma-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in gamma-ray sensitivity are reported for cells within the same UV complementation group.     

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and γ-rays: Association with a reduced ability to repair potentially lethal damage

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease characterized by mental and physical retardation, microcephaly, dwarfism, retinitis pigmentosa and a hypersensitivity to sunlight. Cells originating from patients also exhibit, in vitro, a hypersensitivity to UV radiation.Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains (GM739, BOR, CS697, CS698 and KA) and two normal ones (HF19 and GP) to UV- and γ-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to γ-rays has been reported for 4 out of the 5 CS cell strains investigated. However, these CS cell strains are less sensitive to γ-rays than are ataxia telangiectasia cells. The KA cell strain exhibits a normal response to γ-irradiation.Repair of potentially lethal damage (PLD) after UV- and γ-irradiation was investigated by using unfed plateau-cell cultures. Under these conditions, control cells show a great capacity to repair PLD (10- to 30-fold survival increase at 1% survival level). The two CS strains (GM739 and BOR), which are hypersensitive to both UV- and γ-irradiation, exhibit no or only little PLD repair after treatment. In contrast, the normal response of KA cells to γ-rays is associated with a normal PLD repair capability. This latter cell strain exhibits an intermediate sensitivity to UV and shows an intermediate PLD repair capacity.The response of CS cell strains after γ-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in γ-ray sensitivity are reported for cells within the same UV complementation group.     

In vitro radiosensitivity of human diploid fibroblasts derived from women with unusually sensitive clinical responses to definitive radiation therapy for breast cancer

Between January 1985 and December 1986, 811 patients were treated for carcinoma of the breast at the Joint Center for Radiation Therapy by an identical protocol. Of these 811 patients, five patients (0.6%) were identified as having an unusually sensitive clinical response to routine external beam irradiation. This unusual clinical response was characterized by severe skin erythema and edema during the first few weeks of treatment, requiring treatment breaks. Skin fibroblast cell strains were established from these five women as well as from six women with a normal clinical response to breast irradiation (chosen at random from the population of 811 patients). Radiation survival parameters were determined by a colony formation assay from complete survival curves in coded and blinded samples. Cells from the sensitive patients were significantly more sensitive to the cytotoxic effects of radiation in vitro as determined by the parameters D0, D, D10, and n, than were the strains derived from patients with a normal response. We conclude that an unusually severe response to standard fractionated radiotherapy may be associated with greater intrinsic radiation sensitivity of the individual's somatic cells.     

Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.     

Radiation sensitivity of human and murine peripheral blood lymphocytes,stem and progenitor cells

Immunodeficiency is a severe side effect of radiation therapy, notably at high radiation doses. It may also impact healthy individuals exposed to environmental ionizing radiation. Although it is believed to result from cytotoxicity of bone marrow cells and of immunocompetent cells in the peripheral blood, the response of distinct bone marrow and blood cell subpopulations following exposure to ionizing radiation is not yet fully explored. In this review, we aim to compile the knowledge on radiation sensitivity of immunocompetent cells and to summarize data from bone marrow and peripheral blood cells derived from mouse and human origin. In addition, we address the radiation response of blood stem and progenitor cells. The data indicate that stem cells, T helper cells, cytotoxic T cells, monocytes, neutrophils and, at a high degree, B cells display a radiation sensitive phenotype while regulatory T cells, macrophages, dendritic cells and natural killer cells appear to be more radioresistant. No conclusive data are available for basophil and eosinophil granulocytes. Erythrocytes and thrombocytes, but not their precursors, seem to be highly radioresistant. Overall, the data indicate considerable differences in radiosensitivity of bone marrow and blood normal and malignant cell populations, which are discussed in the light of differential radiation responses resulting in hematotoxicity and related clinical implications.     

Ionizing radiation-induced mutant frequencies increase transiently in male germ cells of older mice

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.     

X-ray and UV-radiation sensitivity of circulating lymphocytes in multiple epidermal cancer in relation to previous radiation exposure

The cellular sensitivity to X rays (200 kV, 16 mA) and UV radiation (254 nm) was examined in lymphocytes from three groups of patients with multiple epidermal malignant tumors, selected by their clinical history of carcinogenesis. Eight patients previously exposed to low energy ionizing radiation (less than or equal to 12 kV) had an increased cellular sensitivity to UV radiation as well as X rays compared with 24 age and sex matched controls. This indicates the existence of a cellular cross-sensitivity to UV radiation and ionizing radiation not previously established for human cells. In contrast six patients previously exposed to high energy ionizing radiation (between 25 and 170 kV) had normal cellular response to both UV radiation and X rays, indicating a different biologic effect of low and high energy ionizing radiation. In the third group of patients, previously exposed to therapeutic UV radiation/excess sunlight, the lymphocytes had a normal response to X rays, but an increased sensitivity to UV radiation. The possibility of evaluating the individual risk at radiation exposure is suggested.     

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.     

γH2AX in Bystander Cells: Not Just a Radiation-Triggered Event,a Cellular Response to Stress Mediated by Intercellular Communication

The recent years have witnessed a rapid accumulation of experimental data showing that ionizing radiation elicits a plethora of biological effects in unirradiated cells receiving bystander signals from hit cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. Our group and others found that DNA double-stranded breaks (DSBs), directly measured by the γ-H2AX focus formation assay, accumulate in bystander cells in a number of experimental systems such as human cultured cells, human 3-dimensional tissue models and in mice. In addition, we recently found that various other sources of cell stress, including media from cancerous cells resulted in a DNA damage response (DDR) in normal human cells that is reminiscent of RIBE. These results suggest that the RIBE may be part of a more general stress response, however, the molecular mechanism underpinning the formation of DNA DSBs in bystander cells is still unclear. This extra view points to some possibilities that might explain why DDR in human cells can be observed under bystander conditions.     

"Modeled microgravity" affects cell response to ionizing radiation and increases genomic damage

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.     

Detection of adaptive response at chromosome level.

We have just started the basic study to detect the genetic alterations at chromosome level as a result of radioadaptive response. The assay system is based upon the analysis of loss of heterozygosity (LOH) induced in human lymphoblastoid cell TK6, which were pre-irradiated with low-doses of ionizing radiation (IR) before the challenging irradiation. In our previous study, this analysis was shown to be very sensitive to IR because the radiation-specific hemizygous LOHs (interstitial deletions) were observed after 10 cGy of IR (X-rays or accelerated carbon-ion beam). Here, we would like to introduce our plan how to detect the changes in such radiation-specific LOH patterns by the pre-irradiation of TK6. If we succeed the detection, the radioadaptation assay system can be used for elucidating the biological effects of low-doses of space ionizing radiation. In addition, we are also considering the modification of assay system by introducing the site-specific chromosome breakage (DNA double-strand break) instead of challenging IR. Furthermore, the preliminary results of the experiments using frozen TK6 cells for the preparation of ISS experiments.