Relationship between radiation-induced low-dose hypersensitivity and the bystander effect

Recent advances in our knowledge of the biological effects of low doses of ionizing radiation have shown two unexpected phenomena: a "bystander effect" that can be demonstrated at low doses as a transferable factor(s) causing radiobiological effects in unexposed cells, and low-dose hyper-radiosensitivity and increased radioresistance that can be demonstrated collectively as a change in the dose-effect relationship, occurring around 0.5-1 Gy of low-LET radiation. In both cases, the effect of very low doses is greater than would be predicted by conventional DNA strand break/repair-based radiobiology. This paper addresses the question of whether the two phenomena have similar or exclusive mechanisms. Cells of 13 cell lines were tested using established protocols for expression of both hyper-radiosensitivity/increased radioresistance and a bystander response. Both were measured using clonogenicity as an end point. The results showed considerable variation in the expression of both phenomena and suggested that cell lines with a large bystander effect do not show hyper-radiosensitivity. The reverse was also true. This inverse relationship was not clearly related to the TP53 status or malignancy of the cell line. There was an indication that cell lines that have a radiation dose-response curve with a wide shoulder show hyper-radiosensitivity/increased radioresistance and no bystander effect. The results may suggest new approaches to understanding the factors that control cell death or the sectoring of survival at low radiation doses.     

Combined effect of heptaplatin and ionizing radiation on human squamous carcinoma cell lines

Heptaplatin, cis-malonato [(4R,5R)-4,5-bis (amino-methyl)-2-isopropyl-1,3-dioxolane] platinum(II) (SKI-2053R, Sunpla) is a new platinum derivative with anti-tumor activity comparable to cisplatin on various cancer cell lines. Preclinical studies suggest that it is less nephrotoxic than cisplatin. This study was undertaken to examine the combined effect of heptaplatin and ionizing radiation on two established human squamous carcinoma cell lines (NCI-H520, SQ20B). The cytotoxic activity of heptaplatin was concentration-dependent in both cell lines. When low dose heptaplatin was combined with high dose ionizing radiation, there was an additive cytotoxic effect on NCI-H520 cells (P < 0.05), while a moderate dose of heptaplatin and a low dose of ionizing radiation had an additive cytotoxic effect on the growth of SQ20B cells (P < 0.05). FACS analysis and DAPI staining showed that their additive cytotoxic effects were correlated with the induction of apoptosis. Further studies are warranted using heptaplatin and ionizing radiation in squamous cell carcinoma as a substitute for cisplatin.     

Increased chromosomal radiosensitivity of a Chinese hamster ovary cell line that inducibly expresses the Eco RI restriction endonuclease

We have transfected a Chinese hamster ovary cell line (CHO 6) with a plasmid that inducibly expresses the Eco RI restriction endonuclease gene in the presence of cadmium sulfate (CdSO4). Expression of Eco RI results in DNA double-strand breaks, which can lead to chromosome aberrations. The new line, designated CHO 10, also has a low level of constitutive expression of Eco RI in the absence of CdSO4 without any cytogenetic effect. This suggested that these cells may be efficient at repairing low levels of DNA double-strand breaks. To test this, both cell lines were exposed to ionizing radiation, and aberration yields were analyzed with or without induction of Eco RI. CHO 10 cells showed increased radiosensitivity after G1 irradiation, but after G2 exposure, only doses greater than or equal to 0.4 Gy caused more damage in CHO 10 cells. We conclude that CHO 10 cells can tolerate constitutive expression of Eco RI, but that when the cells are subjected to additional stress, in this case ionizing radiation, they become very sensitive to DNA double-strand breaks.     

Role of apoptosis in low-dose hyper-radiosensitivity

Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557-566, 2004). We also show that enriching populations of MR4 and V79 cells with G(1)-phase cells, to minimize the numbers of G(2)-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G(2)-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G(2)-phase cells harboring unrepaired DNA breaks entering mitosis.     

Tenascin Expression in Vitro and in Vivo: Comparison between Epithelial and Nonepithelial Rat Cell Lines

Tenascin is a novel six-armed extracellular-matrix glycoprotein expressed in association with mesenchymal-epithelial interactions, and its expression is temporally and spatially restricted during organogenesis and carcinogenesis. The distribution and alterations in the expression of fibronectin, laminin, and especially of tenascin, were compared between in vitro and in vivo studies with rat epithelial (hepatocyte-derived) and nonepithelial (sarcoma-derived) cell lines. Immunoprecipitation studies revealed that the production of extracellular-matrix glycoproteins varied among the cell lines. Two ascites-hepatoma-derived cell lines and one sarcoma-derived line were found to synthesize tenascin in vitro. Their major tenascin isoform yielded a molecular weight of 220 kDa under reducing conditions. The other cell lines examined, including all of those derived from normal hepatocytes, were negative for the expression of tenascin. Coculture studies were performed between epithelial and nonepithelial cell lines. No drastic change in tenascin expression was found after coculturing the cells. As an in vivo study, cell lines were transplanted into nude mice. All xenografts of the epithelial lines were associated with a strong positive reaction for extracellular-matrix glycoproteins, and especially for tenasein, in the mouse fibrous stroma adjacent to them. This represents the epithelial induction of stromal tenascin. Whether or not they produced tenascin in vitro, after transplantation none of the epithelial cell lines themselves produced tenascin, whereas both of the nonepithelial cell lines prominently produced tenascin. These findings suggest that, in the process of interactions between epithelial and nonepithelial cells, the expression of tenascin depends on the switch from in vitro to in vivo.     

Cell-density dependent effects of low-dose ionizing radiation on E. coli cells

The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.     

Low-dose radiation hypersensitivity is associated with p53-dependent apoptosis

Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses <50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times postirradiation, we examined the response of human A549 lung carcinoma, T98G glioma, and MCF7 breast carcinoma cell lines exposed to gamma radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (Annexin V binding). We observed that caspase-3 activation and Annexin V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and Annexin V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no hyperradiosensitivity and apoptosis was not detectable by Annexin V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.     

Broad modulation of gene expression in CD4+ lymphocyte subpopulations in response to low doses of ionizing radiation

Gruel, G., Voisin, P., Vaurijoux, A., Roch-Lefèvre, S., Gré goire, E., Maltère, P., Petat, C., Gidrol, X., Voisin, P. and Roy, L. Broad Modulation of Gene Expression in CD4(+) Lymphocyte Subpopulations in Response to Low Doses of Ionizing Radiation. Radiat. Res. 170, 335-344 (2008).To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56(+), CD4(+) and CD8(+) cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4(+) cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4(+) cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4(+) cells could help to understand the mechanisms involved in low-dose response and allow their detection.     

p53-independent downregulation of histone gene expression in human cell lines by high- and low-let radiation

Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status.     

Combination Effect of Epigenetic Regulation and Ionizing Radiation in Colorectal Cancer Cells

Exposure of cells to ionizing radiation (IR) induces, not only, activation of multiple signaling pathways that play critical roles in cell fate determination, but also alteration of molecular pathways involved in cell death or survival. Recently, DNA methylation has been established as a critical epigenetic process involved in the regulation of gene expression in cancer cells, suggesting that DNA methylation inhibition may be an effective cancer treatment strategy. Because alterations of gene expression by DNA methylation have been considered to influence radioresponsiveness, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-aza-dC), on radiosensitivity. In addition, we investigated the underlying cellular mechanisms of combination treatments of ionizing irradiation (IR) and 5-aza-dC in human colon cancer cells. Colon cancer cell lines were initially tested for radiation sensitivity by IR in vitro and were treated with two different doses of 5-aza-dC. Survival of these cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays. The effects of 5-aza-dC along with irradiation on cell growth, cell cycle distribution, apoptosis, and apoptosis-related gene expression were examined. Combination irradiation treatment with 5-aza-dC significantly decreased growth activity compared with irradiation treatment alone or with 5-aza-dC treatment alone. The percentage of HCT116 cells in the sub-G1 phase and their apoptotic rate was increased when cells were treated with irradiation in combination with 5-aza-dC compared with either treatment alone. These observations were strongly supported by increased caspase activity, increased comet tails using comet assays, and increased protein levels of apoptosis-associated molecules (caspase 3/9, cleaved PARP). Our data demonstrated that 5-aza-dC enhanced radiosensitivity in colon cancer cells, and the combination effects of 5-aza-dC with radiation showed greater cellular effects than that of single treatment, suggesting that the combination of 5-aza-dC and radiation has the potential to become a clinical strategy for the treatment of cancer.     

Protein kinase C zeta nuclear translocation mediates the occurrence of radioresistance in friend erythroleukemia cells

Friend erythroleukemia cells require high doses (15 Gy) of ionizing radiation to display a reduced rate of proliferation and an increased number of dead cells. Since ionizing radiation can activate several signaling pathways at the plasma membrane which can lead to the nuclear translocation of a number of proteins, we looked at the intranuclear signaling system activated by Protein Kinases C, being this family of enzymes involved in the regulation of cell growth and death. Our results show an early and dose-dependent increased activity of zeta and epsilon isoforms, although PKC zeta is the only isoform significantly active and translocated into the nuclear compartment upon low (1.5 Gy) and high (15 Gy) radiation doses. These observations are concomitant and consistent with an increase in the anti-apoptotic protein Bcl-2 level upon both radiation doses. Our results point at the involvement of the PKC pathway in the survival response to ionizing radiation of this peculiar cell line, offering PKC zeta for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

Low efficiency of repair of critical DNA damage induced by low doses of radiation

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.     

Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells against apoptosis induced by low doses of ionizing radiation

The initial and rate-limiting enzyme of the oxidative pentose phosphate shunt, glucose-6-phosphate dehydrogenase (G6PD), is inhibited by NADPH and stimulated by NADP(+). Hence, under normal growth conditions, where NADPH levels exceed NADP(+) levels by as much as 100-fold, the activity of the pentose phosphate cycle is extremely low. However, during oxidant stress, pentose phosphate cycle activity can increase by as much as 200-fold over basal levels, to maintain the cytosolic reducing environment. G6PD-deficient (G6PD(-)) cell lines are sensitive to toxicity induced by chemical oxidants and ionizing radiation. Compared to wild-type CHO cells, enhanced sensitivity to ionizing radiation was observed for G6PD(-) cells exposed to single-dose or fractionated radiation. Fitting the single-dose radiation response data to the linear-quadratic model of radiation-induced cytotoxicity, we found that the G6PD(-) cells exhibited a significant enhancement in the alpha component of radiation-induced cell killing, while the values obtained for the beta component were similar in both the G6PD(-) and wild-type CHO cell lines. Here we report that the enhanced alpha component of radiation-induced cell killing is associated with a significant increase in the incidence of ionizing radiation-induced apoptosis in the G6PD(-) cells. These data suggest that G6PD and the oxidative pentose phosphate shunt protect cells from ionizing radiation-induced cell killing by limiting the incidence of radiation-induced apoptosis. The sensitivity to radiation-induced apoptosis was lost when the cDNA for wild-type G6PD was transfected into the G6PD(-) cell lines. Depleting GSH with l-BSO enhanced apoptosis of K1 cells while having no effect in the G6PD(-) cell line     

"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.