Mourcin, F., Grenier, N., Mayol, J-F., Lataillade, J-J., Sotto, J-J., Hérodin, F. and Drouet, M. Mesenchymal Stem Cells Support Expansion of In Vitro Irradiated CD34 Cells in the Presence of SCF, FLT3 Ligand, TPO and IL3: Potential Application to Autologous Cell Therapy in Accidentally Irradiated Victims. Radiat. Res. 164, 1–9 (2005).

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34 cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34 cells. In vitro irradiated (4 Gy ⁶⁰Co γ rays) or nonirradiated mobilized peripheral blood CD34 cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor thrombopoietin interleukin 3 FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34 cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34 (4.9-fold), CD34 (3.8-fold), CD34 /Thy-1 (8.1-fold), CD41 (12.4-fold) and MPO (50.6-fold), although at lower levels than in nonirradiated CD34 cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for autologous cell therapy applied to accidentally irradiated victims.

Kashiwakura, I., Inanami, O., Abe, Y., Takahashi, T. A. and Kuwabara, M. Different Radiosensitive Megakaryocytic Progenitor Cells Exist in Steady-State Human Peripheral Blood. Radiat. Res. 164, 10–16 (2005).

CD34 antigen is a novel marker for human hematopoietic stem/progenitor cells. In the present study, two cell fractions, CD34^low and CD34^high, were prepared from steady-state human peripheral blood on the basis of CD34 antigen expression. The colony-forming unit megakaryocytes (CFU-Meg) contained in each cell fraction were compared for X-radiation sensitivity and cytokine action. The content of CD34 CD45 cells in the CD34^low and CD34^high cell fractions was 74.8% and 88.8%, respectively, and the frequency of thrombopoietin (TPO)-supported CFU-Meg in the CD34^low cell fraction was 1.9 times higher than that in CD34^high. The CFU-Meg in CD34^high were more radiosensitive than those in CD34^low, indicating that steady-state human peripheral blood contains different types of CFU-Meg. However, no significant differences were observed between cell fractions in the radiation survival curves of CFU-Meg stimulated by TPO plus cytokines except granulocyte colony-stimulating factor (G-CSF). TPO plus interleukin 3 was the optimal combination for survival of both types of CFU-Meg after X irradiation. The present study also demonstrated that TPO plus G-CSF is able to increase the survival of irradiated CD34^low CFU-Meg. These results suggest that two megakaryocytic progenitor populations with different radiosensitivity and cytokine responses are found in steady-state human peripheral blood.

Ding, L-H., Shingyoji, M., Chen, F., Hwang, J-J., Burma, S., Lee, C., Cheng, J-F. and Chen, D. J. Gene Expression Profiles of Normal Human Fibroblasts after Exposure to Ionizing Radiation: A Comparative Study of Low and High Doses. Radiat. Res. 164, 17–26 (2005).

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G₁-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.

Guo, W-F., Lin, R-X., Huang, J., Zhou, Z., Yang, J., Guo, G-Z. and Wang, S-Q. Identification of Differentially Expressed Genes Contributing to Radioresistance in Lung Cancer Cells using Microarray Analysis. Radiat. Res. 164, 27–35 (2005).

Radiotherapy has played a key role in the control of tumor growth in many cancer patients. It is usually difficult to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. The response of tumor cells to radiation is believed to be accompanied by complex changes in the gene expression pattern. It may be possible to use these to sensitize radioresistant tumor cells and improve radiocurability. Based on the biological effects of ionizing radiation, in the present study, we developed one oligonucleotide microarray to analyze the expression of 143 genes in cells of two lung cancer cell lines with different radiosensitivities. Compared to NCI-H446 cells, expression of 18 genes significantly increased the basal levels in the radioresistant A549 cells, in which eight genes were up-regulated and 10 genes were down-regulated. In A549 cells irradiated with 5 Gy, 22 (19 up-regulated and three down-regulated) and 26 (eight up-regulated and 18 down-regulated) differentially expressed genes were found 6 and 24 h after irradiation, respectively. In NCI-H446 cells, the expression of 17 (nine up-regulated and eight down-regulated) and 18 (six up-regulated and 12 down-regulated) genes was altered 6 and 24 h after irradiation, respectively. RT-PCR was performed, and we found that MDM2, BCL2, PKCZ and PIM2 expression levels were increased in A549 cells and decreased in NCI-H446 cells after irradiation. Genes involved in DNA repair, such as XRCC5, ERCC5, ERCC1, RAD9A, ERCC4 and the gene encoding DNA-PK, were found to be increased to a higher level in A549 cells than in NCI-H446 cells. Antisense suppression of MDM2 resulted in increased radiosensitivity of A549 cells. Taken together, these results demonstrate the possibility that a group of genes involved in DNA repair, regulation of the cell cycle, cell proliferation and apoptosis is responsible for the different radioresistance of these two lung cancer cells. This list of genes may be useful in attempts to sensitize the radioresistant lung cancer cells.

Iizuka, D., Inanami, O., Matsuda, A., Kashiwakura, I., Asanuma, T. and Kuwabara, M. X Irradiation Induces the Proapoptotic State Independent of the Loss of Clonogenic Ability in Chinese Hamster V79 Cells. Radiat. Res. 164, 36–44 (2005).

The clonogenic ability (reproductive cell death) of Chinese hamster V79 cells was measured after treatment with X radiation and a newly developed anti-cancer drug, 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd, TAS106). Amplification in the loss of clonogenicity was observed compared to that obtained for cells exposed to X rays alone. Addition of benzyloxycarbonyl-val-ala-asp-fluoromethylketone (Z-VAD-FMK), a broad-spectrum caspase inhibitor, attenuated the increased lethality, but the dose–response curve obtained was found to merely revert to that obtained for cells exposed to X rays alone. Flow cytometric analysis showed that the number of cells arrested at the G₂/M phase by X irradiation was decreased by co-treatment with TAS106, and instead the number of cells in the sub-G₁ phase increased. Western blot analysis proved that TAS106 treatment down-regulated the expression of the G₂/M arrest-related proteins cyclin B1, phospho-CDC2 and WEE1. From these results, it was concluded that (1) no apoptosis was included in the dose–response curve obtained from cells exposed to X rays alone, (2) X radiation induced a potentially apoptotic (proapoptotic) state in cells independent of the loss of their clonogenic ability, and (3) TAS106 enhanced the loss of their clonogenic ability by converting the proapoptotic cells to apoptotic cells through the abrogation of arrest at the G₂/M phase.

Olwell, P. M., Cottell, D. C., Ní Shúilleabháin, S., Maderna, P., Seymour, C., Mothersill, C. and Lyng, F. M. Cytoskeletal Reorganization and Altered Phagocytotic Ability in Primary Cultures of Rainbow Trout Hemopoietic Tissue Exposed to Low-Level Ionizing Radiation. Radiat. Res. 164, 45–52 (2005).

It has long been known that the hematopoietic tissue of mammals is one of the most radiosensitive tissues. In vitro studies on prawns have also shown that low doses of radiation have an extremely deleterious effect on cells cultured from this animal's blood-forming tissues. This raises questions about the relative effects of radiation in animals of different species. One of the most important aquatic animals, from both an economic and an ecological point of view, is the fish. With this in mind, primary cultures of the blood-forming tissues of rainbow trout were exposed to radiation followed by a morphological comparison between control and irradiated cultures. The cultured cells were characterized as macrophages after incubation with apoptotic human polymorphonuclear leukocytes and were classified as phagocytotic leukocytes. These cells were found in two morphological forms, stretched and rounded. It was shown that there was a commensurate increase in the number of stretched cells after irradiation. Radiation was also shown to cause a dose-dependent increase in the amounts of apoptosis in these cells over time. The phagocytotic efficacy of these cells was shown to inhibited by the exposure to low doses of radiation.

Cabuy, E., Newton, C., Joksic, G., Woodbine, L., Koller, B., Jeggo, P. A. and Slijepcevic, P. Accelerated Telomere Shortening and Telomere Abnormalities in Radiosensitive Cell Lines. Radiat. Res. 164, 53–62 (2005).

We examined telomere maintenance in cells of 11 primary fibroblast cell lines with differing genetic defects that confer sensitivity to ionizing radiation. These included cell lines derived from patients with ataxia telangiectasia, Nijmegen breakage syndrome, Fanconi anemia, defective Artemis, DNA ligase I and DNA ligase IV, an immunodeficient patient with a defect in DNA double-strand break repair, and a patient diagnosed with xeroderma pigmentosum who, in addition, showed severe clinical sensitivity to ionizing radiation. Our results, based on Southern blot, flow-FISH and Q-FISH (quantitative FISH) measurements, revealed an accelerated rate of telomere shortening in most cell lines derived from the above patients compared to cell lines from normal individuals or a cell line isolated from a heterozygotic parent of one radiosensitive patient. This accelerated telomere shortening was accompanied by the formation of chromatin bridges in anaphase cells, indicative of the early loss of telomere capping function and in some cases low levels of chromosome abnormalities in metaphase cells. We also analyzed telomere maintenance in mouse embryonic stem cells deficient in Brca1, another defect that confers radiosensitivity. Similarly, these cells showed accelerated telomere shortening and mild telomere dysfunction in comparison to control cells. Our results suggest that mechanisms that confer sensitivity to ionizing radiation may be linked with mechanisms that cause telomere dysfunction.

Laurent, C., Pouget, J-P. and Voisin, P. Modulation of DNA Damage by Pentoxifylline and α-Tocopherol in Skin Fibroblasts Exposed to Gamma Rays. Radiat. Res. 164, 63–72 (2005).

Previous in vivo studies showed the combination pentoxifylline (PTX) and α-tocopherol was highly efficient in reducing late radiation-induced skin damage. The present work aimed at investigating the molecular and cellular mechanisms involved in the effects of this combination. Primary cultures of confluent dermal fibroblasts were γ-irradiated in the presence of PTX and trolox (Tx), the water-soluble analogue of α-tocopherol. Drugs were added either before or after radiation exposure and were maintained over time. Their antioxidant capacity and their effect on radiation-induced ROS production was assessed together with cell viability and clonogenicity. DNA damage formation was assessed by the alkaline comet assay and by the micronucleus (MN) test. Cell cycle distribution was also determined. The combination of PTX/ Tx was shown to reduce both immediate and late ROS formation observed in cells after irradiation. Surprisingly, decrease in DNA strand breaks measured by the comet assay was observed any time drugs were added. In addition, the micronucleus test revealed that for cells irradiated with 10 Gy, a late significant increase in MN formation occurred. The combination of PTX/Tx was shown to be antioxidant and to decrease radiation-induced ROS production. The observed effects on DNA damage at any time the drugs were added suggest that PTX/Tx could interfere with the DNA repair process.

Pinto, M., Prise, K. M. and Michael, B. D. Evidence for Complexity at the Nanometer Scale of Radiation-Induced DNA DSBs as a Determinant of Rejoining Kinetics. Radiat. Res. 164, 73–85 (2005).

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp–5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET α particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9 ± 0.5 h and a slow component with a half-life of 16 ± 9 h. For α particles, a fast component with a half-life of 0.7 ± 0.4 h and a slow component with a half-life of 12 ± 5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37 ± 0.12) relative to low-LET radiation (0.22 ± 0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.

Spees, W. M., Evelhoch, J. L., Thompson, P. A., Sloop, D. J. and Ackerman, J. J. H. Defining the pH_i-Hyperthermia Sensitivity Relationship for the RIF-1 Tumor In Vivo: A ³¹P MR Spectroscopy Study. Radiat. Res. 164, 86–99 (2005).

This study quantifies the enhancement of the therapeutic efficacy of hyperthermia resulting from an acutely acidified and accurately monitored intracellular pH (pH_i) in a mouse tumor model in vivo. Metabolic manipulation of the physiology of RIF-1 tumor (subcutaneous, on the hind flanks of female C3H/HeJ mice) achieved by i.p. bolus injection of glucose (glycolytic tumor acidification) or 3-O-methylglucose (non-glycolytic tumor acidification) was monitored by ³¹P magnetic resonance (³¹P MR) prior to, during and up to 1 h after localized hyperthermia. The pre-hyperthermia ³¹P MR-observable metabolic parameter that correlates most strongly with thermal sensitivity is pH_i. Thermal sensitivity increases linearly with decreasing pH_i regardless of the mechanism (glycolytic or non-glycolytic) of metabolic manipulation. The quantitative relationship is described by log₁₀(SF)/EQ43 = 0.0079 pH_i,preHT −0.0606 (R = 0.63, P < 0.0001), where EQ43 is the thermal heat dose delivered to the tumor (in units of equivalent minutes at 42.5°C), pH_i,preHT is the intracellular pH immediately prior to hyperthermia, and SF is the surviving fraction. The therapeutic enhancement is not as dramatic as expected based upon previously reported in vitro studies but is generally consistent with other in vivo studies. The method still represents a viable strategy for enhancing the therapeutic efficacy of hyperthermia, especially when used in combination with other therapeutic modalities.

Deckhut Augustine, A., Gondré-Lewis, T., McBride, W., Miller, L., Pellmar, T. C. and Rockwell, S. Animal Models for Radiation Injury, Protection and Therapy. Radiat. Res. 164, 100–109 (2005).

Current events throughout the world underscore the growing threat of different forms of terrorism, including radiological or nuclear attack. Pharmaceutical products and other approaches are needed to protect the civilian population from radiation and to treat those with radiation-induced injuries. In the event of an attack, radiation exposures will be heterogeneous in terms of both dose and quality, depending on the type of device used and each victim's location relative to the radiation source. Therefore, methods are needed to protect against and treat a wide range of early and slowly developing radiation-induced injuries. Equally important is the development of rapid and accurate biodosimetry methods for estimating radiation doses to individuals and guiding clinical treatment decisions. Acute effects of high-dose radiation include hematopoietic cell loss, immune suppression, mucosal damage (gastrointestinal and oral), and potential injury to other sites such as the lung, kidney and central nervous system (CNS). Long-term effects, as a result of both high- and low-dose radiation, include dysfunction or fibrosis in a wide range of organs and tissues and cancer. The availability of appropriate types of animal models, as well as adequate numbers of animals, is likely to be a major bottleneck in the development of new or improved radioprotectors, mitigators and therapeutic agents to prevent or treat radiation injuries and of biodosimetry methods to measure radiation doses to individuals.