Wilson, W. R., Hicks, K. O., Pullen, S. M., Ferry, D. M., Helsby, N. A. and Patterson, A. V. Bystander Effects of Bioreductive Drugs: Potential for Exploiting Pathological Tumor Hypoxia with Dinitrobenzamide Mustards. Radiat. Res. 167, 625–636 (2007).

Tumor hypoxia is an important therapeutic target, and it can potentially be exploited by hypoxia-activated prodrugs. However, physiological hypoxia in normal tissues is a limitation. One solution would be to confine activation to severely (pathologically) hypoxic tissue, using hypoxia-activated prodrugs that provide a bystander effect through diffusion of the activated cytotoxin to adjacent regions at intermediate oxygen concentrations (associated with partial radioresistance). To evaluate this requirement, we identified five hypoxia-activated prodrugs with at least 10-fold higher potency against a cell line (A549-P540^puro) overexpressing human cytochrome P450 reductase (P450R) relative to A549-Lo21 cells with 200-fold lower P450R activity. Bystander killing by these hypoxia-activated prodrugs was tested in anoxic multicellular layer co-cultures of these two cell lines. Cytotoxic potency against A549-Lo21 cells was unaffected by the presence of A549-P450^puro cells for tirapazamine and RSU-1069 but increased more than 10-fold for the aziridinyldintrobenzamide CB 1954, more than 14-fold for the corresponding nitrogen mustard SN 23862, and 15-fold for its water-soluble analog SN 23816. The cytotoxic extracellular metabolites resulting from hypoxic nitroreduction of CB 1954 and SN 23862 by A549-P450^puro cells were identified by LC/MS and bioassay methods. For SN 23862, these included the 2-amine metabolite, previously, identified as the bystander metabolite from aerobic activation by the E. coli nfsB nitroreductase, but also novel di-reduced metabolites. Cytotoxicity of SN 23862 to A549-P450^puro cells was inhibited by lower concentrations of oxygen than for tirapazamine. The combination of selective activation under severe hypoxia with an efficient bystander effect identifies the dinitrobenzamide mustards for further development as hypoxia-activated prodrugs.

Asaka-Amano, Y., Takiguchi, Y., Yatomi, M., Kurosu, K., Kasahara, Y., Tanabe, N., Tatsumi, K. and Kuriyama, T. Effect of Treatment Schedule on the Interaction of Cisplatin and Radiation in Human Lung Cancer Cells. Radiat. Res. 167, 637–644 (2007).

This study was designed to determine the effects of the treatment schedule on the interaction between cisplatin and radiation. Cells of a human squamous cell lung cancer cell line were treated with cisplatin and radiation using three treatment protocols: 1-h exposure to cisplatin immediately followed by irradiation (A), 4-day continuous exposure to cisplatin immediately followed by irradiation (B), and 1-h exposure to cisplatin followed by irradiation after a 4-day interval (C). The interactions were assessed by isobologram, cell cycle distribution and apoptosis. The combination resulted in a additive effect in every protocol. Cell cycle accumulation at G₂/M phase before irradiation was observed in Protocols B and C, whereas no cell cycle shift in the limited time course was noted in Protocol A. Although a 4-day continuous exposure to cisplatin and a 1-h exposure to cisplatin followed by a 4-day interval before irradiation caused significantly increased apoptosis, an additional increase in apoptosis after irradiation was not observed in Protocols B and C, whereas Protocol A showed an additional increase. Despite a cell cycle shift favoring radiation sensitivity, the drug–radiation interactions in Protocols B and C were additive, possibly because of negative effects including induction of a durable G₂/M-phase arrest and suppression of apoptosis by cisplatin.

Topsch, J., Scholz, M. and Mueller-Klieser, W. Radiobiological Characterization of Human Tumor Cell Multilayers after Conventional and Particle Irradiation. Radiat. Res. 167, 645–654 (2007).

The goal of this study was to establish planar multilayers from human tumor cells (WiDr and SiHa) as a model for irradiation of solid tumors. In addition to using conventional X rays (250 kV) as a reference standard, multilayers were tested for their suitability in cell survival studies with heavy-ion irradiation (¹²C⁶ ) in the plateau and the extended Bragg peak with a scanned ion beam. Multilayers of both cell lines showed decreased survival compared to the corresponding monolayers after both X and heavy-ion irradiation. This multicellular sensitization effect is in contrast to the multicellular resistance or contact effect commonly described in the literature. Flow cytometry measurements showed an arrest of irradiated SiHa cells in G₂/M phase. In contrast to the transient arrest of the monolayers, the multilayers stayed in a prolonged arrest. After Bragg-peak irradiation of monolayers, the arrest time was increased by 12–24 h, and more cells were arrested than with X rays. For multilayers, there were no differences between G₂ arrest after X rays and heavy ions for the entire observation period.

Horikawa-Miura, M., Matsuda, N., Yoshida, M., Okumura, Y., Mori, T. and Watanabe, M. The Greater Lethality of UVB Radiation to Cultured Human Cells is Associated with the Specific Activation of a DNA Damage-Independent Signaling Pathway. Radiat. Res. 167, 655–662 (2007).

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.

Blickwedehl, J., McEvoy, S., Wong, I., Kousis, P., Clements, J., Elliott, R., Cresswell, P. Liang, P. and Bangia, N. Proteasomes and Proteasome Activator 200 kDa (PA200) Accumulate on Chromatin in Response to Ionizing Radiation. Radiat. Res. 167, 663–674 (2007).

Proteasome activator 200 kDa (PA200) forms nuclear foci after exposure of cells to ionizing radiation and enhances proteasome activity in vitro. Within cells, it is unclear whether PA200 responds to radiation alone or in association with proteasomes. In the present study, we identified three forms of cellular PA200 (termed PA200i, ii and iii) at the mRNA and protein levels. Neither PA200ii nor PA200iii appears to associate with proteasomes. All detectable PA200i is associated with proteasomes, which indicates that PA200i and proteasomes function together within the cell. Consistent with this idea, we find that exposure of cells to radiation leads to an equivalent accumulation of both PA200i and core proteasomes on chromatin. This increase in PA200 and proteasomes on chromatin is not specific to the stage of cell cycle arrest since it occurs in cells that arrest in G₂/M and cells that arrest in G₁/S after exposure to radiation. These data provide evidence that PA200 and proteasomes function together within cells and respond to a specific radiation-induced damage independent of the stage of cell cycle arrest.

Mukaida, N., Kodama, S., Suzuki, K., Oshimura, M. and Watanabe, M. Transmission of Genomic Instability from a Single Irradiated Human Chromosome to the Progeny of Unirradiated Cells. Radiat. Res. 167, 675–681 (2007).

Ionizing radiation can induce chromosome instability that is transmitted over many generations after irradiation in the progeny of surviving cells, but it remains unclear why this instability can be transmitted to the progeny. To acquire knowledge about the transmissible nature of genomic instability, we transferred an irradiated human chromosome into unirradiated mouse recipient cells by microcell fusion and examined the stability of the transferred human chromosome in the microcell hybrids. The transferred chromosome was stable in all six microcell hybrids in which an unirradiated human chromosome had been introduced. In contrast, the transferred chromosome was unstable in four out of five microcell hybrids in which an irradiated human chromosome had been introduced. The aberrations included changes in the irradiated chromosome itself and rearrangements with recipient mouse chromosomes. Thus the present study demonstrates that genomic instability can be transmitted to the progeny of unirradiated cells by a chromosome exposed to ionizing radiation, implying that the instability is caused by the irradiated chromosome itself and also that the instability is induced by the nontargeted effect of radiation.

Day, T. K., Zeng, G., Hooker, A. M., Bhat, M., Scott, B. R., Turner, D. R. and Sykes, P. J. Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose. Radiat. Res. 167, 682–692 (2007).

Adaptive responses are induced by stress such as X radiation and result in a lower than expected biological response. Two-dose adaptive response experiments typically involve a low priming dose followed by a subsequent high radiation dose. Here, we used a sensitive in vivo chromosomal inversion assay to demonstrate for the first time an adaptive response when a low dose (0.01–1 mGy) was given several hours after a high 1000-mGy radiation dose. The adaptive responses in this study were of similar magnitude to the two-dose adaptive responses previously observed in this test system when the low dose was given first. A chromosomal inversion adaptive response was also induced by two 1000-mGy doses and when a 1-mGy dose was preceded or followed by a dose of 0.01 mGy, but not by two 4000-mGy doses. This is also the first example of an adaptive response when both doses are low. Our data agree with previous reports of an on-off mechanism of adaptive response. The induction of an adaptive response by a low dose after a high damaging dose provides evidence that the mechanisms underlying radiation adaptive responses are not due to prevention of damage induced by the high dose but to modulation of the cellular response to this damage.

Nakano, M., Kodama, Y., Ohtaki, K., Nakashima, E., Niwa, O., Toyoshima, M. and Nakamura, N. Chromosome Aberrations do not Persist in the Lymphocytes or Bone Marrow Cells of Mice Irradiated In Utero or Soon after Birth. Radiat. Res. 167, 693–702 (2007).

Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (≤0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated when ≥6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53^−/− mice and 1.4% in p53 ^/− mice) compared to the frequency in the p53^−/− mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

Yoshida, K., Hirabayashi, Y., Wada, S., Watanabe, F., Watanabe, K., Aizawa, S. and Inoue, T. p53 (TRP53) Deficiency-Mediated Antiapoptosis Escape after 5 Gy X Irradiation Still Induces Stem Cell Leukemia in C3H/He Mice: Comparison between Whole-Body Assay and Bone Marrow Transplantation (BMT) Assay. Radiat. Res. 167, 703–710 (2007).

Mice exposed to a lethal dose of radiation were repopulated with heterozygous p53 ^/− (TRP53 ^/−) bone marrow cells and then exposed to doses of 1, 3 and 5 Gy 1 month later. This resulted in the transplanted bone marrow-specific diseases other than competitively induced nonhematopoietic neoplasms. Interestingly, the present study showed a high frequency of stem cell leukemia, i.e., leukemias characterized by a lack of differentiation due also to p53 deficiency, even after 5 Gy irradiation. The frequencies of stem cell leukemias (and those of total hematopoietic malignancies) were 16% (24%) at 1 Gy and 45% (75%) at 3 Gy. Furthermore, markedly high incidences of stem cell leukemias were observed at 5 Gy in p53 ^/− mice, i.e., 87% (100%) in the transplantation assay and 60% (83.3%) in the whole-body assay, whereas a conventional whole-body assay induced only 14% in wild-type mice. The high incidence of stem cell leukemias observed in this study using heterozygous p53-deficient mice agrees with results of a previous study of homozygous p53-deficient mice and is consistent with the high frequency of loss of heterozygosity in the p53 wild-type allele observed in leukemias. This suggests that the target cells for radiation-induced stem cell leukemias may be p53-deficient hematopoietic stem cells.

Boice, J. D., Jr., Mumma, M. T. and Blot, W. J. Cancer and Noncancer Mortality in Populations Living Near Uranium and Vanadium Mining and Milling Operations in Montrose County, Colorado, 1950–2000. Radiat. Res. 167, 711–726 (2007).

Mining and milling of uranium in Montrose County on the Western Slope of Colorado began in the early 1900s and continued until the early 1980s. To evaluate the possible impact of these activities on the health of communities living on the Colorado Plateau, mortality rates between 1950 and 2000 among Montrose County residents were compared to rates among residents in five similar counties in Colorado. Standardized mortality ratios (SMRs) were computed as the ratio of observed numbers of deaths in Montrose County to the expected numbers of deaths based on mortality rates in the general populations of Colorado and the United States. Relative risks (RRs) were computed as the ratio of the SMRs for Montrose County to the SMRs for the five comparison counties. Between 1950 and 2000, a total of 1,877 cancer deaths occurred in the population residing in Montrose County, compared with 1,903 expected based on general population rates for Colorado (SMR_CO 0.99). There were 11,837 cancer deaths in the five comparison counties during the same 51-year period compared with 12,135 expected (SMR_CO 0.98). There was no difference between the total cancer mortality rates in Montrose County and those in the comparison counties (RR = 1.01; 95% CI 0.96–1.06). Except for lung cancer among males (RR = 1.19; 95% CI 1.06–1.33), no statistically significant excesses were seen for any causes of death of a priori interest: cancers of the breast, kidney, liver, bone, or childhood cancer, leukemia, non-Hodgkin lymphoma, renal disease or nonmalignant respiratory disease. Lung cancer among females was decreased (RR = 0.83; 95% CI 0.67–1.02). The absence of elevated mortality rates of cancer in Montrose County over a period of 51 years suggests that the historical milling and mining operations did not adversely affect the health of Montrose County residents. Although descriptive correlation analyses such as this preclude definitive causal inferences, the increased lung cancer mortality seen among males but not females is most likely due to prior occupational exposure to radon and cigarette smoking among underground miners residing in Montrose County, consistent with previous cohort studies of Colorado miners and of residents of the town of Uravan in Montrose County.

Parveen, B., Preston, D. L., Doody, M. M., Hauptmann, M., Kampa, D., Alexander, B. H., Petibone, D., Simon, S. L., Weinstock, R. M., Bouville, A., Yong, L. C., Freedman, D. M., Mabuchi, K., Linet, M. S., Edwards, A. A., Tucker, J. D. and Sigurdson, A. J. Retrospective Biodosimetry among United States Radiologic Technologists. Radiat. Res. 167, 727–734 (2007).

Measurement of chromosome translocations in peripheral blood lymphocytes has been used to quantify prior exposure to ionizing radiation, including for workers exposed to low, chronic doses. We assessed translocation frequencies in a subset of U.S. radiologic technologists to substantiate ionizing radiation dose estimates developed for 110,418 technologists who worked between 1916 and 1984. From 3,441 cohort members known to have begun working before 1950, we selected a sample of 152, stratified by estimated cumulative dose, oversampling from higher-dose categories and excluding persons with a prior cancer diagnosis, a personal or family history of chromosomal instability disorders, or a current history of smoking. Estimates of film-badge dose ranged from less than 10 cSv to more than 30 cSv. Blood samples, obtained in 2004, were analyzed by fluorescence in situ hybridization (FISH) whole chromosome painting by simultaneously labeling chromosomes 1, 2 and 4 in red and 3, 5 and 6 in green. Translocations were scored in 1800 well-spread metaphase cells and expressed per 100 cell equivalents (CE) per person. Linear Poisson regression models with allowance for overdispersion were used to assess the relationship between estimated occupational red bone marrow absorbed dose in cGy and translocation frequency, adjusted for age, gender and estimated red bone marrow absorbed dose score from personal diagnostic procedures. We observed 0.09 excess translocations per 100 CE per cGy red bone marrow dose (95% CI: −0.01, 0.2; P = 0.07), which is similar to the expected estimate based on previous cytogenetic studies (0.05 excess translocations per 100 CE per cGy). Despite uncertainty in the estimates of occupational red bone marrow absorbed doses, we found good general agreement between the doses and translocation frequencies, lending support to the credibility of the dose assessment for this large cohort of U.S. radiologic technologists.

Pierce, D. A., Vaeth, M. and Shimizu, Y. Selection Bias in Cancer Risk Estimation from A-Bomb Survivors. Radiat. Res. 167, 735–741 (2007).

We consider the possible bias in cancer risk estimation from A-bomb survivors due to selection of the cohort by survival. The paper considers both relevant information from the data and basic theoretical issues involved. The most direct information from the data comes from making various restrictions on the dose–distance range, partly to reduce differential selection and partly just to reduce the magnitude of the selection. These analyses suggest that there are no serious biases, but they are not conclusive. Theoretical considerations include laying out more explicitly than usual just how biases could result from the selection. This involves heterogeneities in the ability to survive acute effects, in baseline and radiogenic cancer rates, and most importantly the correlation between survival-related and cancer-related heterogeneities. Following on this, idealized modeling is used to quantify the extent of possible bias in terms of the assumed values of the magnitude of these heterogeneities and their correlation. It is indicated that these values would need to be very large to introduce substantial bias. Based on all these considerations, it seems unlikely that the bias in cancer risk estimation could be large in relation to other uncertainties in generalizing from what is seen among A-bomb survivors; in particular, indications are that the bias in relative risks is unlikely to be as large as 0.05 to 0.07. For solid cancer this would correspond to bias in the excess relative risk at 1 Sv of at most about 15–20%.