Zips, D., Petersen, C., Junghanns, S., Eicheler, W., Brüchner, K. and Baumann, M. Selection of Genetically Distinct, Rapidly Proliferating Clones does not Contribute to Repopulation during Fractionated Irradiation in FaDu Squamous Cell Carcinoma. Radiat. Res. 160, 257–262 (2003).

Acceleration of clonogen repopulation during fractionated irradiation after about 3 weeks has been demonstrated previously in FaDu human squamous cell carcinoma in nude mice (Petersen et al., Int. J. Radiat. Oncol. Biol. Phys. 51, 483–493, 2001). Selection of genetically distinct, rapidly proliferating clones might contribute to this phenomenon. To address this question, three sublines (R1–R3) were established from FaDu tumors that recurred locally after fractionated irradiation. The tumors were retransplanted and irradiated under clamp hypoxia with single doses or with 18 × 3 Gy within 18 days or 36 days, followed by graded top-up doses. The results were compared with data obtained after the same treatment schedules in the parental tumor line. Histologies, tumor volume doubling times, and potential doubling times of FaDu sublines R1–R3 were not different from those of the parental line. The radiation dose required to control 50% of the tumors (TCD₅₀) after single-dose irradiation of 37–38 Gy was the same for the FaDu sublines R1–R3 and the parental tumor. The top-up TCD₅₀ values for the FaDu sublines R1–R3 after 18 fractions within 36 days were 14–17 Gy higher than those after 18 fractions within 18 days, indicating significant repopulation. The magnitude of this effect was not significantly different between the sublines R1–R3 or between these sublines and the parental FaDu tumors. The results indicate that selection of genetically distinct, rapidly proliferating clones does not contribute to the acceleration of repopulation during fractionated irradiation in poorly differentiated FaDu tumors.

Morel, E., Dublineau, I. and Griffiths, N. M. Effect of Radiation on cAMP, cGMP and Ca² _i Pathways and their Interactions in Rat Distal Colon. Radiat. Res. 160, 263–272 (2003)

The secretory response implicated in the intestinal response to luminal attack is altered by radiation. The cAMP, cGMP and Ca² _i pathways leading to secretion as well as the interactions between the cAMP pathway and the cGMP or Ca² _i pathway were studied in the rat distal colon 4 days after a 9-Gy abdominal X irradiation, when modifications mainly occurred. The secretory response in Ussing chambers and cAMP and cGMP accumulation in single isolated crypts were measured. The muscarinic receptor characteristics were determined in mucosal membrane preparations. The secretory response by the cAMP pathway (stimulated by vasoactive intestinal peptide or forskolin) and the cAMP accumulation in crypts were decreased (P < 0.05) after irradiation. The weak secretory response induced by the cGMP pathway (stimulated by nitric oxide or guanylin) was unaltered by radiation, and the small amount of cGMP determined in isolated crypts from the control group became undetectable in the irradiated group. Inducible NOS was not involved in the hyporesponsiveness to VIP after irradiation (there was no effect of an iNOS inhibitor). The secretory response by the Ca² _i pathway (stimulated by carbachol) was unaffected despite a decreased number and increased affinity of muscarinic receptors. The non-additivity of VIP and carbachol co-stimulated responses was unmodified. In contrast, VIP and SNP co-stimulation showed that NO enhanced the radiation-induced hyporesponsiveness to VIP through a reduced accumulation of cAMP in crypts. This study provides further understanding of the effect of ionizing radiation on the intracellular signaling pathways.

Heinloth, A. N., Shackelford, R. E., Innes, C. L., Bennett, L., Li, L., Amin, R. P., Sieber, S. O., Flores, K. G., Bushel, P. R. and Paules, R. S. ATM-Dependent and -Independent Gene Expression Changes in Response to Oxidative Stress, Gamma Irradiation, and UV Irradiation. Radiat. Res. 160, 273–290 (2003).

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G₁-, S- and G₂-phase cell cycle checkpoint functions in response to γ irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to γ irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G₁ checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either γ radiation (5 Gy), oxidative stress (75 µM t-butyl-hydroperoxide), or UV radiation (7.5 J/m²), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1.

Mårtensson, S. Nygren, J. Osheroff, N. and Hammarsten, O. Activation of the DNA-Dependent Protein Kinase by Drug-Induced and Radiation-Induced DNA Strand Breaks. Radiat. Res. 160, 291–301 (2003).

The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation (¹³⁷Cs γ rays and N⁷ ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.

Kita, K., Sugaya, S., Zhai, L., Wu, Y. P., Wano, C., Chigira, S., Nomura, J., Takahashi, S., Ichinose, M. and Suzuki, N. Involvement of LEU13 in Interferon-Induced Refractoriness of Human RSa Cells to Cell Killing by X Rays. Radiat. Res. 160, 302–308 (2003).

Culture of human cells with human interferon α and β (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF^r and F-IF^r cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.

Nazarov, I. B., Smirnova, A. N., Krutilina, R. I., Svetlova, M. P., Solovjeva, L. V., Nikiforov, A. A., Oei, S-L., Zalenskaya, I. A., Yau, P. M., Bradbury, E. M. and Tomilin, N. V. Dephosphorylation of Histone γ-H2AX during Repair of DNA Double-Strand Breaks in Mammalian Cells and its Inhibition by Calyculin A. Radiat. Res. 160, 309–317 (2003).

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called γ-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that γ-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of γ-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of γ-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the γ-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, γ-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of γ-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from γ-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and γ-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

Shao, C., Furusawa, Y., Aoki, M. and Ando, K. Role of Gap Junctional Intercellular Communication in Radiation-Induced Bystander Effects in Human Fibroblasts. Radiat. Res. 160, 318–323 (2003).

Involvement of gap junctional intercellular communication (GJIC) in bystander responses of confluent human fibroblasts irradiated with a carbon-ion beam was investigated. It was found that the lower the radiation dose, the higher the yield of radiation-induced micronuclei per nuclear traversal, suggesting the existence of bystander effects. This low-dose sensitivity was increased when GJIC was enhanced by treating cells with 8-Br-cAMP, but it was partly reduced by treating cells with DMSO, an effective scavenger of reactive oxygen species (ROS). Moreover, no low-dose sensitivity was observed when cells were treated with 100 µM lindane, an inhibitor of GJIC. The survival of irradiated cells was increased by DMSO but was not influenced significantly by cAMP or lindane. On the other hand, G₁-phase arrest was detected in the irradiated cells, and it was enhanced by cAMP. In contrast, this arrest was reduced or almost eliminated by DMSO or lindane, respectively, even when cells were irradiated with such a high dose that each cell received five nuclear traversals on average. Thus the bystander responses occurred after both low-dose and relatively high-dose irradiation. Our results indicated that both GJIC and ROS contributed to the radiation-induced bystander effect, but gap junctional channels might play an essential role by modulating the release of radiation-induced signaling factors.

Kellerer, A. M. and Chen, J. Comparative Microdosimetry of Photoelectrons and Compton Electrons: An Analysis in Terms of Generalized Proximity Functions. Radiat. Res. 160, 324–333 (2003).

A current discussion on mammography screening is focused on claims of high relative biological effectiveness (RBE) of mammography X rays compared to conventional 200 kV X rays. An earlier assessment in terms of the electron spectra of these radiations has led to the conclusion that the RBE is bound to be less than 2, regardless of specific model assumptions and the microdosimetric properties of electrons. The present study extends this result in terms of the microdosimetric proximity function, t(x), for electrons, which is essentially the spatial auto-correlation function of energy within particle tracks. If pairs of DNA lesions, e.g. chromosome breaks or deletions, bring about the observed damage, the value t(x) determines for a specified radiation the relative frequency of pairs of lesions a distance x apart. The effectiveness of the radiation is thus proportional to an average of the values of t(x) over the distances, x, for which lesions can combine. The analysis suggests that 15 keV electrons can have a low-dose relative biological effectiveness (RBE_M) of 1.6 relative to 40 keV electrons if the interaction distances do not exceed about 1 µm. An extension of the concept, the reduced proximity function, t_Δ(x), permits the inclusion of models with an energy threshold, such as Δ = 100 eV, 500 eV or 2 keV, for the formation of each of the DNA lesions. This makes it possible to assess the potential impact of the Auger electrons which accompany most photoelectrons, but only a minority of the Compton electrons. It is found that the Auger electrons could make photoelectrons substantially more effective than Compton electrons at energies below 10 keV but not at energies above 15 keV. The conclusions obtained for the RBE of 15 keV electrons relative to 40 keV electrons will be roughly representative of the RBE of mammography X rays relative to conventional 200 kV X rays.

Razskazovskiy, Y., Debije, M. G., Howerton, S. B., Williams, L. D. and Bernhard, W. A. Strand Breaks in X-Irradiated Crystalline DNA: Alternating CG Oligomers. Radiat. Res. 160, 334–339 (2003).

Direct ionization of crystalline d(CGCGCGCG) and d(CGCGCGCGCG) oligomers produces 3′- and 5′-phosphate-terminated fragments as the main strand breakage products detectable by ion-exchange chromatography. The nature of the base has no effect on the probability of strand breakage at the given site. The yields of 3′-phosphates are systematically lower than the yields of the 5′-phosphates originating from the same cleavage site, pointing to the possible presence of unidentified products with sugar remnants attached to the 3′-end. These results show that direct ionization is efficient at producing single-strand breaks in DNA and its action is relatively indiscriminate with respect to base sequence.

Krivokapić, A., Hole, E. O. and Sagstuen, E. EPR and ENDOR Study of Crystalline Cytosine·HCl Doped with 5-Methylcytosine. Radiation-Induced Radical Formation and Hole Transfer. Radiat. Res. 160, 340–354 (2003).

Radical formation and hole transfer were investigated in crystals of cytosine·HCl (C·HCl) doped with 0–1.1 mol-% 5-methylcytosine·HCl (5MC·HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3αH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3αH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3αH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2′deoxycytidine hydrochloride (CdR·HCl), 5′dCMP and 3′CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC·HCl doped into crystals of C·HCl give rise to a considerable yield of 3αH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3αH radicals in the crystalline cytosine derivatives CR, CdR·HCl, 5′dCMP and 3′CMP and suggests that the 3αH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C·HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3αH radical is more stable than the primary cytosine radicals. The relative amounts of the 3αH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3αH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3αH radical being less exposed to recombination than other cytosine radicals.

Marfak, A., Trouillas, P., Allais, D. P., Calliste, C. A., Cook-Moreau, J. and Duroux, J. L. Mechanisms of Transformation of the Antioxidant Kaempferol into Depsides. Gamma-Radiolysis Study in Methanol and Ethanol. Radiat. Res. 160, 355–365 (2003).

In this study, we irradiated the antioxidant kaempferol in ethanol and methanol solutions with γ rays at doses ranging from 0.2–20 kGy. NMR and ES-MS spectroscopy were used to identify radiolysis products. Two depsides, {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) methyl acetate and {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) ethyl acetate, were the major compounds of kaempferol degradation in methanol and in ethanol, respectively. Other products formed in low concentrations were identified as [4-hydroxyphenyl](oxo) methyl acetate, [4-hydroxyphenyl](oxo) ethyl acetate, and depside {2-[(4′-hydroxybenzoyl)oxy]-4,6-dihydroxyphenyl}(oxo) acetic acid. The formation of the latter was observed in both solvents. We propose degradation mechanisms that suggest that ^·CH₂OH and CH₃^·CHOH, produced by solvent radiolysis, react with the 3-OH kaempferol group because of its high H-donor capacity. π-Electron delocalization in the flavonoxy formed after the first H-transfer leads to C-ring opening and consequently to the formation of depsides. G calculation of the degradation products and of ^·CH₂OH and CH₃^·CHOH radicals confirmed the proposed mechanism of kaempferol radiolysis. The rate constants for the reaction between kaempferol and these free radicals were also calculated. Formation of depside has also been observed in many studies of the oxidation of flavonoids; those studying human metabolism have suggested similar redox transformation of flavonols. The antioxidant activities of radiolysis products were evaluated and compared to those of kaempferol.

Lind, B. K., Persson, L. M., Edgren, M. R., Hedlöf, I. and Brahme, A. Repairable–Conditionally Repairable Damage Model Based on Dual Poisson Processes. Radiat. Res. 160, 366–375 (2003).

The advent of intensity-modulated radiation therapy makes it increasingly important to model the response accurately when large volumes of normal tissues are irradiated by controlled graded dose distributions aimed at maximizing tumor cure and minimizing normal tissue toxicity. The cell survival model proposed here is very useful and flexible for accurate description of the response of healthy tissues as well as tumors in classical and truly radiobiologically optimized radiation therapy. The repairable–conditionally repairable (RCR) model distinguishes between two different types of damage, namely the potentially repairable, which may also be lethal, i.e. if unrepaired or misrepaired, and the conditionally repairable, which may be repaired or may lead to apoptosis if it has not been repaired correctly. When potentially repairable damage is being repaired, for example by nonhomologous end joining, conditionally repairable damage may require in addition a high-fidelity correction by homologous repair. The induction of both types of damage is assumed to be described by Poisson statistics. The resultant cell survival expression has the unique ability to fit most experimental data well at low doses (the initial hypersensitive range), intermediate doses (on the shoulder of the survival curve), and high doses (on the quasi-exponential region of the survival curve). The complete Poisson expression can be approximated well by a simple bi-exponential cell survival expression, S(D) = e^–aD bDe^–cD, where the first term describes the survival of undamaged cells and the last term represents survival after complete repair of sublethal damage. The bi-exponential expression makes it easy to derive D₀, D_q, n and α, β values to facilitate comparison with classical cell survival models.

Tanaka, S., Tanaka, I. B. III., Sasagawa, S., Ichinohe, K., Takabatake, T., Matsushita, S., Matsumoto, T., Otsu, H. and Sato, F. No Lengthening of Life Span in Mice Continuously Exposed to Gamma Rays at Very Low Dose Rates. Radiat. Res. 160, 376–379 (2003).

Late effects of continuous exposure to ionizing radiation are potential hazards to workers in radiation facilities as well as to the general public. Recently, low-dose-rate and low-dose effects have become a serious concern. Using a total of 4000 mice, we studied the late biological effects of chronic exposure to low-dose-rate radiation as assayed by life span. Two thousand male and 2000 female 8-week-old specific-pathogen-free (SPF) B6C3F1 mice were randomly divided into four groups (one nonirradiated control and three irradiated). Irradiation was carried out for approximately 400 days using ¹³⁷Cs γ rays at dose rates of 21 mGy day^–1, 1.1 mGy day^–1 and 0.05 mGy day^–1 with total doses equivalent to 8000 mGy, 400 mGy and 20 mGy, respectively. All mice were kept under SPF conditions until they died spontaneously. Statistical analyses showed that the life spans of mice of both sexes irradiated with 21 mGy day^–1 (P < 0.0001) and of females irradiated with 1.1 mGy day^–1 (P < 0.05) were significantly shorter than those of the control group. Our results show no evidence of lengthened life span in mice continuously exposed to very low dose rates of γ rays.