Schmidt-Ullrich, R.K., Dent, P., Grant S., Mikkelsen, R.B. and Valerie, K. Signal Transduction and Cellular Radiation Responses.

Exposure of cells to ionizing radiation results in complex cellular responses resulting in cell death and altered proliferation states. The underlying cytotoxic, cytoprotective and cellular stress responses to radiation are mediated by existing signaling pathways, activation of which may be amplified by intrinsic cellular radical production systems. These signaling responses include the activation of plasma membrane receptors, the stimulation of cytoplasmic protein kinases, transcriptional activation, and altered cell cycle regulation. From the data presented, there is increasing evidence for the functional links between cellular signal transduction responses and DNA damage recognition and repair, cell survival, or cell death through apoptosis or reproductive mechanisms.

Georgakilas, A.G., Haveles, K.S., Sophianopoulou, V., Sakelliou, L., Zarris, G. and Sideris, E.G. Alpha-Particle-Induced Changes in the Stability and Size of DNA.

The effect of α-particle radiation on the thermal stability and size of calf thymus DNA molecules in deoxygenated aqueous solutions was investigated by thermal transition spectrophotometry, pulsed-field gel electrophoresis, and standard agarose gel electrophoresis. The thermal transition of DNA from helix to coil was studied through analysis of the UV A₂₆₀ absorbance. The results obtained for α particles of mean LET of 128 keV μm⁻¹ reveal a dual dose response: a tendency for thermal stability of the DNA helix at “low” doses, followed by an increasing instability at higher doses. The same phenomenon was observed for the mean molecular weight of DNA molecules exposed to α particles. The results reported here for α particles in the low-dose region of 0–16 Gy are consistent with our previous hypothesis of inter- and intramolecular interactions of a covalent character in γ-irradiated DNA molecules in the dose region of 0–4 Gy.

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. I. Fragment Size Distribution and Evaluation of Breakage Probability.

Incorporation of ¹²⁵I-dC into a defined location of a double-stranded oligodeoxynucleotide was used to investigate DNA breaks arising from decay of the Auger electron-emitting isotope. Samples of the oligodeoxynucleotide were also labeled with ³²P at either the 5′ or 3′ end of either the ¹²⁵I-dC-containing (so-called top) or opposite (bottom) strand and incubated in 20 mM phosphate buffer or the same buffer plus 2 M dimethylsulfoxide at 4°C during 18–20 days. The ³²P-end-labeled fragments produced by ¹²⁵I decays were separated on denaturing polyacrylamide gels, and the ³²P activity in each fragment was determined by scintillation counting after elution of fragments from the gel. The relative fragment size distributions were then normalized on a per decay basis and converted to a distribution of single-strand break probabilities as a function of distance from the ¹²⁵I-dC. The results of three to five experiments for each of eight possible combinations of labels and incubation conditions are presented as a table showing the relative numbers of ³²P counts in different fragments as well as graphs of normalized fragment size distributions and probabilities of breakage. The average numbers of single-strand breaks per ¹²⁵I decay are 3.3 and 3.7 in the top strand and 1.3 and 1.5 in the bottom strand with and without dimethylsulfoxide, respectively. Every ¹²⁵I decay event produces a break in the top strand, and breakage of the bottom strand occurs in 75–80% of the events. Thus a double-strand break is produced by ¹²⁵I decay with a probability of approximately 0.8.

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage.

The dramatic chemical and biological effects of the decay of DNA-incorporated ¹²⁵I stem from two consequences of the Auger electron cascades associated with the decay of the isotope: high local deposition of radiation energy from short-range Auger electrons, and neutralization of the multiply charged tellurium atom. We have analyzed the extensive data reported in the companion paper (Radiat. Res. 153, 000–000, 2000), in which DNA breakage was measured after ¹²⁵I decay in a 41-bp oligoDNA. The experimental data collected under scavenging conditions (2 M dimethylsulfoxide) were deconvoluted into two components denoted as radiation and nonradiation, the former being attributed to energy deposition by Auger electrons. The contribution of the components was estimated by adopting various assumptions, the principal one being that DNA breakage due to the radiation mechanism is dependent on the distance between the decaying ¹²⁵I atom and the cleaved deoxyribosyl unit, while the nonradiation mechanism, associated with neutralization of the multiply charged tellurium atom, contributes equally at corresponding nucleotides starting from the ¹²⁵I-incorporating nucleotide. Comparison of the experimental data sets collected under scavenging and nonscavenging (without dimethylsulfoxide) conditions was used to estimate the radiation-scavengeable component. Our analysis showed that the nonradiation component plays the major role in causing breakage within 4–5 nucleotides from the site of ¹²⁵I incorporation and produces about 50% of all single-stranded breaks. This overall result is consistent with the relative amounts of energy associated with Auger electrons and the charged tellurium atom. However, the nonradiation component accounts for almost four times more breaks in the top strand, to which the ¹²⁵I is bound covalently, than in the bottom strand, thus suggesting an important role of covalent bonds in the energy transfer from the charged tellurium atom. The radiation component dominates at the distances beyond 8–9 nucleotides, and 36% of the radiation-induced breaks are scavengeable.

Daşu, A. and Denekamp, J. Inducible Repair and Intrinsic Radiosensitivity: A Complex but Predictable Relationship?

Two groups have proposed a simple linear relationship between inducible radioresistance in a variety of mammalian cells and their intrinsic radiosensitivity at 2 Gy (Lambin et al., Int.J. Radiat. Biol. 69, 279–290, 1996; Alsbeih and Raaphorst, unpublished results, 1997). The inducible repair response (IRR) is quantified as a ratio, α_S/α_R, i.e. the slope in the hypersensitive low-dose region, α_S, relative to the α_R term of the classical linear-quadratic formula. These proposals imply that the intrinsic radiosensitivity at clinically relevant doses is directly linked to the cell’s ability to mount an adaptive response as a result of exposure to very low doses of radiation. We have re-examined this correlation and found that the more extensive data set now available in the literature does not support the contention of a simple linear relationship. The two parameters are correlated, but by a much more complex relationship. A more logical fit is obtained with a log-linear equation. A series of log-linear curves are needed to describe the correlation between IRR and SF2, because of the spectrum of α/β ratios among the cell lines and hence the confounding effect of the β term at a dose of 2 Gy. The degree of repair competence before irradiation starts could also be a major factor in the apparent magnitude of the amount of repair induced. There appears to be a systematic difference in the data sets from different series of cell lines that have been obtained using flow cytometry techniques in the laboratory in Vancouver and using dynamic microscope imaging at the Gray Laboratory. We suggest that the use of a brief exposure to a laser beam in flow cytometry before the cells are irradiated might itself partially induce a stress response and change the DNA repair capacity of the cells. The clinical consequences of the relationship for predicting the benefits of altered fractionation schedules are discussed.

Chomentowski, M., Kellerer, A. M. and Pierce, D. A. Radiation Dose Dependences in the Atomic Bomb Survivor Cancer Mortality Data: A Model-Free Visualization.

The standard approach to obtaining nominal risk coefficients for radiation-related cancer involves fitting linear or linear-quadratic dose–response functions. This is usually complemented by a more direct visualization where the data are subdivided into distinct dose categories and the effect level is quantified for each of these categories. Such model-free computations, however, can be quite dependent on the arbitrary choice of the cutpoints in dose. The method proposed here largely avoids this arbitrariness by choosing a dose category width—constant on a log scale—to obtain the desired degree of smoothing, and then superimposing results for all placements of the resulting log-dose grids. The method is applied to leukemia and solid cancer mortality of the A-bomb survivors.

Park, H.J., Lyons, J.C., Griffin, R.J., Limm, B.U. and Song, C.W. Apoptosis and Cell Cycle Progression in an Acidic Environment after Irradiation.

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G₂/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G₁ and S phases while the rest of the cells underwent apoptosis in G₂/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G₂/M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4–12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G₂/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G₁, S and G₂/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G₂/M-phase arrest.

Ghosh, J.C., Izumida, Y., Suzuki, K., Kodama, S. and Watanabe, M. Dose-Dependent Biphasic Accumulation of TP53 Protein in Normal Human Embryo Cells after X Irradiation.

The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21^WAF1/CIP1) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.

Schwartz, J.L., Jordan, R., Sun, J., Ma, H. and Hsie, A.W. Dose-Dependent Changes in the Spectrum of Mutations Induced by Ionizing Radiation.

We examined the influence of dose on the spectrum of mutations induced at the hypoxanthine guanine phosphoribosyltransferase (Hprt) locus in Chinese hamster ovary (CHO) cells. Independent CHO-K1 cell mutants at the Hprt locus were isolated from cells exposed to 0, 0.5, 1.5, 3.0 and 6.0 Gy ¹³⁷Cs γ rays, and the genetic changes responsible for the mutations were determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. We observed dose-dependent changes in mutation spectra. At low doses, the principal radiation-induced mutations were point mutations. With increasing dose, multibase deletion mutations became the predominant mutation type such that by 6.0 Gy, there were almost three times more deletion mutations than point mutations. The dose response for induction of point mutations was linear while that for multibase deletions fit a linear-quadratic response. There was a biphasic distribution of deletion sizes, and different dose responses for small compared to large deletions. The frequency of large (>36 kb) total gene deletions increased exponentially, implying that they develop from the interaction between two independent events. In contrast, the dose response for deletion mutations of less than 10 kb was nearly linear, suggesting that these types of mutations develop mostly from single events and not the interactions between two independently produced lesions. The observation of dose-dependent changes in radiation-induced mutation spectra suggests that the types of alterations and therefore the risks from low-dose radiation exposure cannot be easily extrapolated from high-dose effects.

Park, S-H., Lee, S-J., Chung, H-Y., Kim, T-H., Cho, C-K., Yoo, S-Y. and Lee, Y-S. Inducible Heat-Shock Protein 70 Is Involved in the Radioadaptive Response.

The thermoresistant (TR) clone of radiation-induced fibrosarcoma (RIF) cells showed an adaptive response, i.e. a reduced effect, after exposure to a higher challenging dose (4 Gy) when the priming dose (1 cGy) was given 4 or 7 h earlier, but RIF cells did not. Since inducible Hsp70 expression was different in cells of these two cell lines, the role of inducible Hsp70 in the adaptive response was examined. When inducible Hsp70 was transfected into RIF cells, the adaptive response was acquired. Transfection of inducible Hsp70 to NIH 3T3 mouse embryo cells also conferred radioresistance to the cells as assayed by clonogenic survival, [³H]thymidine incorporation, and an ELISA cell death detection kit. An increased tendency for the induction of an adaptive response was also observed. Interestingly, basal levels of Ca² -dependent and independent Pkc activities were increased by transfection with inducible Hsp70 compared to those of control vector cells. Irradiation with γ rays induced activation of Pkc within minutes in control vector cells, while transfection with inducible Hsp70 did not. Cellular redistribution to particulate fractions of Pkca, d and z after exposure γ rays also was not detected. Furthermore, radioresistance by transfection with inducible Hsp70, as tested by clonogenic survival, disappeared after pretreatment with Pkc inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and GF109203X. Taken together, these data suggest that radioresistance inducible by Hsp70 is associated with an elevated level of Pkc activity.

Ogawa, A., Griffin, R. J. and Song, C. W. Effect of a Combination of Mild-Temperature Hyperthermia and Nicotinamide on the Radiation Response of Experimental Tumors.

The effect of mild-temperature hyperthermia and nicotinamide individually or combined on tumor radiosensitivity was investigated with SCK tumors grown s.c. in the right hind limbs of A/J mice. An i.p. injection of nicotinamide at 50–250 mg/kg slightly enhanced the cell killing caused by 10–20 Gy of ionizing radiation as determined by the in vivo/in vitro tumor excision assay. Treatment of tumors with mild-temperature hyperthermia at 41.5°C for 60 min prior to tumor irradiation was significantly more effective than nicotinamide and the combination of nicotinamide and hyperthermia was far more effective than nicotinamide or hyperthermia alone in enhancing radiation-induced cell killing. Radiation-induced tumor growth delay was enhanced by a factor of 1.2 by 50 mg/kg nicotinamide, 2.1 by hyperthermia, and 3.6 by the combination of nicotinamide and hyperthermia. Taking these results and those of our previous studies together, we conclude that mild-temperature hyperthermia increases tumor blood flow and oxygenation and that combining mild-temperature hyperthermia and nicotinamide is more effective than either of these alone in increasing tumor radiosensitivity.

Matsubara, J., Turcanu, V., Poindron, P. and Ina, Y. Immune Effects of Low-Dose Radiation: Short-Term Induction of Thymocyte Apoptosis and Long-Term Augmentation of T-Cell-Dependent Immune Responses.

We and others have shown that low-dose X or γ irradiation of mice leads to an increase in their survival after a subsequent lethal high-dose irradiation. The greatest increase in radioresistance appears at a fixed window of dose and time, e.g. 8 weeks after 5–10 cGy or 2 weeks after 50 cGy preirradiation. We show that low-dose irradiation induces thymocyte apoptosis with a maximal level at 6 h postirradiation that returns to background levels after 24 h. At the same time, we observed no morphological alteration of splenocytes and no early modification of the intensity of T-cell-dependent immune responses as measured by plaque-forming cell (PFC) counts. Nevertheless, we found that PFCs were increased 2 weeks after 50 cGy irradiation, which is the same time at which mice expressed the optimal increase in survival after a second lethal irradiation. We also examined thymocyte apoptosis and spleen PFCs in mice subjected to other stress-inducing pretreatments. Our results emphasize the existence of a lag time between the time of low-dose irradiation in vivo and the appearance of radioresistance. A mechanism that interconnects an environmental stimulus with the response of the animal is proposed based on the evidence presented here and reported in the literature.

Coppes, R.P., Roffel, A.F., Zeilstra, L.J.W., Vissink, A. and Konings, A.W.T. Early Radiation Effects on Muscarinic Receptor-Induced Secretory Responsiveness of the Parotid Gland in the Freely Moving Rat.

Although the salivary glands have a low rate of cell turnover, they are relatively radiosensitive. To study the possible mechanism behind this inherent radiosensitivity, a rat model was developed in which saliva can be collected after local irradiation of the parotid gland without the use of anesthetics or stressful handling. Saliva secretion was induced by the partial muscarinic receptor agonist pilocarpine (0.03–3 mg/kg) with or without pretreatment with the β-adrenoceptor antagonist propranolol (2.5 mg/kg), or the full muscarinic receptor agonist methacholine (0.16–16 mg/min), and measured during 5 min per drug dose before and 1, 3, 6 and 10 days after irradiation. The maximal secretory response induced by pilocarpine plus propranolol was increased compared to that with pilocarpine alone but did not reach the level of methacholine-induced secretion, which was about five times higher. One day after irradiation a decrease in maximal pilocarpine-induced secretion was observed (−22%) using the same dose of pilocarpine that induces 50% of the maximal response (ED₅₀), in both the absence and presence of propranolol, indicating that the receptor–drug interaction was not affected by the radiation at this time. The secretory response to methacholine 1 day after irradiation, however, was normal. At day 3 after irradiation, the maximal methacholine-induced secretion was also affected, whereas pilocarpine (±propranolol)-induced maximal secretion decreased further. At day 6 after irradiation, maximal secretory responses had declined to approximately 50% regardless of the agonist used, whereas ED₅₀ values were still unaffected. No net acinar cell loss was observed within the first 10 days after irradiation, and this therefore could not account for the loss in function. The results indicate that radiation does not affect cell number or receptor–drug interaction, but rather signal transduction, which eventually leads to the impaired response. We hypothesize that the early radiation effect, within 3 days, may be membrane damage affecting the receptor–G-protein signal transfer. Later critical damage, however, is probably of a different nature and may be located in the second-messenger signal transduction pathway downstream from the G protein, not necessarily involving cellular membranes.

Almasan, A. Cellular Commitment to Radiation-Induced Apoptosis.

The basic elements of the machinery of programmed cell death (apoptosis) are built into all mammalian cells and are conserved evolutionarily from nematodes to humans. The workshop on Commitment to Radiation-Induced Apoptosis at the 11th International Congress of Radiation Research in Dublin, Ireland reviewed recent information regarding the basic molecular mechanisms which are fundamental to the understanding of the process of apoptosis after treatment with ionizing radiation and some other agents.