Cohen-Jonathan, E., Muschel, R. J., McKenna, W. G., Evans, S. M., Cerniglia, G., Mick, R., Kusewitt, D., Sebti, S. M., Hamilton, A. D., Oliff, A., Kohl, N., Gibbs, J. B. and Bernhard, E. J. Farnesyltransferase Inhibitors Potentiate the Antitumor Effect of Radiation on a Human Tumor Xenograft Expressing Activated HRAS.

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.

Pandita, T. K. and Dhar, S. Influence of ATM Function on Interactions between Telomeres and Nuclear Matrix.

The ATM (ataxia telangiectasia mutated) gene product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. The human ATM protein shows similarity to several yeast and mammalian proteins involved in meiotic recombination and cell cycle progression. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia (AT), influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in AT cells is due to altered interactions between the telomeres and the nuclear matrix. These interactions were examined in nuclear matrix halos prior to and after irradiation. A difference was observed in the ratio of soluble and matrix-associated telomeric DNA between cells derived from AT and normal individuals. Treatment with ionizing radiation affected the ratio of soluble and matrix-associated telomeric DNA only in the AT cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix seen in AT cells. Fibroblasts from AT individuals transfected with a wild-type ATM gene had corrected telomere–nuclear matrix interactions. In experiments designed to determine whether there is a link between the altered telomere–nuclear matrix interactions and defective telomere movement and clustering, a significant difference was observed in the ratio of soluble compared to matrix-associated telomeric DNA sequences in meiocytes of Atm^–/– and control mice. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix and that alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. This paper summarizes our recent publications on the influence of inactivation of ATM on the interaction of telomeres with nuclear matrix in somatic and germ cells.

Lawrence, T. S., Burke, R., Davis, M. A. and Wygoda, M. R. Lack of Effect of TP53 Status on Fluorodeoxyuridine-Mediated Radiosensitization.

Substantial evidence suggests that TP53 (also known as p53) status can influence the response of cells to chemotherapy and radiation. We wished to determine if TP53 function affected the response of cells to fluoropyrimidines and radiation, a combination used for tens of thousands of patients each year. To assess the role of TP53 in fluoropyrimidine-mediated radiosensitization, we carried out experiments using RKO parental cells (wild-type TP53) and RKO cells overexpressing mutant TP53 (which blocks TP53 function) or expressing E6 (which degrades TP53). We found that TP53 function had no effect on the ability of fluorodeoxyuridine to increase radiation sensitivity. These findings are consistent with the hypothesis that the late G₁-phase checkpoint, which is mediated by TP53, is not crucial to radiosensitization. Rather, the ability of cells to progress in to S phase in the presence of the drug, which is independent of TP53, is more closely associated with increased radiation sensitivity.

Niehoff, L. B. and Uckun, F. M. Role of PRKCM (PKCμ) in Radiation-Induced Increase of JUN Proto-oncogene mRNA Levels in B-Lineage Lymphoid Cells.

Exposure of cells to ionizing radiation results in both activation of protein kinase C (PRKC, also known as PKC) and induction of transcription of the JUN proto-oncogene. PRKC plays a pivotal role in radiation-induced JUN expression, since inhibition of PRKC abrogates the JUN signal. However, the specific PRKC isoforms involved in radiation-induced elevation of JUN mRNA levels have not been identified. Here we demonstrate that in DT40 B-lineage lymphoid cells, the μ isoform of PRKC (PRKCM) is critical for the response of JUN to ionizing radiation. The zinc chelator, 1,10-phenanthroline, abrogated induction of JUN after exposure to ionizing radiation, indicating that this PRKCM-mediated response is also dependent on zinc.

Pötter, T., Göhde, W., Wedemeyer, N. and Köhnlein, W. Keratinocytes Exposed to Ultraviolet Radiation Reveal Three Down-regulated Genes with Potential Function in Differentiation and Cell Cycle Control.

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIβ-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIβ and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

Murray, D., Rosenberg, E. and Allalunis-Turner, M. J. Protection of Human Tumor Cells of Differing Radiosensitivity by WR-1065.

We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol®), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of γ radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently (∼1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA–drug associations.

Debije, M. G., Strickler, M. T. and Bernhard, W. A. On the Efficiency of Hole and Electron Transfer from the Hydration Layer to DNA: An EPR Study of Crystalline DNA X-Irradiated at 4 K.

The aim of this project was to gain an improved understanding of how the efficiency of hole and electron transfer from the solvation layer to DNA decreases as a function of distance from DNA. The packing of DNA in crystals of known structure makes it possible to calculate the degree of DNA hydration with a precision that is significantly greater than that achievable for amorphous samples. Previous work on oligodeoxynucleotide crystals has demonstrated that the efficiency of free radical trapping by DNA exposed to ionizing radiation at 4 K is relatively insensitive to base sequence, conformation, counterion, or base stacking continuity. Having eliminated these confounding variables, it is now possible to ascertain the degree of radical transfer that occurs from ionized water as a function of DNA hydration (Γ, in mol water/mol nucleotide). EPR is used to measure the hydroxyl radical concentration in crystals irradiated at 4 K. From a lack of hydroxyl radicals trapped in the inner hydration mantle, we determine that hole transfer to DNA is complete for water molecules located within 8 Å. This corresponds to Γ = 9−11 and indicates that hole transfer is 100% (as efficient as direct ionization of DNA) for water molecules adjacent to DNA. Beyond ∼8 Å (Γ > 10), hydroxyl radicals are observed; thus deprotonation of the water radical cation is seen to compete with hole transfer to DNA as soon as one water intervenes between the ionized water and DNA. The boundary for 0% hole transfer is projected to occur somewhere between 15 and 20 waters per nucleotide. Electron transfer, on the other hand, is 100% efficient across the entire range studied, 4.2 ≤ Γ ≤ 15.6.

Hannig, J., Zhang, D., Canaday, D. J., Beckett, M. A., Astumian, R. D., Weichselbaum, R. R. and Lee, R. C. Surfactant Sealing of Membranes Permeabilized by Ionizing Radiation.

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to γ radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 ± 3 min was determined between exposure to 160 Gy of γ radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.

Pierce, D. A. and Preston, D. L. Radiation-Related Cancer Risks at Low Doses among Atomic Bomb Survivors.

To clarify the information in the Radiation Effects Research Foundation data regarding cancer risks of low radiation doses, we focus on survivors with doses less than 0.5 Sv. For reasons indicated, we also restrict attention mainly to survivors within 3,000 m of the hypocenter of the bombs. Analysis is of solid cancer incidence from 1958–1994, involving 7,000 cancer cases among 50,000 survivors in that dose and distance range. The results provide useful risk estimates for doses as low as 0.05–0.1 Sv, which are not overestimated by linear risk estimates computed from the wider dose ranges 0–2 Sv or 0–4 Sv. There is a statistically significant risk in the range 0–0.1 Sv, and an upper confidence limit on any possible threshold is computed as 0.06 Sv. It is indicated that modification of the neutron dose estimates currently under consideration would not markedly change the conclusions.

Dublineau, I., Ksas, B. and Griffiths, N. M. Functional Changes in the Rat Distal Colon after Whole-Body Irradiation: Dose–Response and Temporal Relationships.

The aim of this study was to determine the acute radiation response of the rat distal colon by in vivo and in vitro measurements of the functions of the colon over a range of radiation doses. Rats received a whole-body irradiation of 2 to 12 Gy and were studied from 1 to 7 days after exposure. In vivo water and electrolyte absorption was measured by insertion of an agarose cylinder in the colon of anesthetized rats. In vitro transepithelial electrical parameters (potential difference, short-circuit current, transepithelial conductance) were measured in Ussing chambers in basal and agonist-stimulated conditions. In vivo and in vitro functional studies were completed by standard histological analyses. The majority of functional modifications appeared at 4 days after exposure. At this time, a dose-dependent decrease in absorption of water and sodium/chloride ions in the colon was noted. In contrast, a twofold increase in potassium secretion was observed for every radiation dose studied. The response to secretagogues was attenuated at doses >8 Gy. Modifications of basal transepithelial electrical parameters together with marked histological alterations were observed at 4 days with the higher doses (≥10 Gy). In conclusion, these results show that functions of the colon are affected by irradiation and may contribute to diarrhea induced by ionizing radiation.

Meyer, R. E., Braun, R. D., Rosner, G. L. and Dewhirst, M. W. Local 42°C Hyperthermia Improves Vascular Conductance of the R3230Ac Rat Mammary Adenocarcinoma during Sodium Nitroprusside Infusion.

The effect of sodium nitroprusside-induced hypotension on the perfusion of the R3230 adenocarcinoma during local 42°C hyperthermia was studied using a combination of intravital microscopy and laser Doppler flowmetry. Fischer 344 rats were implanted with dorsal skin flap window chambers containing the R3230Ac tumor and allocated to three treatment groups (34°C with nitroprusside, 42°C with nitroprusside, and 42°C with 0.9% saline). After baseline observation at 34°C, tumors were locally heated to 42°C using a water bath and either 0.9% saline or nitroprusside sufficient to reduce blood pressure 20% below pretreatment baseline was infused. Nitroprusside at 34°C decreased tumor vascular conductance 40% with no effect on the diameter of arterioles entering the tumor. The diameter of arterioles entering 42°C heated tumors increased 35% independent of blood pressure change. Saline at 42°C had no effect on tumor vascular conductance; however, nitroprusside at 42°C increased tumor vascular conductance 55%. Local 42°C tumor heating, combined with a moderate reduction in blood pressure with nitroprusside, overrides the vascular steal effect associated with reduced perfusion pressure alone and results in improved tumor perfusion. Observations of the effect of vasodilator substances on normothermic tumor perfusion cannot be extrapolated to situations where moderate hyperthermia is used.

Griffin, R. J., Ogawa, A. and Song, C. W. A Novel Drug to Reduce Tumor Perfusion: Antitumor Effect Alone and with Hyperthermia.

We have investigated the feasibility of enhancing damage induced by hyperthermia in SCK murine tumors by reducing tumor blood perfusion using a new agent, KB-R8498. Within several minutes of an i.v. injection, the tumor perfusion was reduced to less than 20% of the control value, and it recovered to 40–70% of the control value by 1 h after injection. The perfusion in normal tissues decreased or increased soon after drug administration depending on the tissue type. However, by 1 h after drug treatment, perfusion in five of the seven tissues examined had returned to the control level. The tumor pH was also reduced after i.v. drug administration. Control tumors grew to four times the initial volume in 6 days. Tumors that were heated at 42.5°C for 60 min were delayed in growth by 4 days compared to control tumors. There was a growth delay of 14 days when an i.v. injection of KB-R8498 was given and the tumors were heated at 42.5°C either immediately or 1 h later. In drug-alone studies, the tumor growth was delayed by 4 days when the drug was infused continuously at a rate of 30–50 mg/kg day^–1 for 7 days or about 2 days when mice were treated with five daily injections of 30 mg/kg KB-R8498.

Nakasono, S. and Saiki, H. Effect of ELF Magnetic Fields on Protein Synthesis in Escherichia coli K12.

Escherichia coli K12 was used as a model system to determine whether ELF magnetic fields (MFs) are a general stress factor. The cells were exposed to ELF MFs (5–100 Hz) at a maximum intensity of 14 mT r.m.s. for circularly polarized MFs and 10 mT r.m.s. for vertically polarized MFs. The response of the cells to the MFs was estimated from the change in protein synthesis by using 2D PAGE. Approximately 1,000 proteins were separated on the 2D gels. The stress-responsive proteins such as CH10, DNAK, CH60, RECA, USPA, K6P1 and SODM were identified from the SWISS-2DPAGE database on the 2D gels. These proteins respond to most stress factors, including temperature change, chemical compounds, heavy metals, and nutrients. When the bacterial cells were exposed to each MF at 5–100 Hz under aerobic conditions (6.5 h) or at 50 Hz under anaerobic conditions (16 h) at the maximum intensity (7.8 to 14 mT r.m.s.), no reproducible changes were observed in the 2D gels. Changes in protein synthesis were detected by 2D PAGE with exposure to heat shock (50°C for 30 min) or under anaerobic conditions (no bubbling for 16 h). Increases in the levels of synthesis of the stress proteins were observed in heat-shocked cells (CH60, CH10, HTPG, DNAK, HSLV, IBPA and some unidentified proteins) and in cells grown under anaerobic conditions (DNAK, PFLB, RECA, USPA and many unidentified proteins). These results suggest that 2D PAGE is sufficient to detect cell responses to environmental stress. The high-intensity ELF MFs (14 mT at power frequency) did not act as a general stress factor.

Hofer, M., Mazur, L., Pospíšil, M., Weiterová, L. and Znojil, V. Radioprotective Action of Extracellular Adenosine on Bone Marrow Cells in Mice Exposed to Gamma Rays as Assayed by the Micronucleus Test.

The frequency of micronucleated polychromatic erythrocytes (PCEs) in mouse bone marrow was assessed after administration of dipyridamole and/or adenosine monophosphate (AMP) to nonirradiated mice or to mice irradiated 15 min later with a sublethal dose of 6.5 Gy γ rays. In nonirradiated mice, the administration of the drugs increased the frequency of micronucleated PCEs significantly (by 108%). In contrast, in irradiated mice, the number of radiation-induced micronucleated PCEs was significantly decreased if the mice had been pretreated with dipyridamole or AMP alone (by 24% after administration of each of the compounds) and in particular after administration of the drugs in combination (by 36%).