The pathway involving Bre1-dependent monoubiquitination of histone H2B lysine 123, which leads to Dot1-dependent methylation of histone H3 lysine 79 (H3K79me2), has been implicated in survival after exposure to ionizing radiation in Saccharomyces cerevisiae . We found that depletion of mammalian homologs of Bre1 compromises the response to ionizing radiation, leading to increased radiosensitivity and a G 2 /M checkpoint defect. The deficiency in Bre1a/b function was also associated with increased sensitivity to crosslinking drugs and defective formation of Rad51 foci in mouse cells, suggesting a defect in homologous recombinational repair analogous to that seen in Saccharomyces . In budding yeast, H3K79me2 is important for the recruitment of the checkpoint signaling protein Rad9 to sites of double-strand breaks (DSBs). However, in mammalian cells, 53BP1 (the Rad9 ortholog) in addition to H3K79me2 recognizes a different residue, H4K20me2, and some studies argue that it is H4K20me2 and not H3K79me2 that is the preferred target for 53BP1. We show here that depletion of Bre1b specifically reduced dimethylation of H3K79 without affecting dimethylation of H4K20. Thus our data suggest that the observed defects in the radiation response of Bre1a/b-deficient cells are associated with reduced H3K79me2 and not with H4K20me2.

Song, S., McCann, K. E. and Brown, J. M. Radiosensitization of Yeast Cells by Inhibition of Histone H4 Acetylation. Radiat. Res. 170, 618–627 (2008). Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51 -mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (γ-H2A) induction after irradiation but a reduced rate of loss of γ-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G 1 and G 2 checkpoints after exposure to ionizing radiation, suggesting that G 1 and G 2 checkpoint activation is independent of histone H4 acetylation.

Spicakova, T., McCann, K. and Brown, J. M. A Role for Lsm1p in Response to Ultraviolet-Radiation Damage in Saccharomyces cerevisiae . Radiat. Res. 170, 411–421 (2008). A genome-wide screen in Saccharomyces cerevisiae identified LSM1 as a new gene affecting sensitivity to ultraviolet (UV) radiation. Lsm1p is a member of a cytoplasmic complex composed of Lsm1p–7p that interacts with the yeast mRNA degradation machinery. To investigate the potential role of Lsm1p in response to UV radiation, we constructed double mutant strains in which LSM1 was deleted in combination with a representative gene from each of three known yeast DNA repair pathways. Our results show that lsm1 Δ increases the UV-radiation sensitivity of the rad1 Δ and rad51 Δ mutants, but not the rad18 Δ mutant, placing LSM1 within the post-replication repair/damage tolerance pathway (PRR). When combined with other deletions affecting PRR, lsm1 Δ increases the UV-radiation sensitivity of the rev3 Δ, rad30 Δ and pol30-K164R mutants but not rad5 Δ. Furthermore, the UV-radiation sensitivity phenotype of lsm1 Δ is partially rescued by mutations in genes involved in 3′ to 5′ mRNA degradation, and mutations predicted to function in RNA interactions confer the most UV-radiation sensitivity. Together, these results suggest that Lsm1p may confer protection against UV-radiation damage by protecting the 3′ ends of mRNAs from exosome-dependent 3′ to 5′ degradation as part of a novel RAD5- mediated, PCNA-K164 ubiquitylation-independent subpathway of PRR.

Game, J. C., Birrell, G. W., Brown, J. A., Shibata, T., Baccari, C., Chu, A. M., Williamson, M. S. and Brown, J. M. Use of a Genome-Wide Approach to Identify New Genes that Control Resistance of Saccharomyces cerevisiae to Ionizing Radiation. Radiat. Res. 160, 14–24 (2003). We have used the recently completed set of all homozygous diploid deletion mutants in budding yeast, S. cerevisiae , to screen for new mutants conferring sensitivity to ionizing radiation. In each strain a different open reading frame (ORF) has been replaced with a cassette containing unique 20-mer sequences that allow the relative abundance of each strain in a pool to be determined by hybridization to a high-density oligonucleotide array. Putative radiation-sensitive mutants were identified as having a reduced abundance in the pool of 4,627 individual deletion strains after irradiation. Of the top 33 strains most sensitive to radiation in this assay, 14 contained genes known to be involved in DNA repair. Eight of the remaining deletion mutants were studied. Only one, which deleted for the ORF YDR014W (which we name RAD61 ), conferred reproducible radiation sensitivity in both the haploid and diploid deletions and had no problem with spore viability when the haploid was backcrossed to wild-type. The rest showed only marginal sensitivity as haploids, and many had problems with spore viability when backcrossed, suggesting the presence of gross aneuploidy or polyploidy in strains initially presumed haploid. Our results emphasize that secondary mutations or deviations from euploidy can be a problem in screening this resource for sensitivity to ionizing radiation.

Vordermark, D., Menke, D. R. and Brown, J. M. Similar Radiation Sensitivities of Acutely and Chronically Hypoxic Cells in HT 1080 Fibrosarcoma Xenografts. Radiat. Res. 159, 94–101 (2003). It has been suggested that chronically hypoxic tumor cells may be more radiosensitive than acutely hypoxic or even aerobic cells. In the present study we have used the fact that chronically, but not acutely, hypoxic cells that are transformed with a vector containing an enhanced green fluorescent protein (EGFP) driven by a hypoxia-responsive promoter become green (high EGFP) at low oxygen concentrations and can be viably sorted from transplanted tumors in vitro. We showed that the fluorescence of HT 1080 human fibrosarcoma cells stably transfected with this vector increases constantly with decreasing O 2 concentrations (<2%, longer than 1 h, half maximum ∼0.2% for longer than 8 h), and that cells subjected to repeated cycles of hypoxia/reoxygenation (simulating acutely hypoxic cells) showed only background fluorescence. To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP (“chronically hypoxic”) and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro. We found that the survival of high-EGFP cells after irradiation was not affected by clamping, suggesting that all, or almost all, of these cells were fully (chronically) hypoxic. Also, the survival of the low-EGFP cells irradiated under clamped conditions (acutely hypoxic cells) was not significantly different from that of the high-EGFR cells (chronically hypoxic) cells irradiated under nonclamped (or clamped) conditions. We therefore conclude that, at least in this tumor model, the radiation sensitivity of chronically hypoxic cells is similar to that of the acutely hypoxic cells.

The presence of hypoxic cells in human tumors is thought to be one of the principal reasons for the failure of radiation therapy. Intensive laboratory and clinical efforts to overcome tumor hypoxia have focused on oxygenating, radiosensitizing or killing the maximally radioresistant fraction of tumor cells. This "hypoxic fraction" dominates the single-dose radiation response, irrespective of the oxygenation status of the remainder of the tumor cell population. However, at doses that are typical of those delivered in a daily radiotherapy protocol, we show that the tumor response is highly dependent upon the cells at oxygen levels intermediate between fully oxygenated and hypoxic (0.5-20 mm Hg). For most tumors, these cells are more important than the radiobiologically hypoxic cells in determining treatment outcome after 30 fractions of 2 Gy. We also show that under conditions of diffusion-limited hypoxia, the impact of full reoxygenation between fractions is much smaller than previously realized. Together, the results imply that tumor hypoxia plays a more significant role in determining the outcome of fractionated radiotherapy than previous measurements and assumptions of hypoxic fractions have indicated. Therefore, the concept of a hypoxic fraction in human tumors is less meaningful when pertaining to a fractionated radiotherapy regimen, and should not be expected to be useful for predicting tumor responses in the clinic. This implies the need to characterize tumor oxygenation in a manner that reflects the true oxygenation status of all the tumor cells, not just the ones most refractory to the effects of ionizing radiation. Furthermore, effective therapeutic agents must have the ability to specifically sensitize or kill those cells at intermediate levels of oxygen in addition to the radiobiologically hypoxic cells.

Severe combined immunodeficient (scid) murine cells, which are defective in both repair of DNA double-strand breaks and V(D)J recombination, are deficient in DNA-dependent protein kinase (DNA-PK), a protein which forms an activated complex with the DNA end-binding Ku proteins (p80 and p70) upon association with damaged DNA. Xrs 5 cells are deficient in the Ku p80 protein and also fail to form an active DNA-PK repair complex. Since both scid and xrs cells are defective in the same protein complex, we compared the kinetics of chromosome repair in scid cells to results published previously for xrs 5 cells. C.B-17 cells, scid cells and scid cells complemented with a single human chromosome 8 were irradiated with 6 Gy and allowed to repair from 0-24 h before fusion to HeLa cells for chromosome condensation. Breaks and dicentrics were visualized by fluorescence in situ hybridization. All cells had the same initial amount of chromosome damage, but scid cells had a slower rate of rejoining, more unrejoined breaks and more dicentrics than C.B-17 and scid cells with human chromosome 8. The scid cells appear to respond differently than xrs 5 cells, despite both cells lacking an essential component of the same DNA repair complex.

This review describes molecular cytogenetic approaches to genetic analysis including fluorescence in situ hybridization, primed in situ labeling and comparative genomic hybridization. It also summarizes the applications of this technology to physical mapping, cancer diagnosis and prognostication and in radiation biology.

Knowledge about the oxygenation of human tumors and its importance in the response to radiotherapy is crucial to the effort to develop improved treatment methods for radiotherapy. The measurement of oxygenation of human tumors and correlations with response to radiotherapy were the subjects of a recent workshop sponsored by the National Cancer Institute. The following methods for measuring oxygen or hypoxia, or a parameter related to either, were presented: polarographic oxygen electrodes, the comet and alkaline elution assays for radiation-induced DNA damage, nitroimidazole binding assays, hemoglobin saturation assays, magnetic resonance spectroscopy, electron spin resonance spectroscopy, phosphorescence imaging, and an assay for tumor interstitial pressure. The electron spin resonance, alkaline elution, and phosphorescence imaging methods have not been used in human tumors. The comet assay, nitroimidazole binding assays, magnetic resonance spectroscopy, cryospectroscopy, and near-infrared spectroscopy have been employed in human tumors, but correlations to treatment response have not been made. Polarographic measurements have indicated that the presence of hypoxia correlates with a poor response to radiotherapy in cervical cancers, but additional data are needed on early-stage disease, and with long-term follow-up on local control and survival. If these confirm the correlation between hypoxia and poor response to radiotherapy, additional tumor sites should be studied. Future clinical trials of treatments that sensitize, exploit, or kill hypoxic cells should identify and include the individual patients with hypoxic tumors. Fundamental unanswered questions regarding the assessment of tumor oxygenation concern the need for invasive procedures, the spatial resolution needed for prediction of response to radiotherapy, the importance of reoxygenation, differences between tumors in rates and degrees of reoxygenation, whether measurements made during a course of therapy are of value, and correlations among methods and with other predictive assays such as intrinsic radiosensitivity and potential doubling time.

A long-standing controversy in radiation cytogenetics is the precise mechanism for the formation of chromosome exchanges. The classical breakage-and-reunion hypothesis suggested by Stadler and developed by Sax was challenged in 1959 by Revell, who proposed that radiation produced an "unstable lesion" which could interact with another such lesion to form an exchange. A difference between the predictions of these two theories is that the breakage-and-reunion hypothesis would allow the two broken ends of a chromosome to join with contemporary broken ends in different chromosomes, thereby producing nonreciprocal exchanges involving more than two chromosomes. This would not occur according to the Revell theory, which demands pairwise commital to the exchange. The ability to "paint" a whole chromosome using fluorescence in situ hybridization allows a discrimination between reciprocal and nonreciprocal chromosome exchanges. We scored metaphases in AG1522 human fibroblasts irradiated in G 1 phase with 6 Gy and hybridized to chromosomes 1, 4, or 8. Of the complete exchanges involving one of these chromosomes, 26% were found to possess either one or three centromeres in the exchange pair. Since we could rule out any significant contribution of complex exchanges (i.e., those involving more than one break per chromosome), these one- and three-centromere exchange pairs must have arisen from a nonreciprocal exchange. Because an equal number of nonreciprocal exchanges would be expected to have a total of two centromeres, this suggests that approximately 50% of all exchanges at this dose were nonreciprocal. These data support the breakage-and-reunion hypothesis and are incompatible with the standard form of the exchange hypothesis of Revell.

SCCVII carcinomas and RIF-1 sarcomas were transplanted and grown in the unirradiated (control tumors) or preirradiated tissue of mice. Tumor oxygenation was measured when the tumors were in the size range <tex-math>$50-150\ {\rm mm}^{3}$</tex-math> using three different methods. The tumors growing in tissues preirradiated with 20 Gy were significantly more hypoxic than the control tumors for both tumor types. Radiobiologically hypoxic fractions of these tumors were approximately 10 times higher than those of the controls. The relative <tex-math>$[{}^{3}{\rm H}]\text{misonidazole}$</tex-math> binding of the tumors transplated into irradiated tissue was approximately five times (P < 0.01) higher in SCCVII tumors or approximately two times (P < 0.05) higher in RIF-1 tumors than that of the controls. The <tex-math>$p{\rm O}_{2}$</tex-math> histographs measured directly by a polarographic microelectrode were more hypoxic (P < 0.001) and changed from bimodal to unimodal in pattern in the tumors transplanted into irradiated tissue compared to those of the controls. The increase of the relative <tex-math>$[{}^{3}{\rm H}]\text{misonidazole}$</tex-math> binding in the tumors transplanted into irradiated tissue compared to that of the controls was similar to the increase of the cumulative frequency of <tex-math>$p{\rm O}_{2}$</tex-math> below 10.0 mm Hg for both the SCCVII and the RIF-1 tumors. The data suggest that the majority of tumor cells with <tex-math>$p{\rm O}_{2}$</tex-math> values below 5.0 mm Hg were clonogenic in the SCCVII tumors while only a small portion of tumor cells with <tex-math>$p{\rm O}_{2}$</tex-math> below 5.0 mm Hg were clonogenic in the control RIF-1 tumors. Increased hypoxia caused by preirradiation of the tumor bed is a useful model in evaluating the efficacy of various treatments affected by tumor oxygenation levels. The model is also useful in evaluating techniques for measuring oxygen levels.

Korbelik and Skov (Radiat. Res. 119, 145-156, 1989) have reported that cis-diamminedichloroplatinum (II) (cisplatin) shows substantial preferential radiosensitization of hypoxic cells in vitro at low radiation doses (1-4 Gy), and that the interaction seen with low doses of radiation is greatly diminished at high radiation doses. If such an interaction occurred with fractionated irradiation in vivo, it would be extremely important to radiation therapy, since the sensitizer enhancement ratios achievable in the low-dose region are higher than those achievable with current hypoxic cell radiosensitizers. We have tested this possibility in an experimental mouse tumor using fractionated irradiation under conditions in which the response of the tumor was determined by either its aerobic or its hypoxic cells. RIF-1 tumors were irradiated with 10 fractions of 1-4 Gy every 12 h with cisplatin given either as 12 mg/kg once before the first radiation dose or as 1.2 mg/kg at various times prior to each radiation dose. The tumors were irradiated with or without a clamp applied 2-3 min before each radiation dose. The effectiveness of the treatments was assayed by regrowth delay. Cisplatin caused a similar regrowth delay when used alone in both clamped and nonclamped tumors and produced a similar additive or supra-additive interaction when used with the 10 fractionated radiation schedule whether the tumors were hypoxic or aerobic. Our data suggest that cisplatin does not show any preferential radiosensitization of hypoxic cells with low-dose multifraction irradiation in this tumor, although a clear schedule-dependent interaction between the drug and radiation was seen for both aerobic and hypoxic tumors.

The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes. In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied. While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233. For a 90-min hypoxic exposure to 40 μM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes. Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes-drug toxicity decreased slightly with increasing medium volume-this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume. These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.

Nicotinamide has been shown to sensitize tumors to radiation in preference to normal tissues. We have extended our studies to examine the mechanism responsible for this radiosensitization, using the EMT6 tumor model. Our results confirm that nicotinamide (1000 mg/kg) significantly enhances the radiation damage in this tumor when given as a single intraperitoneal injection 90 min before irradiation. The data also show that nicotinamide does not directly sensitize hypoxic cells to radiation either in vitro or in vivo. Excising tumors immediately after irradiation and exposing them to nicotinamide (7 mM) for 24 h similarly failed to increase the radiation damage, implying that nicotinamide does not inhibit the repair of radiation-induced potentially lethal damage. Nicotinamide did, however, produce a decrease in the binding of $[{}^{14}{\rm C}]\text{-misonidazole}$ in tumors, consistent with a reduction in the degree of tumor hypoxia. There was also an increase in mean tumor cell fluorescence of Hoechst 33342 in nicotinamide-treated mice compared to that of controls, suggesting that the increase in tumor oxygenation was probably a consequence of an increase in tumor blood perfusion.

The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of rapair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.

Stable monolayers of contact-inhibited C3H 10T 1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T 1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.

The objective of this study was to characterize the extent of and mechanisms involved in radiosensitization by 2-nitroimidazoles in multifraction schedules using low doses per fraction. For this purpose, contact-inhibited monolayers of C3H 10T 1/2 cells were given 1.7 Gy every 12 h and plated 12 h after the last dose received to allow full repair of potentially lethal damage (PLD). Severe hypoxia was obtained by a 1-h gassing procedure at room temperature immediately before each irradiation. No toxicity occurred as a consequence of multiple exposures to 5 mM misonidazole (MISO) or SR 2508 (2508) during the deoxygenation procedure. Experimental conditions during the pregassing and irradiation (presence of drug and gas mixture) were appropriately manipulated to test for the different mechanisms of radiosensitization demonstrated by nitroimidazoles. A very low oxygen enhancement ratio (OER) results under these conditions (1.34). Exposure to 5 mM MISO or 2508 during the deoxygenation and irradiation of hypoxic cells resulted in greater radiosensitization than could be accounted for by oxygen-mimetic sensitization alone (MISO and 2508 enhancement ratios were greater than the OER). Pregassing cells with N 2 in the presence of 5 mM drug sensitized cells which were subsequently irradiated under aerobic conditions (drug free), indicating the occurrence of the "preincubation effect" (which does not require hypoxia or the drug's presence during the irradiation). Thus, for the hypoxic irradiations, the preincubation effect could account for the greater sensitization by nitroimidazoles than by oxygen. The presence of 5 mM drug only during the irradiation of aerobic cells produced radiosensitization in both multifraction and single-dose experiments with delayed plating. This sensitization has been previously shown to involve reduced PLD repair. Finally, maximum radiosensitization occurred in the multifraction schedule when a transient period of hypoxia with drug preceded an aerobic irradiation with drug present, thus combining the benefits of both the preincubation effect and PLD repair inhibition. This work demonstrates the possibility that effects other than oxygen-mimetic radiosensitization could be largely responsible for the sensitization seen in multifraction schedules, particularly when the OER is already low and only transient periods of hypoxia occur.

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses >1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.