Gamma-radiation (GR) triggers a unique gene expression profile associated with cell death compared to proton radiation (PR) in mice in vivo.

Proton radiation (PR) therapy offers a number of potential advantages over conventional (photon) gamma-radiation (GR) therapy for cancer, due to a more localized delivery of the radiation dose. However, the pathophysiological effects following PR-exposure are less well characterized than those of GR-exposure and the molecular changes associated with the acute apoptotic effects in mice in vivo following PR have not been elucidated. Previous studies have estimated the RBE of protons for various in vivo and in vitro endpoints at between 1.1 and 1.3. We assumed an RBE of 1.1 for the endpoints to be evaluated in these studies. Based on this assumption, ICR mice were treated with whole-body doses of GR (1.1 and 7.0 Gy) and PR (1.0 and 6.4 Gy) that were expected to represent RBE-weighted doses. The bone marrow, thymus, spleen and GI-tract were isolated and processed for histology and immunohistochemistry. The apoptotic responses varied greatly between GR and PR in a tissue- and dose-dependent manner. Surprisingly,cell death in the splenic white pulp was consistently lower in PR-treated animals compared to animals treated with GR. This was in spite of an increased presence of damaged DNA following PR as determined by staining for gammaH2AX and phospho-ATM. Interestingly, both PR and GR triggered nuclear accumulation of p53 and no significant differences were found in the majority of the known pro-apoptotic p53-target genes in the spleens of treated mice. However, GR uniquely triggered a pro-apoptotic expression profile including expression of the pro-apoptotic, p53- and interferon stimulated target gene Bcl-G. In contrast to PR, GR may, in a cell type specific manner, trigger a more diverse non-random stress-response that mediates apoptosis partially independent of the extent of DNA damage.

Hsp25-induced radioresistance is associated with reduction of death by apoptosis: involvement of Bcl2 and the cell cycle.

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.

Influence of apoptosis on the enhancement of radiotoxicity by ouabain.

BACKGROUND: The Na+, K(+)-ATPase inhibitor ouabain enhances the toxicity of irradiation and we have previously demonstrated that the drug suppresses repair capacity. The influence of ouabain on apoptosis is not known and is examined in this study. MATERIALS AND METHODS: Seven human cell lines of defined TP53 status were irradiated with 60Co-gamma irradiation in the presence and absence of 10(-7) M ouabain. Cell survival was determined by the clonogenic assay, apoptosis by acridine orange staining and cell cycle delays by flow cytometry. RESULTS: The ouabain-induced enhancement of radiotoxicity, expressed as the ratio of SF2's, is independent of TP53 status and ranges from 1.1 to 2.8 depending upon cell line. Ouabain prolongs the irradiation-induced G2 delay in TP53 mutant tumor cell lines by a factor greater than 2, but not in the normal lung fibroblast L132, where the cell recovery is not altered in the presence of ouabain. Twenty hours post irradiation, ouabain enhances apoptosis induced by irradiation by factors of 1.3 to 1.7 depending on the cell line. CONCLUSION: Ouabain preferentially enhances the radiotoxicity in tumor cells irrespective of TP53 status. In the pattern of DNA damage responses which are influenced by ouabain we show that the G2 cell cycle delay is prolonged and that early apoptosis events are upregulated in TP53 wild type and TP53 mutant cells. It is concluded that apoptosis plays a significant role in the enhancement of radiotoxicity by ouabain.

[Antimitotic compounds as modifiers of the radiation lesion of cultured cells]. / Antimitoticheskie soedineniia kak modifikatory luchevogo porazheniia kul'tiviruemykh kletok.

The features of antimitotic substances as radioprotectors were studied. In vitro experiments have demonstrated that taxol revealed radioprotective features concerning the process of polymerization of irradiated microtubules. These results were the basis for the use of taxol and some other substances with high affinity for cytoskeleton proteins as potential radiomodificators in vivo. Experiments with cultivated fibroblasts revealed that colchicine significantly enhances radioactive injuries of cells while taxol and phalloidin manifest their radioprotective features.

Evidence for an altered kinetics of DNA excision-repair in cells infected by herpes simplex virus type 1.

In cells infected with herpesviruses a series of host cell nuclear changes can be observed in a temporal sequence. Such changes include chromosome aberrations. The precise mechanism by which virus infection produces chromosome damage is not known, but we have previously reported that herpes simplex virus type 1 (HSV-1) induces a significant number of single-stranded breaks in the host cell DNA at early hours of infection and in a time-dependent fashion. Here, it is reported that HSV-1-infected cells subjected to irradiation with ultraviolet light, show an altered kinetics in the normal process of DNA excision-repair at early hours of infection.

[The mitotic cycle and DNA repair in UV-irradiated murine neuroblastoma cells]. / Mitoticheskii tsikl i reparatsiia DNK v UF-obluchennykh kletkakh neiroblastomy myshei.

A correlation has been shown between a reduced rate of movement of UV-irradiated neuroblastoma cells from G1 into S phase, an essential increase of cells in S phase while progressing through the cell cycle, and a defect in free DNA synthesis on a damaged template. These indices may reflect one and the same cell response to the UV light.

Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction.

Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakage were decreased significantly by preceding photodynamic treatment, suggesting that hyperthermia and photodynamic treatment have a common target for the induction of K+ leakage. Several lines of reasoning indicate that this common target is band 3. A model is thus proposed for the observed potentiation of hyperthermically induced K+ leakage by photodynamic treatment, in which photo-oxidation of band 3 results in increased sensitivity to subsequent thermal denaturation. These phenomena may be of more general significance, as photodynamic treatment and hyperthermia interacted synergistically with respect to K+ leakage with L929 fibroblasts also.

After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase.

Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in the G2-phase (G2-block), and, second, the cellular content of the O6-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in the G2-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked in G2 and the time period the cells persisted in G2 increased with the radiation dose. About 24 h past release from the G2-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into the G2-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of the G2-phase, but not before or during the S-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.

Post-treatment interactions of photodynamic and radiation-induced cytotoxic lesions.

The interaction of chloroaluminum phthalocyanine-sensitized photodynamic treatment and gamma-irradiation was studied in confluent murine L929 fibroblasts. When the cells were given the combined treatments and immediately subcultured for determination of cell survival by colony formation, the data indicate independent actions of each modality. However, when subculture was delayed for 1 h, a substantial fraction of cells treated with a sub-lethal dose of PDT followed by 5 Gy gamma-radiation detached from the monolayer. Most of these detached cells were no longer clonogenic. The mode of photosensitized cell killing was found to be different from that of ionizing radiation-induced cell killing. Photosensitized cell killing was accompanied by morphological changes in the cells and extensive DNA degradation within one hour following the treatment. When chloroaluminum phthalocyanine pretreated cells were exposed to a sublethal fluence of light (6 kJ/m2) and a lethal dose of gamma-radiation (5 Gy), DNA degradation was enhanced, and about 20% of the cell population appeared to undergo the type of cell death typical of photodynamic treatment. Thus, although different initial lethal lesions are induced by photodynamic treatment and by ionizing radiation, interactions may occur during processing of the damage.

RNA synthesis and stability in UV-irradiated and nonirradiated mouse L cells.

In mouse L cells, relatively low doses of UV light (e.g., about 35 J/m2) induced the rapid breakdown of the molecules of many RNA species transcribed shortly before irradiation. This included 28S, 18S, 5.8S, and 5S rRNA, U1, U2, U3, U4, and U5 small nuclear RNA, but not the main band of transfer RNAs or 7SL RNA. At higher UV doses, an RNA band that contains tRNAleu was also degraded rapidly after UV irradiation. RNA molecules synthesized long before irradiation (e.g., 22 h for small RNAs, 4 h for large rRNAs) were not affected. Our results suggest that the maturation and/or assembly into fully mature ribonucleoprotein particles of several small RNA species is not completed 4 h after transcription. The effect of UV radiation occurred in mouse L cells, but not in human HeLa or KB cells. In a previous report, L cells were transformed by DNA transfection with two mouse U1b RNA genes, named U1.1 and U1.2. We observed now that, in L cells transformed with the U1.2 gene, the ratio of radioactivity in the apparent U1b and U1a RNA precursors after 5 min of labeling was about 20 times higher than a) this ratio in briefly labeled L cells that had been transformed with the U1.1 gene, and b) the ratio of radioactive mature U1b and U1a RNA after 20 h of chase in L cells transformed with the U1.2 gene. These results suggest that very high levels of U1b RNA are transcribed from the exogenous U1.2 gene copies, followed by the rapid degradation of most of these transcripts.

Comparison of DNA-protein cross-links induced by 4'-(9-acridinylamino)-methanesulfon-m-anisidide and by gamma-radiation.

The antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits topoisomerase II activity through the formation of a complex of DNA and covalently bound enzyme which, upon protein denaturation, yields DNA breaks (single strand breaks). In the present study, this complex served as a standard for analysis of radiation-induced DNA-protein cross-links (DPC). Following the treatment of exponentially growing mouse L929 cells with 0-100 ng/ml of m-AMSA for 1 h, a linear dose-dependent increase was found in the amount of DNA retained on nitrocellulose filters during subsequent analysis. This result indicates that the assay can detect DPC that have a single protein bound to each DNA fragment. The results of fractionation of nuclear DNA show that m-AMSA induces 20- to 45-fold more DPC in nuclear matrix-associated DNA than in the majority distal loop DNA, supporting the notion that topoisomerase II is located at the nuclear matrix. The frequency of single strand breaks induced by m-AMSA, which should be equal to the frequency of DPC, was determined by alkaline elution. Results of the alkaline elution assay could be correlated with the percentage of DNA retained on nitrocellulose filters; i.e., 1% DNA retention corresponded to 2560 DPC per log-phase L929 cell, which has been determined to have a DNA content of 22.25 pg. Using this standard curve, DPC induced by gamma-irradiation in air were estimated to be formed at a frequency of 133 DPC/cell/Gy, a frequency approximately 3% that of gamma-ray-induced single strand breaks. The radiation dose response for DPC production was unaffected by the high levels of DPC present in cells previously treated with m-AMSA. In addition, DPC induced by m-AMSA were rapidly reversed after the removal of the drug, in contrast to a slower removal of DPC induced by gamma-radiation. These observations suggest that although m-AMSA and gamma-radiation both preferentially induce DPC with matrix-attached DNA, they produce independent types of DPC.

Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.

An amber codon (TAG) was introduced into the N-terminal coding region of the murine H-2Kb gene. The mutant gene was transfected into mouse L cells, and a clone containing a single unrearranged chromosomally integrated copy of the mutant gene was mutagenized with 254-nm UV radiation. Surviving cells were scored for surface expression of H-2Kb protein with in situ immunoperoxidase staining. Revertants were detected at a frequency of 3 X 10(-6) at a dose of 40 J/m2 (3-5% survival). Revertant genes, cloned by plasmid rescue, contained the expected thymine-to-cytosine transitions at the amber codon. These data show that revertants can be rapidly detected in mammalian cells without selection and provide a basis for the development of mammalian cell lines that could be used to study mutational phenomena. During this study the steady-state level of mRNA was reduced in L cells carrying the amber mutant H-2Kb gene compared with L cells containing a wild-type or revertant H-2Kb gene. This reduction was shown not to be due to transcriptional differences, suggesting that the amber mutation decreases stability of the H-2Kb mRNA.

Effects of hyperthermia on mouse L cells irradiated with fractionated X-rays.

The responses to X-rays and radiosensitizing effects of post-treatments with heat were examined in the cells exposed to different doses of multifractionations. When cells were irradiated with daily doses of 2 Gy the radiation responses changed during multifractionated treatments, giving rise to two components in survival curves. The changes may be due to selective killing of radiosensitive cells or a cell progression through radioresistant phases during multifractionation irradiation as shown in analysis of the cell cycle. However, the survival curves became almost exponential when fractionation was performed with daily doses of 3-4 Gy. Radiosensitization by heat at 43 degrees C for 15 min was detectable in the cells after exposures to fractionated doses of more than 7 Gy with daily doses of 2 Gy, but not after exposures to daily doses of 3-4 Gy. Radiosensitization by heat at 43 degrees C for 30 min was manifest as disappearance of shoulder and increase of the slope in survival curves after irradiation with daily doses of 2 Gy. It was observed as an increase of slope in survival curves when cells were irradiated with daily doses of 3 or 4 Gy. Such differences in the radiosensitization may be due to a difference in the susceptibility to heat among the cells which had been exposed to the different doses and regimens of fractionated irradiation.

Failure to produce greater than additive sister chromatid exchange induction with X rays and BCNU.

In 1984, Tofilon and Deen reported that X irradiation of 9L cells immediately after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) produced greater than additive induction of sister chromatid exchanges (SCEs) compared to the sum of SCEs induced by each agent alone [Radiat. Res. 97, 171-177 (1984)]. We (C.P.S., D.F.D.) repeated experiments conducted by Tofilon and Deen, scored SCEs in a blind manner, and were unable to reproduce the original findings. Instead, we found that X rays and BCNU induced SCEs in an additive manner. The slides prepared in the original study are extant; they were coded and recounted blind. Data obtained in this reevaluation do not substantiate the previous report. We conclude that the original findings were apparently the result of bias introduced in the SCE scoring process. Our experience with the SCE assay emphasizes the need to recode SCE slide preparations and count SCEs blind.

The effects of reduced temperature and/or starvation conditions on the radiosensitivity and repair of potentially lethal damage and sublethal damage in L5178Y-R and L5178Y-S cells.

Potentially lethal damage (PLD) and sublethal damage (SLD) modification in L5178Y-S(LY-S) and L5178Y-R (LY-R) cells was investigated for postirradiation holding in either plateau phase or log phase at either 25 or 37 degrees C. Incubation in both plateau and log phases increased PLD repair (PLDR) at 25 degrees C but increased PLD fixation (PLDF) at 37 degrees C in LY-S cells, with the opposite result (PLDR at 37 degrees C, PLDF at 25 degrees C) in LY-R cells. Conditioned medium (CM from plateau-phase cells) had only a slight effect on the radiosensitivity of log-phase LY-S cells. CM was highly toxic to log-phase LY-R cells even without radiation. Postirradiation incubation of log-phase cells at 25 degrees C increased PLDF between 5.5 and 18 h in LY-R cells, whereas PLDR was completed by 5.5 h in LY-S cells. Flow cytometric data show that the LY-R vs LY-S plateau-phase PLD results are not caused by a differential cell cycle distribution. Although all cell cycle stages are found in the plateau-phase cells, G1 is enriched and cells in all stages must be slowly cycling or stationary. Split doses decrease survival of LY-S cells at 37 degrees C but maintain survival at the single acute total dose level at 25 degrees C (no SLD repair (SLDR)). SLDR is observed at both 25 and 37 degrees C in LY-R cells. The results for both the split dose and delayed plating experiments are best understood in terms of competition between PLD repair and fixation with their relative contributions depending on cellular metabolism, which can be altered by changes in temperature or medium constituents. The opposite PLD results obtained for LY-S and LY-R cells suggest that radiosensitivity in these cells is determined by two different mechanisms.

Antibody-nucleic acid interactions. Antibodies to psoralen-modified RNA as probes of RNA structure.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.

Photodynamic effects of hematoporphyrin-derivative on enzyme activities of murine L929 fibroblasts.

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin-derivative resulted in the inactivation of cytosolic, mitochondrial and lysosomal enzymes and in a decrease in cellular adenosine triphosphate and reduced glutathione concentrations. Comparison of these results with those of previous studies revealed that transmembrane transport systems and DNA repair enzymes are inactivated after much shorter illumination periods than are intracellular enzymes. Although the pattern of photodynamic damage altered by varying the protocol of preincubation with hematoporphyrin-derivative and washing, it appeared that under all experimental conditions the plasma membrane was much more sensitive to photodynamic damage than were the intracellular enzymes. Lysosomal membrane disruption with subsequent detrimental release of lysosomal enzymes has been implicated previously in certain forms of porphyrin-induced photodynamic cell destruction. Cytochemical studies on enzyme localization virtually exclude such a mechanism in hematoporphyrin-derivative-induced cell inactivation in L929 fibroblasts.

Disulfiram as a radiation modifier.

The radiation modifying effect and toxicity of tetraethylthiuram disulfide (disulfiram) have been studied. Disulfiram (DSM) inhibits aldehyde dehydrogenase, dopamine-beta-oxygenase, microsomal mixed-function oxidases and cytochrome P-450 enzymes. It is widely used for aversion therapy in alcoholism. Disulfiram also inhibits tumor formation by several known carcinogens. A biphasic toxicity pattern of DSM is reported in the L-929 mouse fibroblast culture system. Disulfiram is 100 percent toxic at 2 X 10(-7) M (0.05 micrograms per ml), 23 percent toxic at 3 X 10(-7) M (0.1 microgram per ml), and 100 percent toxic again at 3.4 X 10(-6) M (1.0 microgram per ml). The pattern is similar to the biphasic toxicity pattern of DMS's major metabolite, sodium diethyldithiocarbamate (DTC). Reports of both radiation protection and radiation enhancement by DTC exist. Previously, a radioprotective effect by 2 X 10(-6) M DTC (dose modifying factor = 1.26) has been demonstrated in the L-929 cell system. To date, no radiation modifying properties of DSM have been reported. Our investigation of DSM as a radiation modifier at 3 X 10(-7) M (0.1 microgram per ml) did not show significant improvement in survival of irradiated cells treated with DSM relative to the irradiated control group, as determined by absence of a difference in the Do of the two groups. Considering DSM's close structural relationship to DTC, it is possible that DSM may exhibit a radioprotective effect when applied in a different concentration than what was used in our research.