Induction of a senescence-like phenotype in bovine aortic endothelial cells by ionizing radiation.

Treatment of confluent monolayers of bovine aortic endothelial cells (BAEC) with gamma rays resulted in the delayed appearance of cells with an enlarged surface area that were morphologically similar to senescent cells. The majority of these cells stained positively for senescence-associated beta-galactosidase (SA-beta-gal), indicating that these cells are biochemically similar to senescent cells. The incidence of the senescence-like phenotype increased with dose (5-15 Gy) and time after irradiation. Cells with a senescence-like phenotype began to appear in the monolayer several days after irradiation. The onset of the appearance of this phenotype was accelerated by subculturing 24 h after irradiation. This acceleration was not entirely due to stimulation of progression through the cell cycle, since a high percentage of the senescent-like cells that appeared after subculture were not labeled with BrdUrd during the period after subculture. Prolonged up-regulation of expression of CDKN1A (also known as p21(CIP1/WAF1)) after irradiation was noted by Western blot analysis, again suggesting a similarity to natural senescence. Phenotypically altered endothelial cells were present in the irradiated monolayers as long as 20 weeks after irradiation, suggesting that a subpopulation of altered endothelial cells that might be functionally deficient could persist in the vasculature of irradiated tissue for a prolonged period after irradiation.

Oxidative stress-induced apoptosis of endothelial cells.

Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O2*-. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 microM menadione resulted in an apoptotic rate of approximately 5% in capillary EC cultures compared with approximately 45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 microM), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 microM for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.

Determining the optimal block margin on the planning target volume for extracranial stereotactic radiotherapy.

PURPOSE: To determine the block margin that minimizes normal tissue irradiation outside of the planning target volume (PTV) for body stereotactic radiotherapy (Body-SRT) of lung and liver tumors. METHODS AND MATERIALS: Representative patient cases of lung and liver tumors were chosen for analysis. A PTV was constructed for each case and plans were generated which employed an array of block margins ranging from -2.5 mm to 10 mm at isocenter. Plans were generated for cerrobend blocks and for a multileaf collimator. The prescription isodose coverage was renormalized for each case and dose-volume histograms (DVH) and normal tissue complication probabilities (NTCP) were determined for each plan. RESULTS AND CONCLUSION: For the cases studied, the optimal block margin was in the 0.0 mm range. The ranking of plans was identical for both dose-volume based and biological based criteria. The method of blocking had no significant effect on treatment plans. The use of narrow margins for Body-SRT results in normal tissue sparing and creates significant target dose inhomogeneity which may be beneficial for tumor control.

An oxidative stress-mediated death pathway in irradiated human leukemia cells mapped using multilaser flow cytometry.

OCI/AML-2 acute myeloid leukemia cells were found to undergo apoptosis after treatment with y rays from a 137Cs source. Multilaser flow cytometry techniques using probes for live cell function were used to monitor the biochemical changes that occurred prior to the loss of surface membrane integrity. These showed increases in the generation of reactive oxygen species (ROS) and in the glutathione (GSH) content of irradiated cells. An additional population of cells that showed a further increase in ROS and depletion of GSH was seen in irradiated cells but not in controls. This population showed loss of mitochondrial membrane potential (deltapsim), indicative of the mitochondrial permeability transition, and exposure of phosphatidylserine on the cell surface. Increases in intracellular calcium were observed in a proportion of these low-deltapsi(m)/high-ROS cells. Similar findings were seen using the antileukemia drug cytosine arabinoside (ara-C), although cell cycle analysis showed that the loss of deltapsi(m) occurred mainly in G1 phase with ara-C treatment, and mainly in G2 phase with irradiation. Furthermore, the protective effect of overexpression of BCL2 was more pronounced after ara-C treatment than with radiation. Cells of the TP53 (formerly known as p53)-null human AML line OCI M2 showed growth arrest in G2 phase after radiation treatment, with no loss of deltapsi(m) or morphological changes indicative of apoptosis. The flavine-dependent oxidoreductase inhibitor diphenylene iodonium failed to inhibit generation of ROS in irradiated OCI/AML-2 cells, indicating that the mechanism is unlikely to involve the TP53-induced gene PIG3. These results show that oxidative stress can occur in irradiated human leukemia "blasts", and may play a direct role in radiation-induced apoptosis.

Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis.

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G2 cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G2 cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.

Combined antitumor effect of radiation and ibuprofen in human prostate carcinoma cells.

Recent clinical observations indicate that ibuprofen may alleviate the radiation-induced dysuria that almost invariably occurs during radiation therapy for prostate cancer. Because the use of ibuprofen could consequently become common during radiation therapy for prostate cancer, we have been interested in the potential interactions between ibuprofen and ionizing radiation on prostate tumor cells. The effects of gamma-irradiation and/or ibuprofen on PC3 and DU-145 human prostate carcinoma cells were evaluated in vitro using three model systems. Clonogenic survival was determined by plating cells 24 h after treatment of nearly confluent monolayers. Analysis of cell growth, cell detachment, and apoptotic cell death was carried out over a period of up to 9 days after treatment of PC3 and DU-145 monolayers. The effect of ibuprofen and/or radiation was also probed by observing the inhibition of growth of established PC3 and DU-145 colonies that were treated on the 14th day of colony growth. Ibuprofen enhanced the radiation response of prostate cancer cells in all three in vitro models. Both the cytotoxic and radiosensitizing effects of ibuprofen seem to require concentrations that are higher than those reported to inhibit prostaglandin synthesis, suggesting that other molecular mechanisms may be responsible for ibuprofen cytotoxicity.

Continuous detection of radiation or metal generated hydroxyl radicals within core chromatin particles.

PURPOSE: The aim of this work was to adapt a recently developed fluorometric method for use in the detection of hydroxyl radical (HO.) generated in the immediate vicinity of chromatin core particles reconstituted from pUC19 plasmid DNA and isolated core histones. MATERIALS AND METHODS: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the fluorescent 7-hydroxy-SECCA after reaction with HO.. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. RESULTS: Electron microscopy and micrococcal nuclease digestion are consistent with successful formation of core particles. No significant differences between core particle formation in the unlabelled and SECCA-labelled samples were detected. Exposure to HO. generated by radiation or copper--ascorbic acid--hydrogen peroxide results in a gradual induction of fluorescence. Studies using dimethyl sulphoxide (DMSO) demonstrate that, unlike HO. produced by radiation, the majority of HO. generated by copper--ascorbic acid--hydrogen peroxide occurs primarily in the immediate vicinity of core particles and DNA and cannot be scavenged. CONCLUSIONS: The present procedure demonstrates the feasibility to quantitate HO. generated by several agents in the immediate vicinity of nucleosomes (chromatin-associated HO.) or associated with specific regions within the genome.

Apoptosis and clonogenic cell death in PC3 human prostate cancer cells after treatment with gamma radiation and suramin.

Suramin is a novel cytostatic/cytotoxic agent that is currently undergoing clinical trials in the treatment of hormone- and chemo-refractory tumors. Its unusual mechanism of action and its activity against prostate cancer raise the possibility that it could be particularly suitable for combined-modality treatment of prostate cancer. PC3 human prostate cancer cells were used as an in vitro model to test the possible interaction between suramin and ionizing radiation. Treatment with gamma radiation resulted in detachment of PC3 cells from the monolayer, and the detached cells exhibited internucleosomal DNA fragmentation characteristic of apoptosis. Low concentration of suramin (50-100 micrograms/ml, 35-70 microM) increased spontaneous as well as radiation-enhanced apoptosis. However, suramin inhibited spontaneous and radiation-enhanced apoptosis at 300 micrograms/ml (210 microM), a concentration that is more commonly used in the clinic. At this concentration suramin inhibited DNA fragmentation induced by chemotherapeutic drugs as well. The effect of suramin on inhibition of DNA fragmentation was reversible if the suramin was removed 24 h after irradiation. Despite inhibition of radiation-induced apoptosis by 300 micrograms/ml suramin (from 5% to 2.9% at 48 h), clonogenic cell death was enhanced by the combination of suramin and radiation. The effects of radiation and suramin on clonogenic cell survival appeared to be additive by isobologram analysis at clinically relevant radiation doses. Continuous exposure to a lower concentration of suramin (100 micrograms/ml) during the clonogenic assay period was as effective in decreasing clonogenic survival as 48 h exposure to 300 micrograms/ml suramin in decreasing clonogenic survival. Our data indicate that, when used in combination with radiation, suramin may be effective at concentrations that are lower than those required for efficacy as a single agent.

Radiation-induced apoptosis in microvascular endothelial cells.

The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary endothelial cells (ECs) to ionizing radiation and bFGF. Apoptosis was assessed by identifying changes in nuclear morphology, recording cell detachment rates and by detecting internucleosomal DNA fragmentation. Withdrawal of bFGF alone induces apoptosis in these monolayers. The magnitude of this apoptotic response depends upon the duration of bFGF withdrawal. Irradiation (2-10 Gy) induces apoptosis in a dose-dependent manner. Radiation-induced apoptosis occurs in a discrete wave 6-10 h after irradiation, and radiation-induced apoptosis is enhanced in cultures that are simultaneously deprived of bFGF. For example, 6 h after 10 Gy, 44.3% (s.e. 6.3%) of cells in the monolayer simultaneously deprived of bFGF exhibit apoptotic morphology compared with 19.8% (s.e. 3.8%) in the presence of bFGF. These studies show that either bFGF withdrawal or ionizing radiation can induce apoptosis in confluent monolayers of capillary endothelial cells and that radiation-induced apoptosis can be modified by the presence of bFGF.

Novel fluorescein-based flow-cytometric method for detection of lipid peroxidation.

The novel property of fluorescein to detect peroxyl radicals is demonstrated. On the basis of this observation, a fluorescein-based, flow-cytometric method to directly and continuously detect free radicals generated in cell membranes during lipid peroxidation has been developed. 5- and 6-Carboxyfluorescein (5-/6-CF) free in solution and fluorescein-labeled polylysine lose their fluorescence gradually upon addition of a peroxyl-radical-generating system (thermal decomposition of 2,2'-azobis(2-amidinopropane) [AAPH]). 5-/6-CF retains its fluorescence when exposed to AAPH in the presence of the peroxyl radical scavenger Trolox. When 5-/6-CF free in solution is incubated with red blood cells exposed to cumene hydroperoxide (CH), a similar loss of fluorescence occurs due to lipid peroxidation on RBC membranes, which is preventable by pretreatment of the cells with Trolox or vitamin E. Undecylamine-fluorescein (C11-fluor), a lipophilic fluorescein conjugate, has been incorporated into the membranes of RBC. Upon addition of CH, a decrease in fluorescence is fluorometrically observed that is proportional to the amount of hydroperoxide added and inhibited by preincubation with Trolox or vitamin E. Flow-cytometric studies are then performed to demonstrate that C11-fluor can monitor free radicals generated during lipid peroxidation on a cell-by-cell basis. When exposed to CH, a time-dependent shift of the flow-cytometric profile toward lower values is observed that is inhibited by Trolox or vitamin E. This approach in conjunction with multiparametric flow cytometry may allow examination of the biologic significance of lipid peroxidation by correlation to other cellular end points on single cells.

Generation of hydroxyl radicals by nucleohistone-bound metal-adriamycin complexes.

A recently developed method has been utilized to demonstrate the generation of hydroxyl radicals (HO.) in the immediate proximity of DNA by copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide. SECCA, a succinylated derivative of coumarin, generates the fluorescent 7-hydroxy-SECCA following reaction with HO.. SECCA was coupled to polylysine or to histone H1 and then complexed to DNA. When HO. was generated in the proximity of DNA by polylysine-coupled iodine-125, which emits short range Auger electrons, 7-hydroxy-SECCA was produced. DMSO was only moderately efficient in reducing the fluorescence induction, demonstrating the "local" generation of HO. in this system. Copper(II)/iron(III)-adriamycin in the presence of ascorbate and hydrogen peroxide generated the fluorescent 7-hydroxy-SECCA both when SECCA was free in solution and when SECCA was DNA-conjugated. With SECCA free in solution, the fluorescence induction was almost eliminated in the presence of HO. scavengers (ethanol, tert-butanol or DMSO) and the relative efficiency of the scavengers in reducing the fluorescence followed their rate constant with HO.. Furthermore, SECCA incubated with a single oxygen-generating compound demonstrated no fluorescence induction. When SECCA was positioned in close proximity to DNA as a SECCA-histone-H1-DNA complex, the relative efficiency of the scavengers in reducing the fluorescence still followed their rate constant with HO.; overall however the scavengers were much less effective in reducing the fluorescence, due presumably to the formation of HO. radical in the immediate vicinity of DNA. These data suggest that copper(II)/iron(III)-adriamycin produces HO. in the presence of ascorbate and hydrogen peroxide whether unbound or bound to DNA and suggest that in the latter case scavengers would not prevent HO. from attacking chromatin. In addition, the ability of DMSO to trap HO. was shown to decrease as the conformation of the H1-DNA complex becomes more compact indicating the strong dependence of the trapping ability on chromatin conformation.

Effect of BSO and etanidazole on neurofilament degradation in neonatal rat spinal cord cultures.

Peripheral neuropathy is the major dose-limiting toxicity of the hypoxic cell sensitiser, etanidazole. Previous work from this laboratory using culture neuronal cell lines suggested that nitroimidazole-induced degradation of neurofilament proteins might be the critical biological event mediating this neurotoxicity. The purpose of the present study was to develop the neurofilament degradation assay in an organotypic spinal cord culture system with the goal of developing strategies for optimising sensitiser efficacy as well as ameliorating nitroimidazole-induced neurotoxicity. Spinal cord cultures were treated with etanidazole and neurofilament protein degradation was analysed by immunoblot analysis. Spinal cord cultures exposed to etanidazole exhibited a dose-dependent loss of parent neurofilament proteins, with concomitant appearance of low molecular weight degradation products. The potential neurotoxic effect of L, S-buthionine sulphoximine (BSO), a compound that enhances the radiosensitising effectiveness of 2-nitroimidazoles, was also screened in this assay system. BSO alone, at concentrations up to 100 microM, did not promote neurofilament degradation. BSO (20 microM) enhanced the effect of etanidazole on neurofilament degradation by a dose-modifying factor of 1.6 +/- 0.5. Since 20 microM BSO is expected to enhance etanidazole radiosensitisation of hypoxic cells by a larger factor, this suggests that a therapeutic gain could be achieved using BSO in combination with etanidazole in radiation therapy.

Tempol prevents impairment of the endothelial cell wound healing response caused by ionising radiation.

It is known that radiation therapy results in some form of damage to the microcirculation. In support of this view, we found that capillary endothelial cells (EC) treated with X-rays (8 Gy) were defective in their ability to recover a denuded area. A scrape wound of 2 mm width was produced in monolayers 30 min after X-ray or sham treatment. After 48 h, the number of cells migrating into each of five successive 125 microns zones from both sides of the original wound were determined. Greater numbers of sham-treated EC entered zones 3 and 4, compared with irradiated cultures, and only sham-treated EC entered the most distant zone 5. We examined actin fibre orientation within migrating irradiated and sham-treated EC using 2-(D-2-aminobutanoic acid)-7-(N6-((((3,6-bis(dimethylamino)xanthylium-9-yl) carboxyphenyl) amino)thioxomethyl)-L-lysine), chloride (NBD)-phalloidin, immunofluorescent microscopy and computer image analysis. After 48 h, sham-treated, but not irradiated EC, contained actin which was orientated perpendicular to the original wound edge. After 6-9 days, only sham-treated EC closed the wounds. Tempol (4 hydroxy-2,2,6,6-tetra methylpiperidine-1-oxyl)(0.5 or 2 mM)), included in the media during irradiation, prevented this wound healing delay, when measured within the first 24 h. In conclusion, radiation treatment of capillary EC results in a wound healing defect. This defect appears to be related to the EC's inability to realign actin. Tempol protects EC from exhibiting a wound healing delay.

Measurement of hydroxyl radicals catalyzed in the immediate vicinity of DNA by metal-bleomycin complexes.

A recently developed sensitive fluorimetric assay has been used to examine whether free hydroxyl radicals (HO.) are generated in the immediate vicinity of DNA by Fe(II)-bleomycin. When aqueous solutions of SECCA (the succinimidyl ester of coumarin-3-carboxylic acid) are irradiated with gamma rays or incubated with Fe(II)-bleomycin or Fe (II)-EDTA in the presence of ascorbate and H2O2, 7-hydroxy-SECCA, a fluorescent product of the interaction of HO. with SECCA, is generated. Studies with catalase and several HO. scavengers indicate that the fluorescence induction is mediated by HO. On the contrary, Cu(II)-bleomycin complexes under similar conditions fail to induce 7-hydroxy-SECCA fluorescence. When SECCA is conjugated to DNA via SECCA-polylysine-DNA complexes and incubated in the same iron-containing systems, the relative ability of the scavengers to reduce the fluorescence again demonstrates the generation of 7-hydroxy-SECCA by HO. However, while the fluorescence is practically eliminated by high concentrations of DMSO (100 mumols dm-3) in the systems with Fe(II) or Fe(II)-EDTA, it is not possible to reduce it similarly in the case of Fe(II)- bleomycin. These data demonstrate the generation of HO. by Fe(II)-bleomycin in the immediate vicinity of DNA. Because the experiments simulate the lifetime of HO. expected in cells, these data suggest that, if such DNA-associated HO. radicals are also produced in vivo by bleomycin, these would not be scavengable by intracellular scavengers and they could interact with chromatin.

Effect of cell cycle stage, dose rate and repair of sublethal damage on radiation-induced apoptosis in F9 teratocarcinoma cells.

There are at least two different modes of cell death after treatment with ionizing radiation. The first is a failure to undergo sustained cell division despite metabolic survival, and we refer to this end point as "classical reproductive cell death." The second is a process that results in loss of cell integrity. This second category includes cellular necrosis as well as apoptosis. Earlier studies in our laboratory showed that the predominant mechanism of cell death for irradiated F9 cells is apoptosis, and there is no indication that these cells die by necrosis. We have therefore used cells of this cell line to reassess basic radiobiological principles with respect to apoptosis. Classical reproductive cell death was determined by staining colonies derived from irradiated cells and scoring colonies of less than 50 cells as reproductively dead and colonies of more than 50 cells as survivors. Cells that failed to produce either type of colony (detached from the plate or disintegrated) were scored as having undergone apoptosis. Using these criteria we found that the fraction of the radiation-killed F9 cells that died by apoptosis did not vary when cells were irradiated at different stages of the cell cycle despite large variations in overall survival. This suggests that the factors that influence radiation sensitivity throughout the cell cycle have an equal impact on apoptosis and classical reproductive cell death. There was no difference in cell survival between split doses and single doses of X rays, suggesting that sublethal damage repair is not a factor in radiation-induced apoptosis of F9 cells. Apoptosis was not affected by changes in dose rate in the range of 0.038-4.96 Gy/min.

A fluorimetric method for the detection of copper-mediated hydroxyl free radicals in the immediate proximity of DNA.

An optical method to detect copper-mediated hydroxyl free radicals generated close to DNA and other biomolecules has been developed. Low-molecular-weight polylysines were labeled with SECCA, a derivative of coumarin that generates the fluorescent 7-OH-SECCA following its interaction with hydroxyl free radicals in aqueous solution. These polylysines were then complexed with DNA to place the detector molecule SECCA in the vicinity of the nucleic acid. Following addition of copper sulfate (0-10 mumol dm-3), free radicals were generated by incubation with ascorbic acid (0-1 mmol dm-3) and hydrogen peroxide (0-1 mmol dm-3). A rapid increase in the induced fluorescence was observed corresponding to the formation of the fluorescent 7-OH-SECCA in the polylysine-nucleic acid complex. This fluorescence was not decreased significantly by addition of high concentrations of hydroxyl free-radical scavengers (DMSO, methanol, ethanol and tert-butanol), but was diminished by addition of relatively low concentrations of EDTA (0.1 mmol dm-3), histidine (0.1 mmol dm-3) or catalase (8.3 x 10(-5) mmol dm-3). On the other hand, when such reaction mixtures were incubated with SECCA molecules that were free in solution or SECCA-labeled polylysine in the absence of DNA, the induced fluorescence was diminished by all hydroxyl free-radical scavengers. The efficiency by which the scavengers reduce the fluorescence increases as their hydroxyl rate constant increases. The data indicate that the detector molecule SECCA can be used to detect copper-mediated hydroxyl free radicals generated close to DNA.

Modulation of radiation-induced apoptosis and G2/M block in murine T-lymphoma cells.

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137Cs gamma irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of gamma radiation. We studied the effect of several pharmacological agents on the radiation-induced G2/M block and DNA fragmentation. The agents which reduced the radiation-induced G2/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G2/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G2/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G2/M block, and to use this effect in the treatment of patients with malignant disease.

Accessibility of nucleic acid-complexed biomolecules to hydroxyl radicals correlates with their conformation: a fluorescence polarization spectroscopy study.

A fluorescence methodology has been developed to examine the relationship between the conformational state of specific biomolecules in simple chromatin models and their accessibility to hydroxyl radicals (OH). Polylysine and histone H1 were labelled with SECCA, the succinimidyl ester of coumarin-3-carboxylic acid, which generates the fluorescent derivative 7-OH-SECCA following its interaction with radiation-induced OH in aqueous solution. The fluorescence induced per unit gamma-ray dose reflecting the accessibility of OH to such SECCA-conjugated biomolecules was recorded. The biomolecules were also labelled with the fluorescent derivative 7-OH-SECCA in trace amounts to study their conformation under identical conditions via fluorescence polarization spectroscopy. When these biomolecules were complexed with a polynucleotide or DNA, a major increase in polarization anisotropy was recorded. Upon salt-induced dissociation of these biomolecules from the nucleic acids, the increase in anisotropy was reversed. The histone H1-DNA complexes also exhibited an initial increase in anisotropy with increasing NaCl concentration (maximum at 100 mmol dm-3) indicating the possible formation of a more compact conformation. The fluctuations in anisotropy were inversely proportional to the recorded fluorescence/Gy. The data indicate a direct correlation between the accessibility of OH to polylysine or histone H1 complexed with nucleic acids and the conformation of these biomolecules.

Quantification of radiation-induced hydroxyl radicals within nucleohistones using a molecular fluorescent probe.

We present a method that specifically records .OH formation within histones and possibly at other sites in irradiated nucleohistone. The approach uses the radiation-induced fluorescence emissions from a chromatin-conjugated .OH detector, SECCA (a succinylated derivative of coumarin), that is converted to a fluorescent derivative, 7-hydroxy-SECCA (7-OH-SECCA), after interaction with .OH in neutral aqueous solutions. It is shown that (a) the fluorescent product 7-OH-SECCA cannot be generated by direct radiation effects after gamma or neutron irradiation of SECCA; (b) when SECCA-labeled histone is complexed with DNA to form nucleohistone, the physical properties of the modified nucleohistone are similar to those of unlabeled nucleoprotein; and (c) after irradiation of SECCA-labeled nucleohistone, a linear induction of the fluorescence signal is observed within the radiation doses examined (0.3-30 Gy). Since the sample remains available for further studies after registration of the optical signal, the current approach should permit the investigator to correlate in a single sample the localization and frequency of .OH formation with the results of other assays.

Pharmacodynamics of prolonged treatment with L,S-buthionine sulfoximine.

PURPOSE: To develop dosing criteria for the use of L-buthionine-S-sulfoximine (active diastereoisomer) as a glutathione depletor in the clinic, using a pharmacodynamic and pharmacokinetic in vitro-in vivo approach. METHODS AND MATERIALS: In vitro: L-buthionine-S-sulfoximine uptake was determined in human glioblastoma cells (T98G) and NIH-3T3 cells using 35S-labeled drug. Dose response relationships were derived for inhibition of glutathione synthesis in CHO cells, and for depletion of glutathione in exponentially growing T98G and CHO cells, as a function of extracellular L-buthionine-S-sulfoximine concentration. Steady-state glutathione levels for CHO and NIH-3T3 cells were measured using an enzymatic assay, while glutathione synthesis rates in CHO cells were determined using a flow cytometric assay. In vivo: L-buthionine-S-sulfoximine biodistribution was determined in male nude mice carrying human glioblastomas (T98G) intracranially, using 35S-labeled drug infused subcutaneously by osmotic pump. Tissue glutathione levels were measured using an enzymatic assay. RESULTS AND CONCLUSION: The observed cellular uptake t1/2 of approximately 55 min, coupled with a previously reported, rapid in vivo clearance of buthionine sulfoximine, suggest that continuous infusion would be preferable to bolus dosing. Effective concentrations of L-buthionine-S-sulfoximine (24 h exposure), required to lower cellular glutathione content to 50% of control (EC50), were under 1 mM for both cell lines. The amount of L-buthionine-S-sulfoximine in tissues (estimated from 35S drug disposition) reached steady state within 8 h and was proportional to the rate of infusion. Brain tumors were depleted to approximately 50% of control glutathione by a infusion rate of 0.25 mumoles/h (25 g mice). At lower infusion rates an increase in glutathione content was noted in certain nude mouse tissues including brain tumor xenografts.