Nuclear magnetic resonance spectroscopy and sensitizer-adduct measurements of photodynamic therapy-induced ischemia in solid tumors.

Dunning R3327-AT prostate carcinomas growing in Fischer X Copenhagen rats were treated with interstitial photodynamic therapy (PDT--15 mg/kg Photofrin II 4 hours before illumination with 630-nm light via four parallelly implanted optical fibers) at different light intensities. Forty to 60 minutes after treatment, 31P-nuclear magnetic resonance spectra of tumors in anesthetized animals were obtained at 2.35 Tesla using surface coil localization. Areas under resonance peaks were normalized to the area under the peak of a phosphorus standard positioned at a fixed distance on the opposite side of the surface coil. Tumor concentrations of phosphomonoesters and phosphodiesters showed no change after tumor light doses up to 3000 J. Phosphocreatine, alpha-adenosine triphosphate (ATP), beta-ATP, and gamma-ATP signals decreased and inorganic phosphate signals increased with increasing light doses. The intratumor pH did not change significantly at these short times after PDT. In other R3327-AT and R3327-H tumor-bearing animals, [3H]misonidazole was administered 30 minutes prior to PDT treatments of both tumors. Twenty-four hours later, the tumors were resected in toto, and levels of retained [3H]misonidazole were determined in lased tumor specimens by liquid scintillation procedures. The amount of [3H]misonidazole activity in tumor tissue (covalently bound after hypoxic reduction) increased with light doses up to 3000 J. Sensitizer-adduct formation was found to correlate with the ratio of the concentration of inorganic phosphate to that of beta-ATP, both of which are presumed measures of tumor oxygenation status. These measurements have high-lighted the heterogenous nature of the oxygenation status of these experimental tumors. The precision of each assay for estimating tumor oxygenation is discussed.

Tolyporphin: a natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo.

Tolyporphin (TP), a porphyrin extracted from cyanobacteria, was found to be a very potent photosensitizer of EMT-6 tumor cells grown both in vitro as suspensions or monolayers and in vivo in tumors implanted on the backs of C.B17/Icr severe combined immunodeficient mice. Thus, during photodynamic treatment (PDT) of EMT-6 tumor cells in vitro, the photokilling effectiveness of TP measured as the product of the reciprocal of D50 (the light dose necessary to kill 50% of cells) and the concentration of TP is approximately 5000 times higher than that of Photofrin II (PII), the only PDT photosensitizer thus far approved for clinical trials. TP almost exclusively localizes in the perinuclear region and specifically in the endoplasmic reticulum (ER), as shown by microspectrofluorometry on single living EMT-6 cells costained with the ER and/or Golgi fluorescent vital probes, 3,3'-dihexyloxacarbocyanine iodide and N-[4,4-difluoro-(5,7-dimethyl-BODIPY)-1-pentanoyl]-D-erythro-sphin gosine (Molecular Probes, Eugene, OR). As a result, the singlet oxygen-mediated photodynamic activity of TP induces an effective inactivation of the acyl CoA:cholesterol-O-acyltransferase, a sensitive marker of ER membrane integrity and alterations of the nuclear membrane. In vivo, with the EMT-6 mouse tumor model, an exceptional effectiveness is also observed as compared to that of PII and other second generation photosensitizers of the pheophorbide class, which are themselves much more potent than PII. The outstanding PDT activity of TP observed in vivo may be due to its unique biodistribution properties, in particular much less extraction by the liver, resulting in a higher delivery to other tissues, including tumor.

Combined radiation-protective and radiation-sensitizing agents. IV: Measurement of intracellular protector concentrations.

Radiosensitization of hypoxic V79 Chinese hamster cells by 0.5 mM misonidazole at approximately 0-4 degrees C is substantially enhanced by pretreating the cells overnight with 0.1 mM buthionine sulfoximine, which lowers the cellular glutathione content to 5% of control values (from 4 mM to approximately 0.2 mM). The enhanced sensitization is reversed by concentrations of exogenous cysteine that are much lower (0.02 mM) than the original glutathione content. Reduced Co-enzyme A affords reversal of the enhancing effect at concentrations of about 1 mM. Sodium ascorbate gives no protection at all even at concentrations of 2 mM. The intracellular concentration of the reducing agents was measured using a spin-through oil technique. There was no diffusion of Co-A (MW greater than 750) or ascorbate (excluded by charge) into the cells. In contrast, cysteine was rapidly concentrated by factors of 4-10, even at the low temperatures used. Extracellular ascorbate's inability to radioprotect argues against electron transfer across the cell membrane as a mechanism for radioprotection. This mechanism could have explained the ability of exogenous thiols to radioprotect in former studies using glutathione, and in the present studies using Co-A. The potential of cysteine to be concentrated by cells poses a problem in the interpretation of "exogenous protection" by non-diffusing thiols, since trace contamination by cysteine could lead to the actual protection observed. Cysteine could also be formed by exchange reactions of exogenous thiols with the disulfide of cysteine, present in all media formulations.

Metabolism induced binding of 14C-misonidazole to hypoxic cells: kinetic dependence on oxygen concentration and misonidazole concentration.

Under conditions of extreme hypoxia, metabolic products of the metabolism of misonidazole bind to cellular molecules at a rate which is linear with time and proportional to the square root of misonidazole concentration. Very small amounts of oxygen reduce the overall rate of binding and cause a change in the dependence on misonidazole concentration from square root (half order) to linear (first order). Because of the known electron affinity of misonidazole, a model is presented whereby the nitro-group is reduced to a radical in a first order reaction. This radical binds to cellular molecules in a slow first order reaction and either disproportionates or dimerizes in a fast second order reaction. Based on the overall effect of oxygen on the kinetics of the rate of binding, the radical is tentatively assumed to be the 3 electron reduction product.

The role of specific reductases in the intracellular activation and binding of 2-nitroimidazoles.

PURPOSE: To determine the relative effectiveness of specific cellular reductases for the activation and binding of 2-nitroimidazoles in vivo. METHODS AND MATERIALS: Monkey kidney cells were transfected with recombinant plasmids to effect intracellular overexpression of P450 reductase and DT-diaphorase. The covalent binding of 2-nitroimidazoles to cellular macromolecules was measured as a function of time of cell incubation at various oxygen concentrations. The effect of allopurinol on cellular binding of radiolabeled 2-nitroimidazoles was also measured. RESULTS: A 1,000-fold overexpression of DT-diaphorase resulted in a small but significant increase in 2-nitroimidazole binding rate. An 80-fold overexpression of cytochrome P450 reductase resulted in a 5-7-fold increase in the binding rate of 2-nitroimidazole. The inhibition of xanthine oxidase by allopurinol had no effect on 2-nitroimidazole binding rates. The amplification of P450 reductase activity within cells was always much larger than the resultant increase in 2-nitroimidazole binding rate, suggesting an enzyme kinetic process less than first order and possibly of 1/2-order. CONCLUSION: These data suggest that cytochrome P450 reductase is the most important enzyme in these cells for reducing 2-nitroimidazoles to intermediates which can covalently bind to cellular macromolecules. Furthermore, since this cellular process demonstrates approximately 1/2-order kinetics, a tissue's capacity for binding 2-nitroimidazole drug in hypoxia should be proportional to the square root of its intracellular P450 reductase level.

Combined radiation-protective and radiation-sensitizing agents. III: Radiosensitization by misonidazole as a function of concentrations of endogenous glutathione or exogenous thiols.

Radiosensitization of V79 Chinese hamster fibroblasts by 0.5 mM misonidazole is a smooth function of endogenous glutathione (GSH) levels as modulated upwards by pre-incubation in medium containing cysteamine, or downwards by pre-incubation in medium containing buthionine sulfoximine. The enhancement ratio (radiation sensitivity in nitrogen/radiation sensitivity in nitrogen +/- sensitizer or thiol) varies from 1.3 at 12 mM to 2.25 at less than 0.1 mM endogenous GSH. The enhanced radiosensitivity of thiol-depleted hypoxic cells is reversed when exogenous thiols are added, and for equivalent ER, the exogenous thiol concentrations are much lower than the endogenous GSH concentrations. Measurement of intracellular drug concentrations amplified rather than diminished the above discrepancy, since intracellular concentrations of cysteamine were lower and glutathione much lower than the extracellular concentrations. Three possible explanations are addressed: an external membrane component of damage is involved, long-range protection to DNA target radicals is possible from outside the cell (e.g., donation of electrons), and (c) endogenous glutathione is not in a free or exchangeable state (e.g., bound).

Preclinical assessment of hypoxic marker specificity and sensitivity.

PURPOSE: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. MATERIALS AND METHODS: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. RESULTS: The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. CONCLUSIONS: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.

Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays.

Tumor cells at low oxygen tension are relatively radioresistant. The hypoxic fraction of individual tumors before, during and after radiotherapy is likely to have prognostic value but its diagnosis still awaits an accurate and acceptable assay. The recent indications that hypoxia can also induce the expression of specific genes and promote a more aggressive tumor phenotype makes its diagnosis even more important. Over 15 years ago, misonidazole, an azomycin-based hypoxic cell radiosensitizer, was found to link covalently to cellular molecules at rates inversely proportional to intracellular oxygen concentration. The use of bioreducible markers to positively label zones of viable hypoxic cells within solid tumors and to predict for tumor radioresistance was proposed. Several hypoxic markers have now been identified and their selective binding within tumors has been measured by both invasive and non-invasive assays. Research from our laboratory has emphasized both mechanistic and preclinical studies associated with nuclear medicine procedures for measuring tumor hypoxia and predicting tumor radioresistance. This report updates radiation oncologists about the status of nuclear medicine hypoxic marker research and development as of mid-1997. While several potential imaging agents have been identified, their testing and validation in appropriate human tumors will require focused research efforts by individual academic departments and, possibly, by clinical trials performed through cooperative groups. Since the prediction of hypoxia in individual tumors could strongly impact radiotherapy treatment planning, the radiation oncology research community is best positioned to execute the validation studies associated with these markers.

The intrinsic radiosensitivity of some human tumor cells throughout their cell cycles.

The intrinsic radiosensitivity of tumor cells is most frequently reported for asynchronous populations, although cell cycle variation in radiosensitivity is known to be significant. Linear-quadratic analyses of survival data for asynchronous human tumor cells show wide variations in the alpha coefficient with smaller variations in the beta coefficient. HT-29 (colon), OVCAR10 (ovary) and A2780 (ovary) tumor cells with alpha coefficients of 0.03, 0.16 and 0.47 Gy(-1), respectively, and square-root of beta coefficients of 0.23-0.27 Gy(-1) for asynchronous populations were amenable to synchronization by mitotic selection. Selection procedures were optimized for each cell line and produced mitotic populations of >90%, approximately 80% and approximately 65% purity for HT-29, OVCAR10 and A2780 cells, respectively. Mitotic cells from each line exhibited similar and maximum radiosensitivities with alpha coefficients of approximately 1.3 Gy(-1) after irradiation with 137Cs gamma rays and after correction for genome multiplicity. Their relative radiosensitivities observed with asynchronous cells were maintained as they progressed through interphase of the cell cycle. All cells in early G1 phase exhibited a marked radioresistance relative to their sensitivity in mitosis, and maximum interphase radiosensitivity was observed near the G1/S-phase boundary. All cells became increasingly radioresistant as they moved through S phase, the effect being most pronounced for OVCAR10 cells and least pronounced for A2780 cells. HT-29 cells remained relatively radioresistant in G2 phase. The different interphase radiosensitivities observed for these cell lines were determined mainly by the single-hit inactivation mechanism. These studies clearly demonstrate the dominant role of single-hit inactivation in determining the intrinsic radiosensitivity of human tumor cells to 137Cs gamma rays, especially at doses of 2 Gy and less.

The effect on the Km for radiosensitization at 0 degree C of thiol depletion by diethylmaleate pretreatment: quantitative differences found using the radiation sensitizing agent misonidazole or oxygen.

Pretreatment of V79- WNRE cells with 150 microM diethylmaleate for 1 hr at 37 degrees C caused a decrease in intracellular glutathione levels to approximately 10-15% of control levels (0.5 vs 5.0 nmol/10(6) cells). The cells could be washed free of diethylmaleate and held at 0 degree C for several hours without toxicity and with no increase in glutathione concentration, although the glutathione concentration rapidly increased to normal levels at higher temperatures. Survival curves were determined as a function of oxygen or misonidazole concentration (the latter in the absence of oxygen). A new "thin-film" technique was used to avoid changes in oxygen concentration because of radiochemical or cellular oxygen consumption. Glutathione depletion itself caused a small but consistent radiosensitization of hypoxic cells (dose enhancement ratio of 1.2). However, glutathione depletion caused a profound change in the radiosensitizing efficiency of misonidazole, with a decrease in Km of about sevenfold from 0.6 to 0.09 mM. In contrast, only a 2.5-fold decrease was found in the Km for radiosensitization by oxygen with diethylmaleate pretreatment. These results suggest a fundamental problem with the conventional theory of radiosensitivity whereby one considers a first-order competition for reaction with target radicals between radical-fixing versus radical-repairing species. It also suggests difficulties in the interpretation of glutathione as the only endogenous protective species.

Protection against light-activated photofrin II killing of tumor cells by nitroimidazoles.

Nitroimidazoles are good quenchers of triplet state porphyrins in chemical systems, thereby inhibiting singlet oxygen formation and type II photodynamic reactions. Photobiological studies were performed with EMT-6 tumor cells in vitro utilizing Photofrin II (PII) in combination with etanidazole (ETAN), misonidazole (MISO), and trifluoromisonidazole (TF-MISO). After short-term (1 h) exposure of cells to PII, 5 mM ETAN and MISO had no effect on photoinactivation while 5 mM TF-MISO had a small but significant protective effect. When the intracellular oxygen level was equilibrated with 0.3% oxygen in the gas phase, all three nitroimidazoles produced significant photoprotection at concentrations as low as 0.3 microM. After long-term (24 h) exposure of cells to PII, all three nitroimidazoles demonstrated large photoprotective effects under both aerobic and 0.3% oxygen conditions. At equal concentrations of nitroimidazole, photoprotection was greatest for the most lipophilic compound (TF-MISO) and least effective for the most hydrophilic compound (ETAN). These studies suggest that nitroimidazoles can quench triplet state porphyrins (within cells) to reduce intracellular concentrations of singlet oxygen, the putative toxin in PII photoinactivation. In addition, after long-term exposures to PII when porphyrins have partitioned into cellular membranes and lipid environments, the lipophilicity of this class of photoprotector correlates with effectiveness in these mammalian cells.

Radiation inactivation of human prostate cancer cells: the role of apoptosis.

Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. Cell viability was measured microscopically with trypan blue staining. Cell survival after various doses was computer-fitted to either a simple linear or a linear-quadratic equation. Twenty-four hours after a 10-Gy dose of 137Cs gamma rays, DNA fragmentation to nucleosome multimers was strongly expressed in only DU-145 cells. In this cell line, when centrifugation at 12,000g for 10 min was used to separate fragmented from large molecular weight DNA, the proportion of DNA in the supernatant increased to a maximum of approximately 17% of the total by 10-12 h after radiation treatment. Cell death 24 h after irradiation measured by trypan blue exclusion assays followed single-hit kinetics up to 80 Gy. The proportion of cells which were labeled with Apoptag displayed single-hit kinetics and yielded the same inactivation coefficient as measured by trypan blue. Together, these data indicate that the rapid (24 h) inactivation of irradiated DU-145 cells results from apoptosis and accounts for about 5% of the single-hit killing measured by clonogenic assay. Temporal studies of radiation-induced killing of DU-145 cells distinguished this rapid mechanism of cell death from the major mechanism (72-144 h). These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.

Oxygen dependency of tumor cell killing in vitro by light-activated Photofrin II.

Asynchronous populations of mouse EMT-6 tumor cells were treated with Photofrin II and exposed to various doses of 630 nm light in slowly stirred suspensions which had been equilibrated with various concentrations of oxygen. Survival curves were generated with cells exposed to 20 micrograms/ml Photofrin II in tissue culture medium for 1 h, a procedure which made it possible to remove more than 50% of the drug by washing. It is expected that under these conditions the drug would be loosely bound to cell surface and plasma membranes and in the cellular cytosol. Survival curves were also generated with cells exposed to 5 micrograms/ml Photofrin II for 20-24 h, a procedure which resulted in greater than 90% of the drug being tightly bound within cells, presumably to cellular lipids and membranes. Oxygen was obligatory for killing cells which had been exposed for both "short term" and "long term" to Photofrin II. After 30-40 min of pregassing cells with nitrogen gas which contained precise levels of oxygen, the concentration required to reduce rates of cell killing to 50% of maximum was approximately 0.5% O2 (gas phase) for short-term drug exposures and less than or equal to 0.05% O2 for long-term drug exposures. Even after pregassing times of 90-120 min prior to light administration, a Km of approximately equal to 0.1% O2 was observed for cells exposed to the drug for the longer time. When the same cells were exposed to 137Cs gamma rays in this irradiation chamber, no change in radiation sensitivity was observed after 30 min of pregassing cells with all oxygen concentrations studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The effectiveness of short-term versus long-term exposures to Photofrin II in killing light-activated tumor cells.

Asynchronous populations of mouse EMT-6 tumor cells were exposed to various doses of 630-nm light in slowly stirred aerobic suspensions after both short-term and long-term exposures to Photofrin II. All survival curves are characterized by a "threshold" light dose below which no cell inactivation occurs followed by a steep light-dose response. Both the shoulder widths and the inactivation curve slopes are functions of Photofrin II concentration. After high doses of light where survival levels are 0.003 and lower, "resistant tails" are observed on some survival curves. Light doses required to inactivate 50% of tumor cell populations were obtained from whole survival curves and their reciprocals (1/D50% survival) used as inactivation "rates". The amount of Photofrin II within cells was measured by a fluorescence assay. Per unit of fluorescence, this photosensitizer is at least 10 times more effective after long-term than after short-term exposures. After long-term exposures, both fluorescence activity and photosensitizing effectiveness are retained in washed cells for several hours. After short-term exposures, a majority of both the fluorescence and photosensitizing activity is lost by multiple washings or stirring in tissue culture medium without drug. These data suggest that the cellular compartments associated with photosensitization after short-term exposures to Photofrin II are probably different from the cellular compartments associated with photosensitization after long-term exposures to the drug. The data are consistent with known properties of the monomeric and oligomeric components of Photofrin II.

Radiation fields backscattered from material interfaces: I. Biological effectiveness.

Confluent cultures of CHO-K1 and CHO-xrs5 cells were irradiated attached to 6 microm Mylar with 137Cs gamma rays and 200 kVp X rays adjacent to scattering materials consisting of polystyrene, glass, aluminum, copper, tin and lead. The absorbed dose in cell nuclei was estimated from measurements of backscattered dose made with a parallel-plate ion chamber with a 5-microm Mylar window and a gas volume whose thickness was equivalent to approximately 2.6 microm of cells or tissue. Cell inactivation after various doses was measured by clonogenic assays after trypsinization and enumeration. Survival curves constructed from data pooled from at least two independent experiments were best fitted to a linear-quadratic (LQ) or a linear equation for CHO-K1 and CHO-xrs5 cells, respectively. An average distance of 9.3+/-1.9 microm from the scattering surfaces to the midline of nuclei for both the cell lines was estimated from electron micrographs of fixed cell sections. The major differences in biological effect observed when the cells were irradiated adjacent to these materials could be largely explained by the differences in the physical dose. Further analyses using the LQ equation suggested additional biological effects with implications for the mechanisms involved. CHO-K1 cells showed a small but consistent increase in the low-dose (alpha-inactivation coefficient) mechanism for both radiations scattered from high-Z material. An increased value of the alpha coefficient suggests an increase in RBE which could be associated with a higher proportion of low-energy and track-end electrons in these fields. The radiation fields which produced maximum single-hit killing in CHO-K1 cells also produced less killing by the quadratic (beta-inactivation coefficient) mechanism. In contrast, when similarly irradiated, CHO-xrs5 cells exhibited significantly lower alpha coefficients of inactivation. The mechanistic basis for this opposite effect of backscattered radiations in these cell lines is as yet unknown.

Condensed chromatin and cell inactivation by single-hit kinetics.

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.

The radiation hypersensitivity of cells at mitosis.

PURPOSE: Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. MATERIALS AND METHODS: Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. RESULTS: Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. CONCLUSIONS: The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

Chemical agents that promote chromatin compaction radiosensitize tumour cells.

PURPOSE: Previous studies indicated that cells whose chromatin is naturally compacted at the time of radiation are hypersensitive to radiation-induced killing, primarily by single-hit inactivation. Some chemicals that are known to promote chromatin compaction in interphase cells are here investigated for their radiosensitizing potential. MATERIALS AND METHODS: Okadaic acid (OA), a protein phosphatase inhibitor, fostriecin (FC), a topoisomerase II inhibitor and trichostatin A (TSA), a histone deacetylase inhibitor, were reported to promote chromatin compaction in mammalian cells. Asynchronous populations of HT-29 (human colon carcinoma) cells were exposed to various concentrations of OA, FC and TSA for various times before irradiation with various doses of Cs-137 gamma-rays and toxicity and radiosensitization were measured. Induced chromatin compaction was visualized by electron microscopy (EM). Histone 1 (H1) and histone 3 (H3) phosphorylation was measured by Western blotting, whole-cell fluorescence microscopy and confocal microscopy. RESULTS: OA and FC produced significant radiosensitization at 2 Gy after short (2 h) exposures. These chemical treatments also produced increased phosphorylation of H3 and increased chromatin compaction as measured by EM. A 2-h exposure of cells to TSA had no effect on cell radiosensitivity, histone phosphorylation or chromatin condensation. However, a 16-h exposure to TSA produced significant radiosensitization, histone phosphorylation and chromatin condensation, presumably by secondary mechanisms. CONCLUSIONS: These data are consistent with the hypothesis that compacted chromatin is a hypersensitive target for radiation killing. Furthermore, the modulation of chromatin conformation by drugs selectively in tumour cells might radiosensitize tumours whose cells are intrinsically radioresistant.

Radiosensitization of tumour cells by cantharidin and some analogues.

PURPOSE: Mammalian cells at mitosis contain chromatin in compacted form and are hypersensitive to ionizing radiation. Previous research had shown some chemicals that induce chromatin compaction within interphase cells act as radiosensitizers. Of these agents, cantharidin (LS-1), which is an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), showed good radiosensitizing activity at non-toxic doses. Cantharidin and 13 additional structural analogues (LS-2-14) were tested for their radiosensitizing activity on tumour cells in vitro. MATERIALS AND METHODS: Twelve of the 14 cantharidin analogues were synthesized in the authors' laboratory. Various concentrations of the drugs were screened for toxicity and radiosensitizing effectiveness with asynchronous DU-145 (human prostate carcinoma) cells. More detailed radiobiological studies of the more potent agents were performed with HT-29 (human colon carcinoma) cells since they could be readily synchronized. The radiosensitization of G1 phase HT-29 cells was measured after a 2-h exposure to the more potent drugs and reductions of the surviving fraction after an acute dose of 2 Gy (SF2Gy) served to estimate their relative effectiveness. The increase in phosphorylation of histone 1 (H1) and histone 3 (H3) induced by these drug exposures was measured by Western blotting of protein extracts. Drug-induced change in chromatin morphology was visualized by electron microscopy, and the alkaline comet assay (which measures DNA single-strand breaks) was employed to measure the radiation sensitivity of cellular chromatin in the drug-treated cells. RESULTS: Of the 14 cantharidin analogues tested, LS-1, LS-2 and LS-5 at concentrations of 3-20 microM showed little or no toxicity, produced elevated levels of H1 and H3 phosphorylation, and effected significant radiosensitization at low radiation dose. The chromatin in tumour cells treated with LS-5 became visibly compacted and its DNA was about 1.6 times more sensitive to radiation-induced strand breakage relative to that of control cells. CONCLUSIONS: The results confirm the authors' earlier studies that showed an increase in tumour cell intrinsic radiosensitivity by exposure to agents that promote chromatin compaction. LS-5 was identified as the optimal radiosensitizing agent of this class of compounds. Radiosensitization was correlated with chromatin compaction and elevated phosphorylation of H1 and H3. The DNA in drug-treated cells exhibited an enhanced sensitivity to radiation-induced single-strand breakage.

Gold microspheres: a selective technique for producing biologically effective dose enhancement.

PURPOSE: To investigate dose enhancement and radiosensitization associated with electrons produced and scattered from gold particles suspended in cells in vitro and with tumour cells growing in vivo irradiated with low-energy photons. MATERIALS AND METHODS: CHO-K1, EMT-6 and DU-145 cells were irradiated with kilovoltage X-ray and Cs-137 beams in slowly stirred suspensions in the presence of various concentrations of gold particles ( 1.5-3.0 microm); cell survival was measured by clonogenic assay. Gold particles were injected directly into EMT-6 tumours growing in scid mice prior to their irradiation. Tumour cell killing was assayed by an in vivo-in vitro technique. RESULTS: Dose enhancement was confirmed by both Fricke dosimetry and cell killing for 100, 140, 200 and 240 kVp X-rays, but not for Cs-137 gamma-rays. For the chemical dosimeter, a dose enhancement (DMF) of 1.42 was measured for 1% gold particle solutions irradiated with 200 kVp X-rays. When rodent and human cells were irradiated in the presence of 1% gold particles, DMF values at the 10% survival level ranged from 1.36 to 1.54, with an overall average value of 1.43. Preliminary attempts to deliver these gold particles to tumour cells in vivo by intra-tumour injection resulted in modest radiosensitization but extremely heterogeneous distribution. CONCLUSIONS: An increased biologically effective dose can be produced by gold microspheres suspended in cell culture or distributed in tumour tissue exposed to kilovoltage photon beams. With the increasing use of interstitial brachytherapy with isotopes that produce low-energy photons, high-Z particles might find a role for significantly improving the therapeutic ratio.