Radiosensitivity Of Mammalian Cells Research Articles

Impact of Amino Acid Substitutions in Two Functional Domains of Ku80: DNA-Damage-Sensing Ability of Ku80 and Survival after Irradiation

ABSTRACTVarious chemotherapeutic drugs, such as etoposide, and ionizing radiation (IR) have been clinically applied for the treatment of many types of animal and human malignancies. IR and chemotheraputic drugs kill tumor cells mainly by inducing DNA double-strand breaks (DSBs). On the other hand, unrepaired or incorrectly repaired DSBs can lead to chromosomal truncations and translocations, which can contribute to the development of cancer in humans and animals. Thus, it is important to clarify the molecular mechanisms underlying the chemosensitivity or radiosensitivity of mammalian cells in order to develop medical treatments and next-generation chemotherapeutic drugs for cancer. Previously, we established and analyzed cell lines stably expressing chimeric constructs of EGFP and the wild-type Ku80 (XRCC5) protein or its mutant protein to which mutations were introduced by the site-directed mutagenesis. We found that the Ku70 (XRCC6)-binding-site mutations (A453H/V454H) of Ku80 and nuclear localization signal (NLS)-dysfunctional mutations (K565A/K566A/K568A) affected the ability to complement etoposide sensitivity. In this study, we examined the radiosensitivity of these cell lines. We found that either or both amino acid substitutions in two functional domains of Ku80, i.e., Ku70-binding-site mutations (A453H/V454H) and NLS-dysfunctional mutations (K565A/K566A/K568A), affect the ability to complement radiosensitivity. Moreover, these mutations in the two domains of Ku80 affect the DSB-sensing ability of Ku80. These information and Ku80 mutant cell lines used might be useful for the study of not only the dynamics and function of Ku80, but also the molecular mechanism underlying the cellular response to IR and chemotherapeutic drugs in mammalian cells.

Hyperthermia alters the interaction of proteins of the Mre11 complex in irradiated cells.

Radiosensitization of mammalian cells by heat is believed to involve the inhibition of repair of DNA double-strand breaks (DSBs). The Mre11 complex (composed of Mre11, Rad50, and Nbs1) is involved in DSB repair and forms foci at sites of radiation-induced DSBs. Heat induces the translocation of a significant amount of Mre11, Rad50, and Nbs1 from the nucleus to the cytoplasm, but little is known about how heat affects the integrity of the proteins still remaining in nuclei, or alters kinetics of formation/disappearance of DNA repair foci in heated, irradiated cells. Here, we show that hyperthermia alters the interaction between proteins of the Mre11 complex in irradiated human melanoma cells and inhibits the formation of repair foci. At various times after X-irradiation and/or heating (2 h at 41.5 or 42.5 °C), the cells were fixed and stained for Mre11, Rad50, and Nbs1. Colocalization of proteins and formation and disappearance of nuclear foci in heated and/or irradiated cells, determined using confocal microscopy, were compared. In heated, irradiated cells, focus formation was inhibited for 2-8 h, and colocalization of the proteins of the Mre11 complex was reduced for 12-24 h post-treatment. Colocalization was recovered in irradiated cells within 24 h after heating at 41.5 °C, but was inhibited longer after heating at 42.5 °C. The decreased colocalization in heated, irradiated cells suggests that there is a decrease in protein interaction, and Mre11 complexes in nuclei disassemble after heating. Such changes could be involved, at least in part, in heat radiosensitization and inhibition of DSB repair. Also, the kinetics of disassembly and reassembly of Mre11 complexes appears to be dependent upon treatment temperature.

The influence of concentration of DNA on the radiosensitivity of mammalian cells

To develop relations that explicitly show the dependence of the linear-quadratic survival parameters alpha and beta on the nuclear volume, average DNA concentration, and degree to which the chromatin is condensed in zones of the nucleus. The microdosimetric-kinetic model of mammalian cell killing is used. The relations indicate an increased tendency for lethal lesions to form by pairwise combination of potentially lethal lesions in regions of relatively greater local DNA concentration. In particular, the value of beta is proportional to the average nuclear concentration of DNA and to a parameter that reflects the condensation of DNA into a relatively concentrated phase, such as occurs in mitotic chromosomes and heterochromatin. These relations indicate local DNA concentration in the nucleus is a significant determinant of three magnitude of beta. Because alpha is composed of a term that is proportional to beta, it is also affected by DNA concentration, generally to equal or less degree. It may be possible to establish a correlation between nuclear size and morphology, as determined from microscope slides prepared from biopsy specimens, with radiation sensitivity and the alpha/beta ratio that could be useful in radiation treatment planning.

The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock.

A synergistic increase in cell killing is observed when a heat-shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat-radiosensitization, is believed to be mediated by inhibition of repair of radiation-induced double strand breaks (DSB) when cells are exposed to temperatures above 42 degrees C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation-induced DSBs are repaired via the non-homologous end-joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA-dependent protein kinase (DNA-PK), a complex involved in NHEJ, appear to be susceptible to a heat-induced loss of DNA-binding activity, with Ku80 representing the heat-sensitive component. Since the heat-induced loss and subsequent recovery of Ku-DNA binding activity correlates well with heat-radiosensitization, a role for Ku80 and NHEJ in heat-radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat-radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5-45.5 degrees C induce a similar amount of aggregation of Ku80 in human U-1 melanoma cells. These data suggest that the time-temperature-dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat-radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80(-/-) mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild-type (WT) cells, we found that heat-radiosensitization was not reduced in the Ku80(-/-) cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat-radiosensitization. Non-involvement of Ku-dependent or Ku-independent NHEJ pathways in heat-radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV-defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation-induced DSBs and as a target for heat-radiosensitization.

Examining the non-homologous repair process following cisplatin and radiation treatments

Purpose : To investigate the extent of non-homologous end-joining (NHEJ) in the mechanism of cisplatin radiosensitization. Materials and methods : Ku80-deficient cells are deficient in the non-homologous DNA double-strand break repair process, while the wild-type MEF cells maintain full mammalian cell repair capabilities. Both cell lines were exposed to clinically applicable doses of cisplatin (1, 3 and 6 μ g ml -1) for 1 h immediately before exposure to 250 kV X-rays. Radiation responses were plotted for each cell line and for all doses of cisplatin to observe relative levels of radiosensitization. Split-dose experiments were also performed on each cell line to measure levels of sublethal damage repair. Results : Radiosensitization was observed in the wild-type cells but not in the Ku80 cells when treated with a combination of 1 μ g ml -1 cisplatin followed by X-rays, implying that the NHEJ pathway may play a large role in cisplatin radiosensitization. Concurrent administration of this cisplatin dose with radiation produced similar levels of radiosensitization. Conversely, 3 and 6 μ g ml -1 cisplatin applied immediately before radiation revealed an increasing resistance to radiation in both cell lines -- possibly due to resistant subpopulations of cells remaining after subsequent lethal doses of cisplatin. Further experiments revealed that the high concentrations of cisplatin did not alter cell cycle distribution. Finally, split-dose experiments revealed that the NHEJ pathway also plays a significant role in sublethal damage repair. Conclusions : The study reveals that clinically applicable doses of cisplatin treatment results in the radiosensitization of mammalian cells due to the inhibition of the operation of NHEJ.

Modulation of the clastogenic activity of gamma-irradiation in buthionine sulphoximine-mediated glutathione depleted mammalian cells

Purpose: Chromosome aberrations (CA) were used as an endpoint to investigate the effect of buthionine sulphoximine (BSO), a potent glutathione-depleting agent, on the radiosensitivity of mammalian cells. The aim was to obtain information about the role of glutathione (GSH) in physicochemical and biochemical processes in irradiated cells. Materials and methods: CA were scored from first cycle metaphases in irradiated BSO-pretreated and untreated samples. BSO exposure was for 10h in mouse bone marrow cells in vivo and 5h for human lymphocytes in vitro. In further experiments fresh blood was irradiated on ice and immediately after irradiation GSH/GSH-ester was added. Results: In both the systems BSO-treated samples showed higher sensitivity to radiation than BSO untreated samples. The frequency of all types of CA increased except exchange aberrations. GSH/GSH-ester treatment given after irradiating the cells at 4 C reduced the frequency of deletions and increased the frequency of exchange aberrations. Conclusions: Data indicate that BSO-mediated GSH depletion increased radiation-induced chromosome aberrations, apart from exchange aberrations. This could be due to reduction in the free-radical scavenging effect of GSH, a failure in rejoining of DNA double-strand breaks, or induction of apoptosis.

RBE of fast neutrons for apoptosis in mouse thymocytes.

We compared apoptosis in mouse thymocytes following exposure to low doses of high linear energy transfer (LET), 62.5-MeV (p-->Be+) fast neutrons and low LET, 4-MeV photons by flow cytometric analysis of hypodiploid cells. The incidence of apoptotic cell death rose steeply at very low radiation doses reaching a plateau of 3 Gy. Both the time course and the radiation dose-response curves were similar for high and low LET radiation modalities. The relative biological effectiveness (RBE) of 1.0 for apoptosis in the mouse thymocyte system contrasts with the much higher value typically seen in many classical systems of clonogenic cell survival and tissue response. This difference suggests that while radiation-induced apoptosis may contribute significantly to loss of susceptible cells at doses of < or = 2 Gy, it appears to have a questionable role in determining the relative intrinsic radiosensitivity of mammalian cells to high and low LET irradiation at clinically relevant levels of cell kill.

Damage to cellular DNA from particulate radiations, the efficacy of its processing and the radiosensitivity of mammalian cells. Emphasis on DNA double strand breaks and chromatin breaks.

For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles. From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of "direct" and "indirect" action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a). Increases in linear energy transfer (LET infinity) up to 100-200 keV micron-1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make significant contributions to the relative biological effectiveness (RBE) of a given radiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Keynote address: Concepts and developments in radiosensitization of mammalian cells

Keynote address: Concepts and developments in radiosensitization of mammalian cells

Radiosensitization of Mammalian Cells in Vitro by Nitroacridines

The nitroacridine nitracrine (1-NC) is a DNA intercalator and a hypoxia-selective, electron-affinic radiosensitizer. Sensitization of Chinese hamster fibroblast cultures at 0 degrees C by the nitro positional isomers of 1-NC has now been compared to help establish structure-activity relationships. The des-nitro analog (E(1) at pH 7 = -899 mV) did not sensitize, suggesting that an electron-affinic chromophore is required. All the nitroacridines (E(1) range -376 to -257 mV) sensitized hypoxic cells with a maximum sensitizer enhancement ratio of about 1.7, but with a 200-fold range in potency. When mean intracellular drug concentrations were compared, 2-, 3-, and 4-NC had potencies which were similar, independent of E(1), and no greater than predicted for non-DNA binding nitroheterocycles. Sensitization by these three isomers occurred at intracellular concentrations likely to saturate the potential intercalation sites on DNA. A large fraction of the radical sites sensitized by O2 are apparently inaccessible to these drugs. It is suggested that sensitization results from electron transfer from migrating transient charge carriers of low reduction potential to immobile bound intercalators. An additional sensitizing mechanism may be available to 1-NC, which was 20 times more potent, a potency not accounted for by E(1), cell uptake, or DNA binding affinity. The dissociation kinetics of the DNA-drug complex was faster for 1-NC than for the other isomers. The higher potency of 1-NC may reflect a short mean residence time (less than 1 ms) in its intercalation site, allowing significant mobility on the DNA within the lifetime of relatively stable radiation-induced target radicals.

Development of a model system to study leukotriene-induced modification of radiation sensitivity in mammalian cells

Leukotrienes (LT) are an important class of biological mediators, for which no information exists concerning their synthesis following a radiation insult, or on their ability to modify cellular response to a subsequent radiation exposure. LT are derived from arachidonic acid, as are prostaglandins, although by a separate enzyme system. Prostaglandins are able to modify radiosensitivity of mammalian cells in vivo and in vitro. In addition, the cytoprotective effect induced by prostaglandins may have significance in cancer therapy since certain breast cancers which secrete elevated levels of prostaglandins are more resistant to therapy than similar tumors without the prostaglandin elevation. The objective of this study was to define a model system in which the metabolic fate of the LT could be monitored, and the effort of LT on the ionizing radiation sensitivity of mammalian cells in vitro could also be characterized.

Radiosensitization of mammalian cells by transition metal complexes containing nitroimidazole ligands

Various transition metal complexes containing nitroimidazoles as ligands have been shown to act as radiosensitizers of hypoxic cells in vitro. Sensitization by cis-Pt(II)Cl 2 (nitroimidazole) 2 complexes is no greater than that which can be demonstrated by the free nitroimidazole ligand alone. These results differ from those previously described for the compound FLAP where an ER of 2.4 was obtained at a concentration of 50 μM. We report that, even using the same treatment technique, the maximum ER that can be achieved is 12. Further, the sensitizing efficiency of FLAP cannot be improved when cells are kept in contact with the compound for 12 hours in air prior to deoxygenation and irradiation. In contrast, Rh(II) complexes show much greater sensitization than can be obtained with the free ligand and these compounds are in turn more efficient sensitizers than the comparable Pt(II) complexes.

Effects of Modifiers of the Yield of Hydroxyl Radicals on the Radiosensitivity of Mammalian Cells at Ultrahigh Dose Rates

Experiments were conducted to investigate the effects of increasing or decreasing the yield of hydroxyl radicals (.OH) reacting with target molecules on the survival of CHO cells irradiated in a thin layer with single 3-nsec pulses of electrons. Dimethyl sulfoxide (DMSO), a known radioprotector, was used as an .OH scavenger. The gas nitrous oxide (N2O), which scavenges hydrated electrons and in the process generates an additional yield of .OH, was used in an attempt to produce sensitization by increasing the amount of .OH-induced cellular damage. It was found that DMSO at high concentration was an effective radioprotective agent in cells equilibrated with nitrogen, air, and N2O and irradiated at ultrahigh dose rates. Sensitization by N2O was observed, but only under certain conditions, specifically, when a high concentration of the .OH scavenger DMSO was also present. The enhancement ratio (ER) for oxygen sensitization was reduced in the presence of DMSO, as was the ER for sensitization by misonidazole. Interpretation of these results according to radiation chemistry considerations will be discussed.

A rapid-mix study on the effect of lipophilicity of nitroimidazoles on the radiosensitization of mammalian cells in vitro.

A liquid flow rapid-mixing apparatus has been used to study the role of lipophilicity (octanol: water partition coefficient, P) in the sensitization of hypoxic V79 cells by nitroimidazoles. Sensitization by seven neutral 2-nitroimidazoles of similar reduction potential but widely differing partition (0.11-77) and one basic 2-nitroimidazole (pKa=8.9; p=8.5 (of free base)) was studied as a function of pre-irradiation contact time ca. 3-40 ms. With increasing P, sensitization occurs at increasingly shorter pre-irradiation contact times. The results suggest that even though factors other than passive diffusion control the sensitization observed with the base Ro 03-8799 it is able to diffuse to the target site faster than midonidazole.

Interaction of Nitroimidazole Sensitizers and Oxygen in the Radiosensitization of Mammalian Cells at Ultrahigh Dose Rates

When CHO cells, equilibrated with 0.44% oxygen, are irradiated with single 3-nsec pulses of electrons from a 600-kV-field emission source, a breaking survival curve is observed. The breaking behavior, believed to be the result of radiolytic oxygen depletion, can be prevented by the presence of a relatively low concentration of the hypoxic cell sensitizer misonidazole; similar results are obtained with metronidazole and Ro-05-9963. The resulting survival curves exhibit a sensitized response similar to that obtained with conventional dose rate radiation for CHO cells under this oxygen concentration. This degree of sensitization is greater than that observed for CHO cells irradiated at ultrahigh dose rates under the same concentration of sensitizer in nitrogen. The data suggest that the nitroimidazole compounds interfere with the radiation chemical oxygen depletion process and that the radiosensitization observed in the nonbreaking survival curve is the consequence of sensitization by both the nitroimidazole and, primarily, the oxygen rather than a direct subsitution for oxygen by the sensitizer. This conclusion is also supported by data obtained in double-pulse experiments. The results are discussed with regard to the mechanisms of the oxygen depletion process and radiosensitization.

Interaction of misonidazole and oxygen in the radiosensitization of mammalian cells

The radiosensitizing effect of misonidazole in the presence of various oxygen concentrations was studied using cultured Chinese hamster ovary (CHO) cells. Survival curves were measured for cells equilibrated with various combinations of oxygen and misonidazole concentrations and irradiated with 50 kVp x-rays. In cases where the two agents independently yield two moderately different enhancement ratios (ER), the combined ER is simply the higher of the two. Only when the two individual ER's are approximately equal is there some degree of increase in the overall ER. An empirical technique was derived, based on the concept of ER-equivalence and the respective K-curves of the two sensitizers, to predict the combinative effect of the agents in radiosensitization. These experimental and theoretical considerations suggest that oxygen and misonidazole share in the same sensitization mechanism and act on the same target site or sites. Since it is likely that there is a distribution of oxygen tension in a tumor sensitizing tumor cells to varying degrees, knowledge of the additive effects of misonidazole and oxygen in radiosensitization may be valuable in understanding the efficacy of the application of electron affinic drugs to cancer radiotherapy.

Rapid-mix studies on the anomalous radiosensitization of mammalian cells by 5-chloro-1-methyl-4-nitromidazole.

Rapid-mix studies on the anomalous radiosensitization of mammalian cells by 5-chloro-1-methyl-4-nitromidazole.

Radiosensitization of Mammalian Cells by Misonidazole and Oxygen: DNA Damage Exposed by Micrococcus luteus Enzymes

When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37/sup 0/C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract.

Effect of Salt Solutions on the Radiosensitivity of Mammalian Cells as a Function of the State of Adhesion and the Water Structure

The radiation isodose survival curve of attached Chinese hamster (V79) cells, subjected to a wide concentration range of salt or sucrose solutions, is characterized by two maxima separated by a minimum. Cells are radioprotected at the maxima (high and low hypertonic salt concentrations) while they are radiosensitized at the minimum (intermediate hypertonic salt concentrations). Both cations and anions can alter the cellular radiosensitivity above and beyond the (osmotic) effect observed for cells treated with sucrose solutions. However, the basic curve shape, except in the case of sulphate salts, remains the same. When these experiments are repeated with single cells in suspension, the isodose survival curve is quite different in that high salt concentrations (greater than 0.9 M) do not protect cells in suspension unlike the case with attached cells. The curve shape is also altered in that the second maximum is absent with many salt solutions. If multicellular spheroids are used for these experiments, the data resemble those for single cell suspensions rather than for attached cells. The radiation survival data for cells in suspension in salt solutions correlate with water proton spin-lattice relaxation time (T1) and, in hypo- and iso-tonic solutions, with cell volume.

Effect of Salt Solutions on the Radiosensitivity of Mammalian Cells

The effects of a wide concentration range of NaCl solutions containing either ouabain, ethanol, para-nitroacetophenone (PNAP), N-ethylmaleimide (NEM), cysteamine or dimethyl sulphoxide (DMSO) on cellular radiosensitivity have been examined. Ouabain and NEM treatment increased the radiosensitivity of V79 Chinese hamster cells, but the action of these chemicals did not depend on the concentration of NaCl. PNAP increased cellular radiosensitivity with increasing NaCl concentration reaching a maximum effect at 0.6 to 0.7 M NaCl. The radioprotective properties of cysteamine, DMSO and ethanol were all strongly dependent on the NaCl concentration in a complex but qualitatively similar manner. DMSO (2.0 M) increased radiation survival of cells after a 1380 rad dose by a factor of about 10(4) when present in 0.075 M NaCl and by a factor of 8.7 when present in 1.2 M NaCl.