Transient failure to dephosphorylate the cdc2-cyclin B1 complex accompanies radiation-induced G2-phase arrest in HeLa cells.

Ionizing radiation causes a division delay in mammalian cells, dominated by a period of G2-phase arrest. The G2- to M-phase transition in dividing mammalian cells is dependent on the kinase activity of the cdc2-cyclin B protein complex. In the present investigation we measured the quantities of these two proteins, the formation of their complex and the kinase activity of the complex as a function of cell age in the cell cycle for irradiated and control mammalian cell populations. The human HeLa S3 cells were synchronized at the G1/S-phase border by double thymidine block and exposed 3 h after release to 1.75 Gy of X rays. Studies of HeLa cells at other laboratories have shown that, for doses of 5 Gy or more, division delay is associated with a suppression of production of cyclin B mRNA. Here we report that, for cells irradiated with low doses, there is a transient failure of the complex to activate which correlates with the duration of radiation-induced G2-phase arrest. The irradiated cells showed an increase in both cyclin B and phosphorylated cdc2 over the levels in control cells, and both persisted for a much longer period than in controls, further confirmation of delay in the activation of the catalytic subunit.

Cellular effects of individual high-linear energy transfer particles and implications for tissue response at low doses.

The energy deposition patterns produced by the radiation environment in space can be quite different from those in conventional radiation environments. Furthermore, conventional radiation biological experiments, using randomly distributed particle tracks, cannot access some variables which may be important in determining the health effects of irradiation. Controlled microbeam irradiation provides the means to investigate the effects and unique energy deposition patterns and cell environment for a variety of end points.

Dose-rate evidence for two kinds of radiation damage in stationary-phase mammalian cells.

Survival based on colony formation was measured for starved plateau-phase Chinese hamster ovary (CHO) cells exposed to 250 kVp X rays at dose rates of 0.0031, 0.025, 0.18, 0.31, and 1.00 Gy/min. A large dose-rate effect was demonstrated. Delayed plating experiments and dose response experiments following a conditioning dose, both using a dose rate of 1.00 Gy/min and plating delays of up to 48 hr, were also used to investigate the alternative repair hypotheses. There is clearly a greater change in survival in dose-rate experiments than in the other experiments. Thus we believe that a process which depends on the square of the concentration of initial damage, and which alters the effect of initial damage on cell survival is being observed. We have applied the damage accumulation model to separate the single-event damage from this concentration-dependent form and estimate the repair rate for the latter type to be 70 min for our CHO cells. Use of this analysis on other published dose-rate studies also yields results consistent with this interpretation of the repair mechanisms.

Multiple components of split-dose repair in plateau-phase mammalian cells: a new challenge for phenomenological modelers.

Split-dose experiments using starved plateau-phase Chinese hamster ovary cells have been used to investigate the kinetics of repair, expressed in terms of enhancement of reproductive survival. The results show two distinct components of repair, one having a characteristic time of just over 1 h for the removal of a lesion, the other, about 18 h. The rate at which each component removes damage and the fraction of the total damage that each removes appear to be independent of the initial amount of damage produced, i.e., dose. This lack of dose dependence is not consistent with some simple models of ionizing radiation damage and repair, such as those which assume that saturation of a repair process, depletion of enzyme pools, or the interaction of pairs of sublesions is responsible for the curvature in the dose-response relationship. However, the relationship between the amounts of each type of damage and dose appears to be consistent with models that assume that only a portion of the initial damage is directly accessible to the repair systems or that the initial damage consists of a mixture of potentially lethal and sublethal lesions.

Adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture.

This paper discusses two phenomena of importance at low doses that have an impact on the shape of the dose-response relationship. First, there is the bystander effect, the term used to describe the biological effects observed in cells that are not themselves traversed by a charged particle, but are neighbors of cells that are; this exaggerates the effect of small doses of radiation. Second, there is the adaptive response, whereby exposure to a low level of DNA stress renders cells resistant to a subsequent exposure; this reduces the effect of low doses of radiation. The present work was undertaken to assess the relative importance of the adaptive response and the bystander effect induced by radiation in C3H 10T(1/2) cells in culture. When the single-cell microbeam delivered from 1 to 12 alpha particles through the nuclei of 10% of C3H 10T(1/2) cells, more cells were inactivated than were actually traversed by alpha particles. The magnitude of this bystander effect increased with the number of particles per cell. An adaptive dose of 2 cGy of gamma rays, delivered 6 h beforehand, canceled out about half of the bystander effect produced by the alpha particles.

Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

Microdosimetry near the trajectory of high-energy heavy ions.

Single-event energy distributions were measured in a 1.3-micron-diameter site as a function of radial distance from the trajectory of high-energy iron ions having an energy of about 600 MeV/amu. It was found that beyond distances of a few micrometers the average lineal energy of the (mostly single) secondary electrons (delta rays) is of the order of 3 keV/micron. This is similar to the value found in a medium irradiated by 170-keV photons. The frequency-mean specific energy for delta rays occurring at large distances from the path of the primary ion exceeds the calculated (radial) absorbed dose by two orders of magnitude.

Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells.

Research to determine the effects of defined numbers of alpha particles on individual mammalian cells is helpful in understanding risks associated with exposure to radon. This paper reports the first biological data generated using the single-particle/single-cell irradiation system developed at Pacific Northwest Laboratory. Using this apparatus, CHO-K1 cells were exposed to controlled numbers of 3.2 MeV alpha particles, and biological responses of individual cells to these irradiations were quantified. Chromosomal damage, measured by the induction of micronuclei, was evaluated after no, one, two, three or five particle traversals. Exposures of up to five alpha particles had no influence on the total numbers of cells recovered for scoring. With increased numbers of alpha particles there was a decrease in the ratio of binucleated to mononucleated cells of 3.5%/hit, suggesting that alpha particles induced dose-dependent mitotic delay. A linear hit-response relationship was observed for micronucleus induction: Micronuclei/binucleated cell = 0.013 +/- 0.036 + (0.08 +/- 0.013) x D, where D is the number of particles. When the estimated dose per alpha-particle traversal was related to the frequency of induced micronuclei, the amount of chromosomal damage per unit dose was found to be similar to that resulting from exposures to alpha particles from other types of sources. Approximately 72% of the cells exposed to five alpha particles yield no micronuclei, suggesting the potential for differential sensitivity in the cell population. Additional studies are needed to control biological variables such as stage of the cell cycle and physical parameters to ensure that each cell scored received the same number of nuclear traversals.

An integrated confocal and magnetic resonance microscope for cellular research.

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of health and disease at a cellular level, particularly when data acquired with different methodologies can be correlated in both time and space. In this Communication, a brief description of a novel instrument capable of simultaneously performing confocal optical and magnetic resonance microscopy is presented, and the first combined images of live Xenopus laevis oocytes are shown. Also, the potential benefits of combined microscopy are discussed, and it is shown that the a priori knowledge of the high-resolution optical images can be used to enhance the boundary resolution and contrast of the MR images.

Microdosimetric measurements of heavy ion tracks.

The biological effectiveness of radiations depends on the spatial pattern of ionizations and excitations produced by the charged particle tracks involved. Ionizations produced by both the primary ion and by energetic delta rays may contribute to the production of biologically relevant damage and to the concentration of damage which may effect the probability of repair. Although average energy concentration (dose) can be calculated using homogeneous track models, the energy is actually concentrated in small volumes containing segments of the ion and delta ray tracks. These local concentrations are studied experimentally using low pressure proportional counters, and theoretically, using Monte Carlo methods. Small volumes near an ion track may be traversed by a delta ray. If they are, the energy deposited will be similar to that produced by a single electron track in a low-energy x-ray irradiation. The probability of a delta ray interaction occurring decreases with the square of the radial distance from the track. The average energy deposited is the product of this probability and the energy deposited in an interaction. Average energy deposited calculated from measured interaction probability is in good agreement with the results of homogeneous track models.

Analyzing the role of biochemical processes in determining response to ionizing radiations.

The interaction of biochemical processes and radiation damage appears to play a major role in determining long-term biological effects. It is responsible for both the removal of radiation-induced alterations in macromolecules and for the time-dependent changes in survival of irradiated cells. Restoration of macromolecules by such means as the rejoining of strand breaks in DNA suggests a variety of possible mechanisms which could lead to the observed enhancement of cell survival. However, even though a number of molecular repair mechanisms have been identified, specific links between any such mechanisms and a subsequent modification of cell survival have proved difficult, if not impossible, to demonstrate. Models of cellular response provide a means of attempting to establish this connection. Although details of radiation chemistry, chromatin structure, enzymatic repair, molecular genetics, and cell cycle kinetics are generally simplified, each individual model incorporates features based on a set of assumed mechanisms. For example, one group of models assumes that all damage is potentially lethal (capable of killing the cell unless it is repaired), while another assumes that part of the damage is sublethal (innocuous until it interacts with other damage). Using split-dose, dose-rate, and delayed-plating techniques, we have demonstrated two distinct components of repair in plateau-phase Chinese hamster ovary cells. One process has a characteristic time of about 1 h; the other, about 18 h. In both cases, the reaction rates and the fractions of damage repaired appear to be independent of the initial amounts of damage produced. These observations suggest that none of the simpler models adequately describes cell inactivation; i.e., reproductive death is inconsistent with all assumptions regarding any of them. Consequently, more-complex models involving combinations of sublethal and potentially lethal damage or multiple-step damage processes may be required. These findings help to define the effects of exposure at low doses and dose rates and to develop an understanding of the underlying biochemical mechanisms involved.

Design of a benchtop alpha particle irradiator.

The design, construction and calibration of a convenient irradiator is described that provides controlled exposure to a uniform external source of well-characterized alpha particles at a dose-rate of 0.99 cGy min-1. The use of a precis- ion photographic shutter allows the accurate delivery of doses as low as 0.01 mGy (1 mrad) to cultured cells. Special features also include a helium environment and reciprocal collimator that permits the use of collimators with different transmission factors to achieve differing dose-rates. A simple method is described to characterize the energy spectrum of alpha particles by use of particle range as measured by track etch.

Kinetic differences between fed and starved Chinese hamster ovary cells.

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. The kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([3H]TdR) autoradiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) The rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [3H]TdR or [125I]UdR. Non-cycling cells in starved cultures accumulate primarily as G1 phase cells. Most cells not in G1 phase had stopped in G2 phase. Very few cells (less than 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G1 or G2 phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.

Monte Carlo simulation of single-cell irradiation by an electron microbeam.

A model is presented for irradiation of a cellular monolayer by an electron microbeam. Results are presented for two possible window designs, cells plated on the vacuum-isolation window and cells plated on Mylar above the vacuum-isolation window. Even for the thicker dual-membrane window that facilitates tissue culture and allows the target cell to be centered relative to the electron beam, the majority of the calculated beam spreading was contained in a volume typical of the mammalian HeLa cell line. None of the 10(4) electrons simulated at 25 keV were scattered into the spatial region occupied by neighbors of the target cell. Dose leakage was largest at 50 keV where the mean energy deposited in all neighbors was 21% of that deposited in the target cell. This ratio was reduced to 5% at 90 keV, the highest beam energy simulated. Lineal energy spectra of energy deposition events scored in the nucleus of the target cell became progressively more like the gamma-ray spectrum as the electron beam energy increased. Hence, our simulations provide strong support for the feasibility of a low-LET, single-cell irradiator.