Biomedical subjects

K M Prise

Publications and source records attributed to K M Prise.

At least 19 recordsLinked to original sources

PubMed2006

ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells.

Radiotherapy is an important treatment for patients suffering from high-grade malignant gliomas. Non-targeted (bystander) effects may influence these cells' response to radiation and the investigation of these effects may therefore provide new insights into mechanisms of radiosensitivity and responses to radiotherapy as well as define new targets for therapeutic approaches. Normal primary human astrocytes (NHA) and T98G glioma cells were irradiated with helium ions using the Gray Cancer Institute microbeam facility targeting individual cells. Irradiated NHA and T98G glioma cells generated signals that induced gammaH2AX foci in neighbouring non-targeted bystander cells up to 48 h after irradiation. gammaH2AX bystander foci were also observed in co-cultures targeting either NHA or T98G cells and in medium transfer experiments. Dimethyl sulphoxide, Filipin and anti-transforming growth factor (TGF)-beta 1 could suppress gammaH2AX foci in bystander cells, confirming that reactive oxygen species (ROS) and membrane-mediated signals are involved in the bystander signalling pathways. Also, TGF-beta 1 induced gammaH2AX in an ROS-dependent manner similar to bystander foci. ROS and membrane signalling-dependent differences in bystander foci induction between T98G glioma cells and normal human astrocytes have been observed. Inhibition of ataxia telangiectasia mutated (ATM) protein and DNA-PK could not suppress the induction of bystander gammaH2AX foci whereas the mutation of ATM- and rad3-related (ATR) abrogated bystander foci induction. Furthermore, ATR-dependent bystander foci induction was restricted to S-phase cells. These observations may provide additional therapeutic targets for the exploitation of the bystander effect.

Astrocytes↗

PubMed2005

A model for radiation-induced bystander effects, with allowance for spatial position and the effects of cell turnover.

Bystander effects, whereby cells that are not directly exposed to ionizing radiation exhibit adverse biological effects, have been observed in a number of experimental systems. A novel stochastic model of the radiation-induced bystander effect is developed that takes account of spatial location, cell killing and repopulation. The ionizing radiation dose- and time-responses of this model are explored, and it is shown to exhibit pronounced downward curvature in the high dose-rate region, similar to that observed in many experimental systems, reviewed in the paper. It is also shown to predict the augmentation of effect after fractionated delivery of dose that has been observed in certain experimental systems. It is shown that the generally intractable solution of the full stochastic system can be considerably simplified by assumption of pairwise conditional dependence that varies exponentially over time.

Bystander Effect↗

PubMed2005

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics.

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.

Cells, Cultured↗

PubMed2004

A Monte Carlo model of DNA double-strand break clustering and rejoining kinetics for the analysis of pulsed-field gel electrophoresis data.

In studies of radiation-induced DNA fragmentation and repair, analytical models may provide rapid and easy-to-use methods to test simple hypotheses regarding the breakage and rejoining mechanisms involved. The random breakage model, according to which lesions are distributed uniformly and independently of each other along the DNA, has been the model most used to describe spatial distribution of radiation-induced DNA damage. Recently several mechanistic approaches have been proposed that model clustered damage to DNA. In general, such approaches focus on the study of initial radiation-induced DNA damage and repair, without considering the effects of additional (unwanted and unavoidable) fragmentation that may take place during the experimental procedures. While most approaches, including measurement of total DNA mass below a specified value, allow for the occurrence of background experimental damage by means of simple subtractive procedures, a more detailed analysis of DNA fragmentation necessitates a more accurate treatment. We have developed a new, relatively simple model of DNA breakage and the resulting rejoining kinetics of broken fragments. Initial radiation-induced DNA damage is simulated using a clustered breakage approach, with three free parameters: the number of independently located clusters, each containing several DNA double-strand breaks (DSBs), the average number of DSBs within a cluster (multiplicity of the cluster), and the maximum allowed radius within which DSBs belonging to the same cluster are distributed. Random breakage is simulated as a special case of the DSB clustering procedure. When the model is applied to the analysis of DNA fragmentation as measured with pulsed-field gel electrophoresis (PFGE), the hypothesis that DSBs in proximity rejoin at a different rate from that of sparse isolated breaks can be tested, since the kinetics of rejoining of fragments of varying size may be followed by means of computer simulations. The problem of how to account for background damage from experimental handling is also carefully considered. We have shown that the conventional procedure of subtracting the background damage from the experimental data may lead to erroneous conclusions during the analysis of both initial fragmentation and DSB rejoining. Despite its relative simplicity, the method presented allows both the quantitative and qualitative description of radiation-induced DNA fragmentation and subsequent rejoining of double-stranded DNA fragments.

Chromosome Breakage↗

PubMed2003

A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation.

The aim of this study was to test whether radiation-induced bystander effects are involved in the response of multicellular systems to targeted irradiation. A primary explant technique was used that reconstructed the in vivo microarchitecture of normal urothelium with proliferating and differentiated cells present. Sections of human and porcine ureter were cultured as explants and irradiated on day 7 when the urothelial outgrowth formed a halo around the tissue fragment. The Gray Cancer Institute charge particle microbeam facility allowed the irradiation of individual cells within the explant outgrowth with a predetermined exact number of (3)He(2+) ions (which have very similar biological effectiveness to alpha-particles). A total of 10 individual cell nuclei were irradiated with 10 (3)He(2+) ions either on the periphery, where proliferating cells are located, or at the centre of the explant outgrowth, which consisted of terminally differentiated cells. Samples were fixed 3 days after irradiation, stained and scored. The fraction of apoptotic and micronucleated cells was measured and a significant bystander-induced damage was observed. Approximately 2000-6000 cells could be damaged by the irradiation of a few cells initially, suggesting a cascade mechanism of cell damage induction. However, the fraction of micronucleated and apoptotic cells did not exceed 1-2% of the total number of the cells within the explant outgrowth. It is concluded that the bystander-induced damage depends on the proliferation status of the cells and can be observed in an in vitro explant model.

Animals↗

PubMed2003

A review of the bystander effect and its implications for low-dose exposure.

Current models for the interaction between ionising radiation and living cells or tissues are based on direct genetic damage produced by energy deposition in cellular DNA. An important observation which has questioned this basic assumption is the radiation-induced bystander response, in which cells which have not been directly targeted respond if their neighbours have been exposed. This response predominates at low doses of relevance to radiation risk analysis (<0.2 Gy) and therefore needs to be fully characterised. The development of microbeams, which allow individual cells within populations to be targeted with precise doses of radiation, has provided a useful tool for quantifying this response. The authors' studies have targeted individual human and mouse cells with counted protons and helium ions and monitored neighbouring cells for the production of bystander responses. Bystander responses have been measured after exposures as low as a single proton or helium ion delivered to an individual cell. An important aspect is that these responses saturate with increasing dose to the single target cell, thus the relative roles of direct and indirect (non-targeted) responses change with dose. Studies with multicellular, tissue-based models are providing evidence that bystander responses may have a complex phenotype involving multiple pathways and the overall response may be a balance between multiple signalling processes and responses to radiation exposure. Current models for radiation risk assume a linear non-threshold response and have generally been extrapolated from high-dose exposures. The involvement of competing processes at low doses may have important consequences for understanding the effects of low-dose exposure.

Adaptation, Physiological↗

PubMed2003

Low-dose studies of bystander cell killing with targeted soft X rays.

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.

Animals↗

PubMed2002

Investigating the cellular effects of isolated radiation tracks using microbeam techniques.

Studies of the effects of radiation at the cellular level have generally been carried out by exposing cells randomly to the charged-particle tracks of a radiation beam. Recently, a number of laboratories have developed techniques for microbeam irradiation of individual cells. These approaches are designed to remove much of the randomness of conventional methods and allow the nature of the targets and pathways involved in a range of radiation effects to be studied with greater selectivity. Another advantage is that the responses of individual cells can be followed in a time-lapse fashion and, for example, processes such as "bystander" effects can be studied clearly. The microbeam approach is of particular importance in mechanistic studies related to the risks associated with exposure to low fluences of charged particles. This is because it is now possible to determine the actions of strictly single particle tracks and thereby mimic, under in vitro conditions, exposures at low radiation dose that are significant for protection levels, especially those involving medium- to high-LET radiations. Overall, microbeam methods provide a new dimension in exploring mechanisms of radiation effect at the cellular level. Microbeam methods and their application to the study of the cellular effects of single charged-particle traversals are described.

Alpha Particles↗

PubMed2002

Quantification of radiation induced DNA double-strand breaks in human fibroblasts by PFGE: testing the applicability of random breakage models.

PURPOSE: To assess the applicability of methods of quantification of double-strand breaks (DSB) based on the random breakage paradigm, measuring yield and distribution of DSB induced by varying radiation quality. MATERIAL AND METHODS: 240 kVp X-rays and (238)Pu alpha-particles were used to induce DSB in AG01522B primary human fibroblasts. DNA molecular weight distributions were resolved by means of three pulsed-field gel-electrophoresis (PFGE) protocols, which, when combined together, allowed separation and quantification of double-stranded fragments between 5.7 Mbp and 12 kbp. Several analytical methods quantified the DSB yields. RESULTS: Data showed significant differences in the fragmentation patterns according to radiation quality. For both X-rays and alpha-particles, it was observed that the shape of the fragmentation profiles deviates from the prediction of a random breakage mechanism. This is in contrast to other studies where sparsely ionizing radiations appeared to distribute breaks uniformly throughout the genome. Deviations from random breakage were more evident after high linear energy transfer (LET) radiation, which showed an excess of breaks <1 Mbp and a deficit in the production of fragments >1 Mbp, a value that could be dose-dependent. CONCLUSIONS: Current methods of DNA fragmentation analysis after induction of DSB may lead to contradictory conclusions on both DSB yields and distributions. This study showed that the application of different DSB quantification methods, derived from random breakage or supported by its concepts, resulted in different radiation biological effectivenesses (RBE) for the induction of DSB, depending on how these methods were employed. To compare experimental results from different laboratories, care should be taken to provide as many details as possible about the application of methods of quantification of DNA damage. For all the methods used, total DSB yields resulted in RBE less than those for mutation induction or reproductive cell death, suggesting that total DSB yields only gave a limited indication of the severity of the inflicted damage. Production of correlated breaks on the chromatin loop structures by single particle-track traversals may explain the deviations observed between experimental data and the predictions of the random breakage paradigm.

Alpha Particles↗

PubMed2002

Double strand break rejoining after irradiation of human fibroblasts with X rays or alpha particles: PFGE studies and numerical models.

When a charged-particle track intercepts the chromatin fibre in DNA of mammalian cells, clustered damage is induced depending on the DNA conformation, local environment and track structure. Intra-track correlated DNA damage may have a higher probability of being mis-repaired or left un-repaired. Fragment size-distributions of DNA double strand breaks (DSBs) induced in primary human fibroblasts by 240 kVp X rays and 238Pu alpha particles (110 keV.micron-1) were resolved using pulsed-field gel electrophoresis (PFGE). By monitoring DSB rejoining kinetics and changes in the fragment size distribution with repair time, the relevance of spatial association of DSBs in determining rejoining kinetics was investigated. Rejoining kinetics appeared bi-phasic and independent of the size of the DNA fragments for both radiation qualities, with high LET radiation-induced DSBs repairing more slowly. Results suggest that local complexity of individual DSBs, rather than spatial association with other breaks is more significant in the determination of rejoining kinetics.

Alpha Particles↗

PubMed2002

The production of single strand and double strand breaks in DNA in aqueous solution by vacuum UV photons below 10 eV.

The mechanisms of break formation in fully hydrated DNA have been investigated using monochromatic photons below 10 eV. This has been achieved by developing a novel 'wet cell' for irradiating DNA in aqueous solution. Our preliminary data show that 7-10 eV photons readily induce strand breaks even though almost all of the energy is absorbed in the water. Therefore, the mechanism for the induction of single and double strand breaks (SSBs and DSBs) most likely involves indirect damage by OH radicals and is substantiated by data from studies in the presence of the OH radical scavenger Tris, which showed a substantial protective effect. The dose-effect curve for DSB induction is seen to be linear, or near-linear, indicating the involvement of 1-hit mediated induction of DSBs. These data point to single-event induction of DSBs being a significant pathway with all radiation types.

DNA↗

PubMed2002

Non-targeted effects of radiation: bystander responses in cell and tissue models.

The standard paradigm for radiation effects in cellular systems has involved direct damage to DNA and in particular, DNA double strand breaks as the triggering lesions leading to mutation, cell death and transformation. Recently, however, a growing body of evidence has reported non-targeted effects, which are not a direct consequence of the initial lesions produced in cellular DNA. These have included bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity. A common observation of these responses is that they dominate at low doses and saturate with increasing dose. Non-targeted effects may therefore have consequences for extrapolation of risk estimates to low doses if these are important in vivo. A range of experimental techniques is being used to study non-targeted responses, including microbeam approaches. Microbeams have considerable advantages in that they allow individual cells and subcellular targets to be selected within populations with precise low doses and, if required, exact dose rates. Recent advances also allow targeting of 3-D cell systems. The mechanisms underlying non-targeted responses appear to involve production of reactive oxygen species and direct cell-to-cell signalling via gap junctional intercellular communication although significant differences exist in different cell types. The triggering lesions for these responses remain unclear however. Some non-targeted responses may be inter-related, for example in the case of bystander responses and instability and may be part of a general stress response system in irradiated populations. Some non-targeted effects may also act as protective mechanisms; if they lead to the removal of potentially damaged cells from the population.

Animals↗

PubMed2002

Bystander-induced apoptosis and premature differentiation in primary urothelial explants after charged particle microbeam irradiation.

The ureter primary explant technique was developed to study bystander effects under in vivo like conditions where stem and differentiated cells are present. Irradiation was performed with a 3He2+ charged particle microbeam available at the Gray Cancer Institute, with high (approximately 2 microns) precision. Tissue sections from porcine ureters were pre-irradiated with the microbeam at a single location with 10 3He2+ particles (5 MeV; LET 70 keV.micron-1). After irradiation, the tissue section was incubated for 7 days, thus allowing the explant outgrowth to form. Total cellular damage (total fraction of micronucleated and apoptotic cells) was measured according to morphological criteria. Apoptosis was also assessed using a 3'-OH DNA end-labelling technique. Premature differentiation was estimated using antibodies to uroplakin III, a specific marker of terminal urothelial differentiation. Results of our experiments demonstrated a significant bystander-induced differentiation and a less significant increase in apoptotic and micronucleated cells. A hypothesis based on the protective nature of the bystander effect is proposed.

Animals↗

PubMed2002

Upgrading of the Gray Laboratory soft X ray microprobe and V79 survival measurements following irradiation of one or all cells with a CK X ray beam of different size.

The X ray microprobe developed at the Gray Laboratory was originally designed to produce carbon K X rays (278 eV) by electron bombardment and focus them to a few hundred nanometers spot by using a circular diffraction grating with increasing line density (zone plate). The very fine focus achieved (< 0.25 micron radius spot) and the highly localised energy deposition of CK X rays (photoelectron range < 7 nm), represent unique tools to investigate modern radiobiological phenomena. Recent improvements have been directed to increase the dose rate (up to approximately 6 Gy.s-1 entrance dose averaged over a typical V79 cell) and to evaluate the possibility of using higher energy photons (AlK of 1.48 keV). The efficiency of the microprobe system has been tested by assessing the clonogenic potential of V79 cells irradiated with CK X ray beams of different sizes (5 and 0.25 microns radius) and investigating the relevance of the spatial distribution of cells for the bystander effect.

Animals↗

PubMed2001

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts.

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts. We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha-particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 micrometer and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell. Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish. This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved.

Alpha Particles↗

PubMed2001

The relationship between the RBE of alpha particles and the radiosensitivity of different mutations of Chinese hamster cells.

The RBE of alpha-particles in different mutations of Chinese hamster cells was determined with the aim of identifying differences in the sensitivity to x-ray and alpha-particle-induced DNA damage. Two parental lines of Chinese hamster cells and four radiosensitive mutants were irradiated with different single doses of x-rays and alpha-particles and clonogenic cell survival was determined. Radiosensitivity to x-rays varied by a factor of 5 between the cell strains whereas sensitivity to alpha-particle irradiation was almost identical among all strains. The RBE is only determined by the sensitivity of the cells towards x-rays. Since cells with different defects of repair or cell cycle control have different radiosensitivities, we conclude that the effects of x-ray irradiation and the RBE are mostly determined by the activity of repair processes.

Alpha Particles↗

PubMed2001

Long-term genomic instability in human lymphocytes induced by single-particle irradiation.

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered. In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing alpha particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha-particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype. These data suggest that instability may be important in situations where even a single particle traverses human cells.

Cations, Divalent↗

PubMed2001

Low-dose hypersensitivity in Chinese hamster V79 cells targeted with counted protons using a charged-particle microbeam.

The Gray Laboratory charged-particle microbeam has been used to assess the clonogenic ability of Chinese hamster V79 cells after irradiation of their nuclei with a precisely defined number of protons with energies of 1.0 and 3.2 MeV. The microbeam uses a 1-microm silica capillary collimator to deliver protons to subcellular targets with high accuracy. The detection system is based on a miniature photomultiplier tube positioned above the cell dish, which detects the photons generated by the passage of the charged particles through an 18-microm-thick scintillator placed below the cells. With this system, a detection efficiency of greater than 99% is achieved. The cells are plated on specially designed dishes (3-microm-thick Mylar base), and the nuclei are identified by fluorescence microscopy. After an incubation period of 3 days, the cells are revisited individually to assess the formation of colonies from the surviving cells. For each energy investigated, the survival curve obtained for the microbeam shows a significant deviation below 1 Gy from a response extrapolated using the LQ model for the survival data above 1 Gy. The data are well fitted by a model that supports the hypothesis that radioresistance is induced by low-dose hypersensitivity. These studies demonstrate the potential of the microbeam for performing studies of the effects of single charged particles on cells in vitro. The hypersensitive responses observed are comparable with those reported by others using different radiations and techniques.

Animals↗