Biomedical subjects

E G Wright

Publications and source records attributed to E G Wright.

At least 19 recordsLinked to original sources

PubMed2001

Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Animals↗

PubMed2001

Induction of endogenous beta-galactosidase by ionizing radiation complicates the analysis of p53-LacZ transgenic mice.

Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-beta-galactosidase activity following whole body irradiation. Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes. The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice.

Animals↗

PubMed2001

Differential post-translational modification of the tumour suppressor proteins Rb and p53 modulate the rates of radiation-induced apoptosis in vivo.

Ionizing radiation induces p53-dependent apoptosis in the spleen, providing a model system to study p53 regulated events in a normal cell type. We have developed an in vivo model that identifies genetic differences in the regulation of p53-mediated apoptosis and addresses whether altered post-translational events in the p53-p21/Rb axis modulate the sensitivity of cells to radiation-induced cell death in vivo. Splenocytes from mice with distinct genetic backgrounds (DBA/2 and C57BL/6) exhibit differences in the rate of apoptosis. Whilst no obvious strain differences in protein levels of Bcl-2 or the cyclin-CDKs were observed, early post-translational regulatory events in the p53-p21/Rb axis showed striking differences in the two mouse strains. Cells from C57BL/6 animals undergo more rapid apoptosis after irradiation resulting from elevated levels and rapid induction of p53, pronounced Rb-cleavage, and the absence of a sustained induction of p21. In contrast, cells from DBA/2 animals have a reduced rate of apoptosis following irradiation with elevated levels of hyperphosphorylated Rb and a sustained induction of the p21 protein that is coincident with the C-terminal phosphorylation of p53. These data suggest that quantitative differences in the level of p21 protein can affect the rate of apoptosis in vivo, consistent with the view that p21 is an anti-apoptotic effector of p53. However, striking differences in the Rb protein-caspase cleavage or hyperphosphorylation-in the same cell type, but in different genetic backgrounds, demonstrates that p53-dependent apoptosis can be modulated in vivo by genetic factors that impinge upon the pro- or anti-apoptotic potential of Rb. In addition, we show that Rb cleavage is p53-dependent and that its phosphorylation status can be uncoupled from p21 expression. This study highlights the possibility that genetic factors can be identified that affect differential sensitivity of cells to ionizing radiation in vivo.

Animals↗

PubMed2001

Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence.

The two prohibitin proteins, Phb1p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.

Animals↗

PubMed2001

Heavy metals of relevance to human health induce genomic instability.

Heavy metals used in medical prostheses or present in water supplies or tobacco can build up in tissues and blood and are well known to produce toxic effects. Normally, legislative controls on the levels of these substances are determined by reference to the acute toxicity data. This paper shows that cadmium and nickel can produce delayed effects in human cells in vitro, which are characteristic of genomic instability. The effects occur even at levels where no acute toxic effects can be demonstrated. Genomic instability can be demonstrated by persistent induction of cytogenetic abnormalities and delayed cell death in progeny of cells many generations after exposure. Formerly, this syndrome has only been definitively proven to occur following irradiation, but in these experiments cell populations exposed for only 1 or 24 hours were expanded over several months, involving eight passages, and the yield of chromosomal aberrations and cell loss due to lethal mutations did not decrease. The consequences of this genomic instability are not yet known but it is possible that many of the systemic symptoms associated with exposure to low concentrations of these metals could involve delayed expression of cellular damage. It is also clear that these effects cannot be predicted from acute toxicity data.

Cadmium↗

PubMed2001

A PCR-based clonal analysis of radiation-induced loss of heterozygosity in haemopoietic stem cells.

CBA mouse strains have been used for many years as a model of radiation-induced acute myeloid leukaemia and the leukaemias in CBA and their F1 hybrids are characterised by a specific loss of heterozygosity involving one homologue of chromosome 2. Previous cytogenetic studies of transplanted irradiated bone marrow, or of bone marrow obtained from irradiated mice significantly before the appearance of leukaemia, have been interpreted as the chromosome 2 deletion being a high frequency, possibly initiating event. However, these studies had not specifically addressed the question of whether the characteristic deletion was induced at a high frequency in stem cells. Using a PCR-based technique, we have studied the induction of chromosome 2 LOH in the progeny of (CBA/H x C57BL/6)F1 stem cells after a potentially leukaemogenic radiation exposure. Whilst chromosome 2 LOH can be induced directly by irradiation and there is a preferential loss of the CBA allele, the frequency is no greater than LOH induced in other chromosomal regions studied. The data do not support radiation-induced deletion involving one homologue of chromosome 2 in long-term repopulating stem cells (<1 in 200) being as high a frequency event as might be inferred by previous cytogenetic studies of total bone marrow.

Animals↗

PubMed2001

In vivo expansion of the endogenous B-cell compartment stimulated by radiation and serial bone marrow transplantation induces B-cell leukaemia in mice.

Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5(+) B cells. This B-cell lineage is established during ontogeny and replenished by the process of self-renewal. Spontaneous and induced leukaemias that frequently affect this lineage are thought to arise as a result of the frequent cell division required to maintain the population throughout adulthood and in response to repeated exposure to environmental antigens. In a series of bone marrow transplant (BMT) experiments performed in B6D2F1 mice, B-cell leukaemia occurred in recipients of serially transplanted syngeneic bone marrow. This study was therefore designed to determine the frequency and phenotype of the observed leukaemia. Male donor cells were initially transplanted into lethally irradiated female hosts and secondary (2 degrees ) BMT was performed at 3 months. At 1, 2, 3 and 16 months following primary (1 degrees ) BMT, and when 2 degrees BMT recipients developed leukaemia, animals were sacrificed and their tissues extensively examined. These analyses confirmed a host-derived CD5(+) transplantable B-cell leukaemia that was initiated in 50% of 1 degrees BMT recipients. With serial passage, the leukaemia became more aggressive and lost CD5 expression, suggesting transformation to a high-grade leukaemia/lymphoma. This previously unreported observation suggests that the combination of radiation and subsequent serial transplantation induces a proliferative stress to the host B-cell compartment that is causative in leukaemic transformation.

Animals↗

PubMed2001

In vivo chromosomal instability and transmissible aberrations in the progeny of haemopoietic stem cells induced by high- and low-LET radiations.

PURPOSE: To study stable and unstable chromosomal aberrations in the haemopoietic cells of CBA/H mice after exposure to both high- and low-LET radiations. MATERIALS AND METHODS: Chromosomal aberrations were scored in the clonal progeny of X-, alpha- or non-irradiated short-term repopulating stem cells using the spleen colony-forming unit (CFU-S) assay, 12 days post-transplantation and in the bone marrow reconstituted by X-, neutron- or non-irradiated exogenous (transplanted) or endogenous (X- or neutron whole-body-irradiated) long-term repopulating stem cells for up to 24 months. RESULTS: Chromosomal instability was demonstrated in 3-6% of cells in all cases. After transplantation of X- or neutron-irradiated bone marrow approximately 8% of cells with stable aberrations were recorded at all times. After 3Gy X- or 0.5 Gy neutron- whole-body irradiation stable aberrations were detected in approximately 17 and 5% of cells respectively. CONCLUSIONS: Chromosomal instability induced in vitro can be transmitted in vivo by transplantation of haemopoietic stem cells exposed to high- or low-LET radiations. Comparable instability can be induced and shown to persist for the remaining lifetime after whole-body irradiation. There was no direct relationship between the expression of stable and unstable aberrations and significant interanimal variation in the expression of both stable and unstable aberrations.

Animals↗

PubMed2001

Individual variation in the production of a 'bystander signal' following irradiation of primary cultures of normal human urothelium.

The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, we have developed an assay where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, allogenic explants or to a clonogenic cell line which has a well characterized and stable response to the bystander signal. The variation in production of the signal from patient to patient can thus be assessed using molecular and cellular endpoints. A study using tissue from over 100 patients and from mouse strains with well characterized responses to low level radiation exposure shows that there is variation in the effect of the signal produced by irradiated tissue from different patients. Gender, smoking status and the existence of a bladder malignancy influence the expression of the signal by normal urothelium. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for combined therapy.

Animals↗

PubMed2001

The Trp53 pathway is induced in vivo by low doses of gamma radiation.

The induction of the Trp53 response after very low doses (0.01-1 Gy) of ionizing radiation was studied in the adult mouse using immunochemical and immunohistochemical methods. We found a detectable response at 0.01 Gy and an increased induction of Trp53 with increasing dose in both radiation-resistant and radiation-sensitive tissues. These results suggest that there is no lower threshold for induction. This response was heterogeneous, since cells that received the same dose had different staining intensities, suggesting that the induction of Trp53 is not based simply on dose-dependent responses to DNA damage. These data also demonstrate the exquisite sensitivity of the Trp53 pathway and show that this response is controlled by cell- and tissue-specific factors that have yet to be defined.

Animals↗

PubMed2000

Chromosomal instability in unirradiated cells induced in vivo by a bystander effect of ionizing radiation.

Using a bone marrow transplantation protocol in which we transplanted a mixture of irradiated and nonirradiated bone marrow cells that were distinguishable by a cytogenetic marker, we have demonstrated chromosomal instability in the progeny of nonirradiated hemopoietic stem cells. This first demonstration of a link between a bystander effect of ionizing radiation and the induction of genomic instability in vivo clearly poses a major challenge to current views of the mechanisms of radiation-induced DNA damage with mechanistic implications for the health consequences of radiation exposure particularly in the context of the induction of malignancy.

Animals↗

PubMed2000

Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles.

PURPOSE: To determine the induction and transmission, to second and third division cells, of complex chromosome aberrations in peripheral blood lymphocytes after exposure to high-LET alpha-particles in vitro. MATERIALS AND METHODS: Separated peripheral blood lymphocytes collected from four healthy donors were irradiated in vitro with either high-LET alpha-particles (121 keV/microm; 0.5 Gy) or low-LET X-rays (250kV constant potential; 3 Gy). Cells were harvested in first, second and third division post-irradiation and chromosome aberrations observed at each cell division were analysed by combining the techniques of FISH and DAPI/Hoechst 33258 harlequin staining. Whole chromosome probes were used for chromosomes 1, 2 and 5, together with a pan-centromeric probe and the resulting chromosome 'painting' patterns were classified according to the Savage and Simpson (S & S) scheme (Savage and Simpson 1994a, Savage and Tucker 1996). RESULTS: A greater proportion of complex chromosome aberrations was observed, defined as involving three or more breaks in two or more chromosomes, relative to total exchanges, after exposure to 0.5 Gy alpha-particles (mean 1 track/cell) than after the high reference dose of 3 Gy X-rays (49-56% and 20-22%, respectively). Qualitatively, alpha-particles induced chromosome aberrations of far greater complexity than those observed after X-rays. This was reflected by both the rapid reduction in the percentage of damaged cells between first and second division indicative of cell death, and the spectrum of aberration types observed in second and third division cells post-irradiation. Regardless of this complexity, 15% of the complexes induced by alpha-particles at first division were potentially transmissible and by third division, transmissible-type complexes, specifically insertions, represented the predominant complex type (65%). CONCLUSION: Transmissible-type complexes were observed, specifically insertions, in both second and third division cells after exposure to high-LET alpha-particles (0.5 Gy) in vitro. The authors predict these cells to be stable and to be capable of persisting through many cell generations. Considering that the induction of complex chromosome aberrations by low-LET radiation is strongly dependent on dose, so that they are expected to be undetectable at environmental exposures, insertions are much more likely to be a characteristic feature of high-LET radiation at all doses. From this the usefulness of insertions as biomarkers of past exposure to environmentally relevant doses of high-LET alpha-particles is supported.

Alpha Particles↗

PubMed2000

Dose- and time-response relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line.

PURPOSE: To investigate the relationship between two well-established delayed effects of ionizing radiation, experiments were conducted to determine the induction and expression of lethal mutations (delayed reproductive death) and chromosomal instability with respect to dose and time in a human immortalized keratinocyte cell line. METHODS: HPV-G cells were gamma- or alpha-irradiated and maintained in culture for up to 72 population doublings. At intervals, measurements were made of cloning efficiency and the cells examined for apoptosis and cytogenetic aberrations. RESULTS: The descendants of cells surviving 1 or 3 Gy gamma-irradiation, but not 0.5 Gy gamma-irradiation, exhibited a reduced colony-forming efficiency. The reduction persisted at a constant rate of 15-20% clonogenic cell loss per population doubling for up to 72 population doublings. Apoptosis was demonstrated in all colonies in the 1 and 3 Gy groups at 30 and 72 population doublings post-irradiation but not in the 0.5 Gy group. A significant persistent reduction in colony-forming ability (approximately 80%) was demonstrated in the progeny of cells irradiated with 0.5 Gy alpha-particles. After 30 population doublings, the proportion of chromosomally aberrant cells was significantly greater than control values for all doses of both high- and low-LET radiations. The major cytogenetic aberrations (chromatid breaks, chromosome fragments and minutes) were consistent with the transmission of chromosomal instability. The expression of instability declined between 30 and 72 population doublings in the 0.5 Gy and 3 Gy gamma-irradiation groups, but persisted up to 72 population doublings in the 1 Gy group. The expression of chromosomal instability was greater in the descendants of alpha-irradiated cells and showed little evidence of reduction with time. CONCLUSIONS: Unstable aberrations characteristic of radiation-induced chromosomal instability may commonly result in apoptosis and account for a component of the delayed reproductive death/lethal mutation phenotype in HPV-G cells. However, the absence of lethal mutations in the descendants of 0.5 Gy gamma-irradiated cells indicates a low-LET threshold effect for this particular endpoint. Overall, and particularly at low doses, there is no direct correlation between the two endpoints, indicating the absence of a simple relationship between these manifestations of radiation-induced genomic instability.

Alpha Particles↗

PubMed2000

Absence of delayed chromosomal instability in a normal human fibroblast cell line after 125I iododeoxyuridine.

PURPOSE: To seek the delayed appearance of chromosomal abnormalities in human fibroblasts exposed to the Auger electron emitter 125I. MATERIALS AND METHODS: Normal untransformed human fibroblasts, HF19, were exposed to a concentration of [I125]IUdR, which allowed the survival of 37% of clonogens. Chromosomal analysis using both conventional Giemsa and fluorescence in situ hybridization (FISH) was undertaken on non-clonal bulk cultures from 2 to 39 days after treatment. RESULTS: The data show a declining level of unstable aberrations in the progeny of HF19 fibroblasts exposed to [I125]IUdR, eventually reaching control levels. CONCLUSIONS: The results provide evidence that [125I]IUdR does not induce ongoing chromosomal instability in long-term culture, and gives further support to the use of Auger-electron emitting radionuclides in the treatment and diagnosis of tumours.

Cell Line↗

PubMed1999

An E mu-BCL-2 transgene facilitates leukaemogenesis by ionizing radiation.

Clonogenic murine B cell precursors are normally ultrasensitive to apoptosis following genotoxic exposure in vitro but can be protected by expression of an E mu-BCL-2 transgene. Such exposures are likely to be mutagenic. This in turn suggests that a level of in vivo genotoxic exposure that usually has minimal pathological consequences might become leukaemogenic when damaged cells fail to abort by apoptosis. If this were to be the case, then the cell type that becomes leukaemic and the chromosomal/molecular changes that occur would also be of considerable interest. We tested this possibility by exposing E mu-BCL-2 and wild-type mice of differing ages to a single dose of X-irradiation of 1-4 Gy. Young (approximately 4-6 weeks) transgenic mice developed leukaemia at a high rate following exposure to 2 Gy but adult mice (4-6 months) did not. Exposure to 4 Gy produced leukaemia in both young and adult transgenic mice but at a higher frequency in the former. Leukaemic cell populations showed clonal rearrangements of the IGH gene but in most cases analysed had immunophenotypic features of an early B lympho-myeloid progenitor population which has not previously been recorded in radiation leukaemogenesis. Molecular cytogenetic analysis of leukaemic cells by banded karyotype and FISH revealed a consistent double abnormality: trisomy 15 plus an interstitial deletion of chromosome 4 that was confirmed by LOH analysis.

Animals↗

PubMed1999

Inherited and inducible chromosomal instability: a fragile bridge between genome integrity mechanisms and tumourigenesis.

Cancer is a multi-step process evolving as the result of the accumulation of a number of mutational events. The growing body of evidence implicating genetic instability as a key feature of this evolutionary process and the risk of malignancy associated with chromosomal instability syndromes highlight the importance of understanding the mechanisms that cells use to maintain the integrity of their genomes. Classic examples of inherited chromosomal instability with cancer predisposition are Bloom's syndrome, ataxia telangiectasia, and Fanconi anaemia, although the mechanisms involved are far from understood. Selected features of these inherited disorders are reviewed to provide a background to the more recently discovered inducible chromosomal instability, a phenotype in which apparently normal cells that have survived ionizing radiation and certain chemical insults may produce descendants exhibiting a high frequency of de novo chromosome aberrations and gene mutations. The phenotype is induced at frequencies considerably greater than conventional mutation frequencies but little is understood of the underlying mechanism(s). To date, chromosomal instability induced by ionizing radiation has been the most extensively studied phenotype and it is evident that the expression of inducible instability has a strong dependence on the type of radiation exposure, the cell type irradiated, and the genetic 'predisposition' of the irradiated cell.

Animals↗

PubMed1999

An improved micro-method for obtaining chromosome preparations from individual haemopoietic colonies.

A robust method for obtaining chromosome preparations from individual haemopoietic colonies in semi-solid media is described. The accumulation of metaphases and the hypotonic treatment of cells were carried out in the culture dish and individual colonies were transferred onto poly-L-lysine-treated slides and fixed stepwise prior to staining. The technique produced high yields of well-spread metaphases facilitating clonal cytogenetic analysis.

Animals↗

PubMed1999

Identification and characterization of three subtypes of radiation response in normal human urothelial cultures exposed to ionizing radiation.

In an attempt to assess genetic variation underlying the variation in human responses to radiation exposure, measurements of apoptosis, necrosis and induction of key proteins were made in primary explant cultures of human normal urothelium and correlated with growth post- exposure to a range of doses of (60)Co. These data were validated by similar experiments using CBA/H and C57/BL6 mouse strains, known to exhibit genetically determined differences in response to radiation. The data for human tissues show a wide variation in response with three broad categories being identifiable. The commonest had a hypersensitive response involving considerable apoptosis in the low dose region, followed by 'induction' of a survival response at higher doses involving the persistence of abnormal cells. The pattern of gene expression was consistent with suppression of apoptosis. The second category showed no induction of survival and considerable necrosis was seen in the progeny. The rarest category showed an extremely hypersensitive low dose response and despite induction of a survival response, the sensitivity to higher doses was very severe. Considerable apoptosis and necrosis were seen in these cultures. In the mouse experiments, strain CBA/H (mice known to exhibit genetic instability post-irradiation) had lower levels of delayed cell death and apoptosis than C57/BL6 mice (which exhibit significantly less instability). It is concluded that there is a variation in response to radiation between human patient cultures which is detectable in this system and which is consistent with a pattern of radiation- induced delayed death/apoptosis correlating with long-term genomic stability. The mouse experiments demonstrate the importance of genetic factors in determining these responses.

Animals↗