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C Mothersill

Publications and source records attributed to C Mothersill.

At least 19 recordsLinked to original sources
PubMed1999

Antigenic characterization of Nephrops norvegicus (L.) hepatopancreas cells.

During this work structural, differentiation and proliferation antigenic markers developed for mammals were applied in paraffin sections of Nephrops norvegicus (L.) hepatopancreas. The purpose was to establish standards for the characterization of invertebrate cells in vitro. Antibody concentration was optimized for quantification of cell proliferation. There are no antibodies specific for crustaceans on the market. An avidin-biotin immunoperoxidase method was used to visualize cell antigen expression. The immunocytochemical results indicate that the epithelium in the Nephrops hepatopancreas digestive tubules does express cytokeratins and proliferating cell nuclear antigen. The results of this work indicate that some mammalian antibodies cross-react with crustacean epitopes. This may facilitate cell characterization of cell types cultured in vitro.

Animals↗
PubMed1999

Effect of docetaxel (Taxotere) on expression of radiation-induced lethal mutations in human cell lines.

PURPOSE: To investigate if docetaxel, ionizing radiation or a combination of both induces delayed cell death in selected human normal or tumour cell lines. MATERIALS AND METHODS: The initial and residual surviving fractions were determined for two malignant human colon cell lines of widely different radiosensitivity (SW 48 and HT29) and for an immortal but non-malignant human keratinocyte line (HaCaT). RESULTS: Lethal mutations were observed only after the treatment of SW48 and HaCaT cells with radiation alone. No lethal mutations were found in the HT29 cell line. No lethal mutations were observed for any cell line treated with Docetaxel and the plating efficiency of progeny was actually increased above the control level. CONCLUSION: In all cases a combination of radiation and docetaxel prevented induction of lethal mutations despite the fact that radiation alone induced lethal mutations in the SW48 and HaCaT cell lines. This suggests that the effects of pre-treating with Docetaxel are in some way blocking or inhibiting the lethal mutation pathway. There appears to be a link between the tendency of the cell line to undergo apoptosis and lethal mutation expression.

Docetaxel↗
PubMed1999

Radiobiological effects of docetaxel (Taxotere): a potential radiation sensitizer.

PURPOSE: To investigate the ability of docetaxel (Taxotere) to radiosensitize human cell lines of differing malignant status, intrinsic radiosensitivity and p53 status. MATERIALS AND METHODS: Cell survival following treatment with drug and/or radiation was determined by colony-forming assay on two malignant human colon cell lines of widely different radiosensitivity (HT29 and SW48). An immortal human keratinocyte line (HaCaT), which does not form tumours in nude mice, was used to assess the effect on non-malignant human epithelium. RESULTS: The experiments indicate that docetaxel had the greatest cytotoxic and radiosensitizing effect on the SW48 (p53wt) cell line. The degree of radiosensitization was not improved by increasing the drug concentration, although the overall cytotoxicity was increased with increasing concentrations of the two agents. Both p53mut lines were less sensitive to the drug, irrespective of their malignant potential. CONCLUSION: The results support a role for Taxotere as a weak radiosensitizer of the three cell lines tested. The p53 status and intrinsic radiosensitivity may be relevant to the mechanism.

Apoptosis↗
PubMed1999

The effect of microcolony size, at time of irradiation, on colony forming ability.

PURPOSE: To investigate the effect of irradiating microcolonies of various sizes on their subsequent ability to form colonies. MATERIALS AND METHODS: The location of individual HPV-G cells in tissue culture flasks was recorded using computerized microscopy. This allowed the cells' positions to be continually revisited, which enabled manual sizing of individual microcolonies both at the time of irradiation (between 0-5 Gy) and following an incubation period during which the microcolonies were assessed for cologenic survival. RESULTS: The experiments indicate that when irradiated as microcolonies, cells have plating efficiencies (PE) that are lower than expected when compared with the prediction from the PE values of cells irradiated individually. CONCLUSION: These data support the conclusions of Mothersill and Seymour (1997b) which challenge the concept of independent survival of certain types of cells following irradiation. The results indicate that interactions between cells can occur even in small aggregates.

Cell Aggregation↗
PubMed1998

The effects of cadmium exposure on the cytology and function of primary cultures from rainbow trout.

Cultured epidermal cells from explants of skin of rainbow trout were used to study the cytological and functional changes following sublethal exposure to cadmium stress. The aim was to develop diagnostic markers for ecotoxicology. Cultures were exposed to the pollutant for 48 h. Cell structural and cytological changes were established by light and electron microscopy. Metabolic alterations were detected by immunohistochemistry. The relation between the initiation of cellular alterations and cadmium concentrations was compared in cultures exposed in commercially-available serum-free and serum-containing medium. The expression of stress proteins (metallothionein and heat shock protein) was also studied. Rainbow trout epithelial cells exposed to cadmium showed typical morphological changes indicative of cell death by apoptosis. Sublethal exposure also resulted in cellular metabolic disturbances with increased deposits of glycogen. Increased melanization was also observed. These changes appeared at lower concentrations of cadmium when cells were exposed in serum-free media than in serum-containing media. Cadmium induced the expression of heat shock proteins but not of metallothioneins. The results broadly confirm in vivo findings for cadmium toxicity and suggest that this in vitro technique may have applications in aquatic toxicology.

Animals↗
PubMed1998

Expression of delayed toxicity and lethal mutations in the progeny of human cells surviving exposure to radiation and other environmental mutagens.

PURPOSE: Delayed expression of lethal mutations in the progeny of cells which survived a toxic insult was first shown for ionizing radiation and is one of the signs of induced genomic instability. The effect appears to be related to DNA strand breakage or repair but not to the physical break itself. To investigate this and the relationship of lethal mutations or delayed death to other instability endpoints, cultures of immortal but non-transformed human keratinocytes were exposed to a range of environmental mutagens or cytotoxic compounds with different DNA damaging properties. METHODS: Delayed expression of damage was assessed by scoring a number of endpoints in the progeny of cells which survived exposure and underwent at least 15 population doublings. Endpoints included delayed apoptosis, cloning efficiency of cells in 'healthy' colonies and expression of the apoptosis regulatory proteins bcl-2 and BAX. RESULTS: The results clearly linked expression of delayed lethal mutations with substances that induced DNA strand breaks. All these substances are known also to induce oxidative stress. The occurrence of delayed damage required a threshold level of toxicity in the initially exposed population, which was remarkably similar for all the effective substances except cadmium. Alkylating agents or microtubule poisons that do not permit repair of DNA damage did not cause any delayed death. CONCLUSION: It is concluded that delayed cell death may be caused by widespread radical damage to DNA which is either signalled, thereby inducing an apoptotic response, or (mis-)repaired yielding a weak or unstable genome. It is likely that the process may be an important factor in determining the long-term response of populations to 'sublethal' levels of environmental mutagens whose mechanism of action includes DNA strand breakage and repair.

Antineoplastic Agents↗
PubMed1998

Mechanisms and implications of genomic instability and other delayed effects of ionizing radiation exposure.

Recently there has been considerable interest in various delayed effects of radiation. These have the common property of showing a high and, in some instances, non-clonal transmission of 'damage' to distant progeny which derive from apparently normal surviving cells and their descendants. This means that conventional analysis and interpretation of long-term radiation damage in terms of mutations induced in DNA at the time of radiation exposure may be incorrect. Several reviews of this area have appeared in recent years which have described the historical development of this field. The aim of this commentary is to highlight areas of discussion, particularly concerning links between the various end-points, and to discuss some of the possible implications of genomic instability for radiation carcinogenesis in general and for the setting of radiation protection action limits in particular.

Animals↗
PubMed1998

Cell-cell contact during gamma irradiation is not required to induce a bystander effect in normal human keratinocytes: evidence for release during irradiation of a signal controlling survival into the medium.

Killing of unirradiated cells by medium from cultures of irradiated cells implies the release of a cytotoxic substance by the irradiated cells. The finding of the gamma-ray-induced cytotoxic effect exclusively in epithelial cells and not in fibroblasts suggested that tissue architecture or cell communication might be important. Normal human keratinocytes and fibroblasts and radiosensitive carcinoma cells were irradiated as single cells, microcolonies of three or four cells, or confluent monolayers. The medium was removed and filtered, and cultures which had never been irradiated were seeded at cloning densities and treated with the medium from the irradiated cells. It was found that the degree of cell-cell contact had no effect on the ability of medium from irradiated epithelial cell cultures to reduce the clonogenic survival of unirradiated cells. Cell density was the only important factor. Inhibition of gap junction intercellular communication using the tumor promoter phorbol myristate acid (PMA), which closes gap junctions, increased killing by the bystander effect when the PMA was added to epithelial cells prior to irradiation. Rescue of epithelial cells exposed to the medium from the irradiated cells was not possible even after only 30 min exposure. This suggests that a signal transduction mechanism may control death or survival by the bystander effect rather than by release of a factor which is directly cytotoxic.

Apoptosis↗
PubMed1998

Development of primary tissue culture techniques for use in radiobiology.

Many of the underlying theories of radiation biology have been developed using a combination of in vitro assays and work with animals. The aim of this dual approach was to develop and test general models using simple methods with in vitro cell lines and then to validate the results by testing their applicability in whole animals. Conversely, observations in whole animals could be modeled using cell lines in the search for mechanisms. In theory this approach is sound, but in many instances easy-to-grow cell lines were chosen which bore little resemblance to any tissue, and validation of the data in animals, if attempted at all, was done in very inbred strains of mice which were therefore genetically homogeneous. This called into question the relevance of the data for addressing questions in radiotherapy and radiation carcinogenesis in humans. In recent years, the development of elegant in situ molecular biology methods and the advances in our understanding of complex tissue biology have led to the development of tissue culture methods which aim to simulate the in vivo characteristics of the intact organ while allowing experimentation in vitro. The aim of this paper is to discuss the use and the limitations of these techniques and models in radiobiology and to indicate where they have changed or are likely to change our views of normal-tissue radiobiology.

Animals↗
PubMed1997

Delayed expression of lethal mutations and genomic instability in the progeny of human epithelial cells that survived in a bystander-killing environment.

It has recently been shown that, when irradiated, human epithelial cells produce a factor or signal in the culture medium that can reduce the clonogenic survival of unirradiated cells. The mechanism is unknown, as is the nature of the signal or substance. In this paper, we show that the medium from these irradiated cells is able to induce delayed effects in the progeny of some cell types that survive the initial exposure to the medium. The initial clonogenic survival of normal human keratinocytes exposed to medium from irradiated parallel cultures is reduced by approximately 40%. If the surviving keratinocytes (60%) are grown to confluence and replated for clonogenic assay, than they still show a reduced plating efficiency, this time of approximately 20% less than the parent line. Similarly treated normal human fibroblasts showed no delayed effects either from a direct dose or from receipt of irradiated medium. The progeny of directly irradiated tumourigenic cell lines have previously been shown to have better clonogenic survival (by a factor of at least two) than unirradiated parallel cultures, and this effect was also found, although it varied depending on the cell line used, when the distant progeny of PC-3 cells or SW48 colon carcinoma cells that received medium only from irradiated progenitors were assayed for expression of delayed lethal mutations. These data suggest that the signal/factor produced in medium by irradiated cells is able to induce genomic instability-type effects in distant progeny.

Carcinoma↗
PubMed1997

Radiation-induced carcinogenesis: studies using human epithelial cell lines.

It has proved difficult to develop suitable models to study radiation-induced carcinogenesis by using human epithelial cells. However, immortalised human epithelial cell lines have proved useful. Unirradiated cells from the human keratinocyte cell line (HPV-G) and the human embryonic lung cell line (L132) were found to be tumourigenic in T-cell-deficient mice; thus, they are not suitable for transformation studies. Human urothelial cell lines (SV-HUC-1, NT11, BC16) and the human thyroid epithelial cell line (HTori-3) were nontumourigenic. The urothelial cell lines were refractory to radiation-induced carcinogenesis, and only one small tumour was observed in 57 mice that received irradiated cells. Whereas tumours were not produced following irradiation of these urothelial cells, changes in anchorage-independent growth were observed after a single dose of 8 Gy gamma-irradiation but not after 2 or 4 Gy. Irradiation of the human thyroid epithelial cell line (HTori-3) in vitro resulted in tumour formation. Passaging of the cells in vitro before injection did not seem to be critical. Some of the cell lines derived from the primary thyroid tumours exhibited p53 mutations in exons 5, 6, 7, and 8, as detected by single-stranded conformational polymorphism (SSCP) analysis. Thus, the human thyroid epithelial cell line (HTori-3) looks promising as a model for investigating the molecular events in radiation-induced carcinogenesis.

Animals↗
PubMed1997

Apoptosis and other effects of radiation in normal human urothelial cells.

In this paper, an attempt is made to identify endpoints that might be of potential use in the quantification of radiation effects in human tissues. Irradiated cultures of cells that are not selected for clonogenic survival but are left in situ to grow after irradiation show a wide variety of morphological and biochemical abnormalities. These include nuclear fragmentation and other evidence of programmed cell death, but they also include a considerable amount of lysis, necrosis, and persistent abnormal growth and function, which are expressed in the progeny of irradiated cells. Induction of proteins associated with stress or shock responses, growth and cell cycle control, and control of apoptosis are also seen and may persist. The dose dependence of these various responses is documented, because it probably determines to a large extent the outcome of radiation exposure in terms of whether a cell dies, divides normally, or develops genomic instability, mutation, and ultimate carcinogenic progression of the progeny. Clearly, a cell that dies presents no further threat to the organism, nor does a fully repaired cell. Therefore, a major challenge facing radiation protection research is to define the population at risk of surviving with damage. The results show that there is a variation in response to radiation between different patient cultures that is detectable in an explant culture system of primary normal human urothelium. The growth pattern and protein expression postirradiation is consistent with apoptosis being a major determinant of low dose response to radiation. This form of death appears to be suppressed at higher doses and, in the majority of subjects, results in the presence of a highly abnormal population of cells, even though the population size is the same whether their progenitors were irradiated or not.

Apoptosis↗
PubMed1997

p53 mutations and protein expression in primary cultures of normal oral mucosa in smokers and non-smokers.

The normal oral mucosa from 77 individuals of known smoking and alcohol-intake history was cultured. After 14-21 days in culture, epithelial cells were stained for p53 expression using immunohistochemistry. PCR-SSCP analysis of the p53 gene was also performed on a random sub-set of these samples (20 non-smokers and 21 smokers). Expression of the stable, non-functional form of p53 protein as detected by the p53-240 antibody was found to be significantly elevated in the cultured oral mucosa of smokers. (P < 0.01). PCR-SSCP analyses indicated a higher level of base changes in smokers than in non-smokers. These observations are consistent with other findings of significantly increased p53 protein expression in the oral mucosa and other tissues of smokers and suggests that p53 mutations may be an early event in smoking-induced oral cancers.

Adolescent↗
PubMed1997

Survival of human epithelial cells irradiated with cobalt 60 as microcolonies or single cells.

Microcolonies of one to >50 cells were irradiated. They were assayed for survival using the Puck and Marcus clonogenic technique and the distant progeny were tested for expression of lethal mutations. The results show that epithelial cell colonies appear to respond as a unit rather than as individual cells to a radiation dose and the uncorrected initial surviving fraction is relatively constant irrespective of the number of cells present at the time the microcolony was irradiated. Irradiation of colonies or monolayers, which were then dispersed, confirmed this and showed slight sparing of the cells irradiated in contact compared with single cells but no sparing effect when the gap junctions were closed. Measurement of apoptosis 2 h post-irradiation showed higher levels in clones derived from cells irradiated in contact but delayed apoptosis in the progeny and lethal mutations appear to be associated with irradiation of single cells. Lethal mutations occurred in the progeny of cells irradiated as single cells for at least 30 cell generations but if cell microcolonies were irradiated the progeny survival showed a complex relationship with progenitor dose. When gap junction intercellular communication (GJIC) was blocked during and immediately post-irradiation using nitrosamines or TPA, cultures regained the initial survival and lethal mutation frequency seen with single cells. It is concluded that the presence of more than one cell in a microcolony at the time of irradiation does result in an altered and possibly a co-ordinated pattern of survival and lethal mutation expression but that inhibition of GJIC can reverse the effects of contact. The results may have implications for investigations of normal tissue response.

Apoptosis↗
PubMed1997

Induction of multiple PCR-SSCPE mobility shifts in p53 exons in cultures of normal human urothelium exposed to low-dose gamma-radiation.

We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma-radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma-radiation from a 60 Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene post-irradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53-related genomic instability.

Culture Techniques↗
PubMed1997

Comparative effects of UV A and UV B on clonogenic survival and delayed cell death in skin cell lines from humans and fish.

The effects of UV radiation on humans and animals are receiving increasing attention and much interest has recently been focused on the environmental effects of UV A and UV B. This study compares the in vitro effects of UV A and UV B on the clonogenic survival of two human skin keratinocyte cell lines, HaCaT which are immortal but not tumorigenic and HPV-G transfected keratinocytes which form non malignant tumours in nude mice. The effects were also studied on an EPC fish cell line. The aim of the work was to establish if similar initial and delayed survival responses occurred in both species. The cells were exposed to ultraviolet lamps emitting maximally at 365 nm (UV A) and 302 nm (UV B). Clonogenic survival was determined at appropriate times post exposure. Results for the initial survival curves show that the HaCaT and HPV-G cells did not show any appreciable difference in their response to UV A but the EPC cells were more sensitive at doses < 3000 Jm-2. The EPC cells were more sensitive to UV B at doses < 200 Jm-2 in comparison to the human HaCaT and HPV-G cells with the HPV-G cells showing the most sensitivity to UV B at doses > 200 Jm-2. The possible contribution of lethal mutations (delayed cell death) to the UV radiation response in the HaCaT and EPC cell lines was examined. The results showed that lethal mutations were expressed in the HaCaT cells following exposure to UV A and UV B but no lethal mutations were expressed in the EPC cells.

Animals↗
PubMed1997

Lethal mutations and genomic instability.

The delayed expression of cell death in progeny of irradiated survivors was christened 'lethal mutations' by Tikvah Alper in 1984. The effect occurs when clones, or populations of cells grown up from irradiated progenitor cells, are replated and reassessed for cloning efficiency or population doubling time. The effect has been shown to be associated with the low dose shoulder region of the survival curve and is due to events occurring in the first two hours post irradiation, i.e. the 'fast repair' period. In this review the lethal mutation data accumulated over the past ten years is discussed in relation to our modern understanding of cellular and molecular events in radiation carcinogenesis and genomic instability research. It is suggested that lethal mutations are associated with a general epigenetic or field effect occurring in all irradiated cells, which makes them more prone to mutations, some of which are lethal. The implications of this for our current approach to risk estimates and therapeutic dose calculation, need to be addressed.

Animals↗