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Biomedical subjects

C Mothersill

Publications and source records attributed to C Mothersill.

At least 91 records · Page 5Linked to original sources
PubMed1992

Endothelial cell proliferation is induced by radiation in cultured explants of human urothelium and oesophageal mucosa.

Normal oesophageal mucosa obtained during upper abdominal surgery or urothelium obtained from kidney transplants was placed in explant culture and exposed to 60Co gamma radiation after 48 h. Cultures were maintained for two to six weeks after exposure and were monitored at various intervals for the development of features associated with malignant transformation. Endpoints examined included proliferation rate, frequency of proliferating cells, cell type distribution and degree of differentiation of the different cell types. The results indicate that following exposure to gamma rays (2.5-10 Gy) an increased overall growth rate of the surviving cells can be observed 2-4 weeks later. Analysis of the results using autoradiography confirms that a higher level of cell proliferation occurs in treated cultures than in the control untreated cultures. When the distribution of different cell types in the culture is examined, the increase in growth can be seen to be due to greatly increased numbers of endothelial cells. These proliferated over the surface of the epithelial cells and are more strongly positive for endothelial cells markers than endothelial cells occurring in control cultures. The degree of differentiation of endothelial cells into capillary like structures is also more apparent in carcinogen treated cultures. Foci expressing both epithelial and endothelial markers also occur. The results suggest that exposure of tissue fragments to radiation stimulates the growth and development of endothelial cells in resulting cell cultures. The effect may be due to a direct action of the treatment on the endothelial cells but it is more likely that it results from a secondary effect mediated by traumatic response of damaged epithelial cells.

Cell Division↗
PubMed1992

All colonies of CHO-K1 cells surviving gamma-irradiation contain non-viable cells.

This paper addresses the problem of the production of defective cells within clones arising from irradiated progenitor cells and is specifically aimed at answering the question of whether lethal mutations result from a generalised effect which lowers the ability of all the progeny to divide successfully or whether it represents a late expressed but unique lethal defect induced by radiation which occurs in some cells only and which causes those cells only to cease dividing. The results obtained from autoradiographic analysis of cells within individual surviving colonies (i.e. containing more than 150 cells) suggests that some cells in all clones are not synthesizing DNA over a 9-h period and that the proportion of non-synthesising cells rises with increasing dose of radiation from less than 3% in the controls to 80-85% after a progenitor dose of 12.5 Gy. Because of the possibility that cells had longer division times post irradiation, these results were repeated using Ki67 antibody labelling, a technique which identifies cells which are in cycle. The results were similar. This suggests the non-labelled cells were not reproducing. Both techniques were also used to look at the % labelling of morphologically abnormal cells in the colonies. The results suggested that up to 35% of these abnormal cells were actively cycling and about 20% were synthesising DNA. Abnormal cells did not appear in subcultures of survivor progeny suggesting that they may have failed to replate successfully and may contribute to the lethally mutated population. The idea that radiation induces a general instability in the cell population was supported by experiments where growth and the plating efficiency of irradiated progeny was measured daily. This revealed that the growth curves deviated from the control by a constant factor suggesting a division probability of about 70% of the control level after a progenitor dose of 10 Gy. The results are discussed in the context of their significance for survival curve analysis and for radiotherapy and radiation protection results.

Animals↗
PubMed1991

Proliferation of normal and malignant human epithelial cells post irradiation.

Fragments of human oesophageal mucosa, urothelium, squamous and adenocarcinoma of the oesophagus and carcinoma of the bladder have been plated in culture and irradiated. The cells growing from the explanted tissues have then been studied for four weeks post irradiation to assess the overall rate of growth from the irradiated explants and the fraction of proliferating cells. The results show that when using cell number as an endpoint it is possible to derive growth curves from this type of data which permit a doubling time to be obtained for the cell population surviving different doses. In an attempt to determine the proliferating fraction of the cell population, cultures were labelled at appropriate intervals with tritiated thymidine and were also stained with Ki-67 antiproliferating antigen. The results show an interesting relationship between the dose response obtained for cell labelling with tritiated thymidine and area of cellular outgrowth. Ki-67 staining when used carefully and analysed as described was a useful indicator of proliferating cells. The results provide a means of determining the post irradiation growth potential of fragments of tissue from human organs and may be important for determined overall response of the tumour bulk to proposed treatment.

Autoradiography↗
PubMed1990

The effect of radiation in combination with carcinogens on the growth of normal urothelium in explant culture.

Radiation is known to be carcinogenic to humans but attempts to demonstrate the process using human tissue culture models have met with little success. In the present study explants were established from urothelium and exposed to radiation and a range of chemical carcinogens, suspected promotor or metabolic agents. The resulting outgrowth was monitored for growth rate, proliferating epithelial fraction and development and differentiation of endothelial cells in culture. The results indicate that enhanced growth of epithelial cells can be seen when cultures are irradiated in the presence of various nitrosamines, benzo(a)pyrene or aniline. Radiation alone reduced the overall growth area measured but several proliferative foci developed on the resulting outgrowth. Their ultrastructural appearance reveals that they carry severe mitochondrial damage and exposure of treated cultures to metabolic inhibitors confirms that their respiration is defective. Endothelial cells proliferated over the surface of the epithelial monolayer and both the number and the degree of differentiation of the endothelial cells increased with increasing dose up to 10 Gy. While the cultures are not immortalised by the treatment, it appears that the epithelial cells have an extended lifespan (division capacity) and that a subpopulation has undergone a number of premalignant changes. Changes in endothelial cell proliferation also occur.

Cell Division↗
PubMed1990

Effect of radiation and other cytotoxic agents on the growth of cells cultured from normal and tumor tissues from the female genital tract.

A technique is presented which allows the response of human gynecological tissue to radiation and cytotoxic drugs to be assessed using a tissue culture explant system. The technique is simple to use and gives results in line with those obtained for human tissues by more complex culture methods. Data are presented showing how the explant technique developed by the group for other tissues can be adapted to yield acceptable results for normal tissue response to radiation. The potential of the technique for use in predictive testing of individual tumor response is then assessed in five cases of gynecological malignancy. It is clear that variations in sensitivity to different radio- and chemotherapy agents and combinations can be detected. The results obtained require clinical validation and it is hoped that this will come over the next few years from evaluation of patient response to treatment using individually optimized, rather empirical therapy.

Adenocarcinoma↗
PubMed1990

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study.

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.

Animals↗
PubMed1990

The effect of radiation and cytotoxic platinum compounds on the growth of normal and tumour bladder explant cultures.

Using an explant tissue culture model developed by this group for use with human surgical and biopsy specimens, data are presented showing the response of normal and tumor bladder urothelium to radiation in combination with cis- and carboplatin. Cellular response is measured after two weeks in culture as a reduction in the extent of outgrowth from the explant. The outgrowth has been shown to be growing and to be epithelial. Results showed that when either drug or radiation is used singly, the tumour is resistant to treatment while the normal cells are severely affected. However, appropriate combinations of either drug with radiation reverse the unfavourable therapeutic ratio and result in higher tumour cell kill. The model may be useful for investigating mechanisms of radiation/chemotherapy action at the cellular level and, if integrated into appropriate clinical trials, may serve as an easy-to-use in vitro test for optimising single agent or combination therapy regimens.

Carboplatin↗
PubMed1989

Enhanced proliferation of cells from human tissue explants following irradiation in the presence of environmental carcinogens.

A method which allows growth of normal human tissue to be studied in vitro is used to investigate possible interactive effects of radiation and environmentally important carcinogens on oesophageal and urothelial cell growth. Carcinogens chosen were selected for their known or suspected effect on the oesophageal mucosa or urothelium in vivo. The results indicate that with carcinogens alone concentrations can be identified that result in increased proliferation of cells. With radiation alone inhibition of cell proliferation occurs at all dose points examined. However, at precise combinations of radiation and carcinogen, greatly enhanced cell proliferation could be detected, suggesting a synergistic interaction between the two agents. The results may have implications for the design and interpretation of experiments aimed at elucidating early or premalignant changes in epithelial tissues and may indicate hitherto unsuspected interactions between radiation and environmentally important carcinogens.

Benzo(a)pyrene↗
PubMed1989

Optimisation of media for the culture of normal human epithelial cells from oesophageal mucosa.

A technique has been developed which allows growth and histology/cytochemistry of primary oesophageal mucosal explant cultures to be monitored over four weeks. The paper describes experiments designed to optimise media and culture conditions. The results suggest that optimal growth can be obtained in media containing RPMI 1640, and 10% horse or newborn calf serum. McCoy's 5A medium or Dulbecco's modified MEM could be substituted for RPMI but Iscove's serum-free medium or Ham's nutrient mixture inhibited growth or promoted fibroblast contamination. The essential additive appeared to be insulin while selenium was highly toxic to the cells. Hydrocortisone or EGF improved growth slightly under some conditions. Neither transferrin nor cholera toxin had any beneficial effect. None of the cell culture flask coating agents improved attachment or growth.

Cell Division↗
PubMed1989

Lethal mutations, the survival curve shoulder and split-dose recovery.

Our group has shown that many of the progeny of cells which survive irradiation, as determined using a clonogenic assay, die out after 8-30 further cell divisions. Correction of conventional survival curves for this extra component of defective cells (termed lethally mutated cells) reduces or eliminates the 'shoulder' which is characteristic of the radiation response of many mammalian cell lines. Since the size of the shoulder is theoretically and experimentally linked with the extent of split-dose recovery, this paper examines the occurrence of lethal mutations following fractionated irradiation using a variety of experimental conditions. The results show that, when curves are corrected for lethal mutations to give residual survival, the size of the single-dose survival-curve shoulder is indeed reduced but the extent of recovery after split-dose irradiation remains the same or, under some conditions, is actually increased. However, the rate of increase in survival with time between doses is reduced over the first 2 h if the data are corrected for lethal mutations, suggesting that early postirradiation repair may be error prone. When a metabolic inhibitor which depletes cellular ATP was used, the single-dose and split-dose curves corrected for lethal mutations were coincident with each other and with the corrected single-dose control curve, all being exponential and with an extrapolation number of one. It is concluded that the mechanisms leading to the production of the primary survival-curve shoulder are different from those leading to split-dose recovery. The results strongly suggest that a mechanism involving induction of repair/resistance by an initial dose of radiation is involved in split-dose recovery.

Adenosine Triphosphate↗
PubMed1988

The effect of glycolysis inhibitors on the radiation response of CHO-K1 cells.

Exposure of CHO-K1 cells to three different inhibitors of glycolysis, prior to treatment with a single dose of ionising radiation, reduced their survival. The effects were concentration-dependent but occurred under all conditions where cells were exposed to the inhibitors prior to irradiation. The results are similar to those obtained by this group when glycolysis was altered using analogues of D-glucose or by blocking the pyruvate----lactate reaction using added lactate or oxamate. They support data from other workers suggesting a role for energy metabolism in the final expression of radiation damage.

Animals↗
PubMed1988

Radiation-induced outgrowth inhibition in explant cultures from surgical specimens of five human organs.

An explant outgrowth technique to determine the radiation response of five different human organs (bladder, oesophagus, colon, breast and thyroid) is described. In each case except thyroid, where malignancies are rare, data are presented for normal and malignant tissue. Results show that variations in response, consistent with those observed in vivo, can be measured. Tumours were in all cases highly resistant to radiation relative to their corresponding normal tissue. Possible reasons for this are discussed. The method may prove useful in the prediction of the radiobiological response for tumour and surrounding normal tissue where post-operative therapy is planned.

Breast↗
PubMed1988

The effect of radiation on the growth of normal and malignant human oesophageal explant cultures pre-treated with bleomycin.

Since all known chemotherapy and radiation treatments affect normal cells to a certain extent, the establishment of favourable differential sensitivities is fundamental to the success of treatment with a particular agent. This type of information can be gained by animal testing and using cultured cells, but ultimately use of the agent in the patient is the only way to determine the response. We have developed a model for testing the response of oesophageal explants from tumour and surrounding normal tissue in the same patient to chemotherapy and radiation, both singly and in combination. The test allows treatment combinations, time and order of administration of agents to the tissue to be accurately controlled. Cytotoxicity, determined by measuring the area of outgrowth from an explant 2 weeks after plating, is the most useful short-term end-point, although many others are possible. Results showing the differential cytotoxicity of bleomycin with and without radiation in squamous and adenocarcinoma of the oesophagus and surrounding normal tissue from the same patient indicate that tumour cells are relatively resistant to radiation alone, that low levels of bleomycin with or without radiation preferentially spare tumour cells and that high levels, in combination with any dose of radiation tested, but not without radiation, spare the normal cells and give a significantly high amount of relative tumour cell kill. Bleomycin must be added to the cells just before or just after irradiation to obtain the normal-tissue sparing effect. The technique may be a useful method for indicating the best approaches to the optimization of combined therapy regimes.

Adenocarcinoma↗
PubMed1988

Differential response of normal and tumour oesophageal explant cultures to radiation.

An in vitro method is described which allows radiation dose response data to be obtained for samples of oesophageal mucosa obtained from patients undergoing resection for adeno or squamous cell carcinoma. Data are obtained using a growth endpoint from explant cultures and may be expressed in terms of absolute growth inhibition or reduced rate of growth. Radiation dose response curves suggest that cell survival is in the range expected for mammalian cells but that, as is found clinically, tumour cells are far more resistant to radiation than normal cells. The technique provides a means of assessing differential radiation response in normal and tumour tissues from the same patient, as it is unusual for both to be amenable to clonogenic assay.

Adenocarcinoma↗
PubMed1987

The influence of lethal mutations on the quantification of radiation transformation frequencies.

Transformation frequencies for gamma irradiated C3H 10T1/2 cells have been analysed, taking account of the occurrence of lethal mutations in these cells. Lethal mutations already noted by these authors in primary thyroid and established CHO K1 cells occur at high levels in C3H 10T1/2 cells and lead, therefore, to considerable underestimates of transformation frequency, particularly at high doses where this is expressed on a per surviving cell basis. The results may help to provide an explanation for the dose response plateau which is seen when these cells are irradiated and transformed foci per surviving cell are scored.

Animals↗