Search PubMed⌕ Search

Biomedical subjects

C Mothersill

Publications and source records attributed to C Mothersill.

At least 73 records · Page 4Linked to original sources
PubMed1997

Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells.

The results reported in this paper show a highly significant fall in cloning efficiency in unirradiated normal and malignant epithelial cell lines receiving medium from irradiated cultures. Medium irradiated in the absence of cells had no effect nor did irradiated medium from a fibroblast line, but irradiated medium from epithelial cells had an extremely toxic effect on unirradiated fibroblasts. Cells from four different cell lines were seeded, using standard techniques, 6 h prior to irradiation with cobalt 60 (Co60). At various times ranging from 1-60 h after irradiation, medium was removed, passed through a 0.22 mu filter and then used to replace the medium from parallel cultures seeded at cloning densities which had not been irradiated. The effect produced by the epithelial cell cultures was dependent on the cell number present at the time of irradiation, suggesting that a cell-derived factor is involved. The effect could be observed using medium taken from irradiated cultures as soon as 30 min/post irradiation. Examination of unirradiated cultures 48 h after receiving irradiated medium revealed the presence of high numbers of apoptotic bodies and other morphological evidence suggesting apoptosis may be a prominent mechanism of cell death responsible for the reduced cloning efficiency.

Cell Line↗
PubMed1997

p53 protein expression and increased SSCP mobility shifts in the p53 gene in normal urothelium cultured from smokers.

This study provides evidence of a significantly (P = 0.018) increased level of expression of the stable conformation of p53 in normal urothelial cells, cultured in vitro from bladder biopsies obtained from normal smokers without malignant disease of any site. With two significant exceptions, non-smokers showed low or no expression of this protein. Past smokers appeared to segregate into high or low p53 expressers, but the expression was not correlated with years since quitting smoking or with pack years smoked. The mean data in this group were not quite significantly different (P = 0.08) from the non-smoker group, due to the wide inter-patient variation. For most of the smoker group, pack years correlated with p53 expression with a mean unit of 1.7 +/- 0.37% p53 per pack year but there was a small group of very heavy smokers who showed lower than expected expression (approximately 0.3-0.8% p53 per pack year). These were statistical outliers (Grubbs test). No explanation could be found for this. Over-expression of p53 protein, often correlates with mutations in the gene, but may also indicate that breakdown of wild-type p53 has slowed. SSCP analysis of the biopsy material was not possible on all patients due to ethical constraints on the amounts of tissue which could be taken but in the cases where it was possible the association between loss of p53 protein function and mobility shifts in p53 exons 5-8 was confirmed with smokers having 3.5 times the number of mobility shifts detected in non-smoker DNA. Thus the results may point to a role for the early abrogation of p53 protein function in bladder carcinogenesis induced by cigarette smoking.

Adult↗
PubMed1997

Further investigation of the response of human uroepithelium to low doses of cobalt-60 gamma radiation.

Explant cultures of normal human uroepithelium were established, exposed to a range of 60Co gamma-ray doses from 0.1-5 Gy and grown for 14 days. Expression of Myc, p53 and Bc12 proteins in the epithelial cells which grew from irradiated explants was measured in situ using immunocytochemistry. The results show that overexpression of Bc12 with low Myc expression correlated with resistance to radiation as shown by the extent of growth detected on day 14. Strong staining for Myc coupled with low or absent Bc12 expression generally correlated with radiosensitivity, although the level of p53 of the culture was critical in these cases. None of the proteins on their own correlated with radiation response. What appeared to be critical was the balance of cells expressing Bc12 and Myc proteins. Building on the results presented in a previous paper which showed a division of cultures from patients into those showing monotonic and nonmonotonic responses, this study presents results for explant cultures from a greater number of patients and attempts to characterize the profile of expression of the above proteins in the uroepithelium of these patients. It shows that high Bc12/Myc ratios were found in cultures which showed a non-monotonic and resistant dose response. Where Myc was the dominant protein in the culture postirradiation, a radiosensitive and monotonic response tended to occur. Since the proteins are being detected in the distant progeny of irradiated cells, it is likely that changes induced by radiation in the cell population are stable. The measurement of these two proteins can be made in cultured biopsy material and may therefore have predictive value in radiotherapy and radiation protection. Both normal and tumor biopsies from bladder mucosa showed similar correlations between Bc12/Myc ratios and growth postirradiation.

Apoptosis↗
PubMed1996

The differential effects of cadmium exposure on the growth and survival of primary and established cells from fish and mammals.

The differential cytotoxic effects of cadmium on fish and mammalian epithelial cells in established and primary culture were assessed by looking at the reduction of the colony-forming ability and reduction in the extent of growth. The influence of medium composition on the toxicity of cadmium was also studied using serum-free and serum-containing media. The experiments using immortalized cell lines showed that mammalian cells were more sensitive than fish cells to cadmium. Both cell types were grown at the same serum concentration. However, using the normal primary system, human epithelial tissue explants showed less sensitivity to cadmium than did similar cultures from rainbow trout. It is likely that cellular mechanisms of cadmium resistance in the different cell types are responsible for these effects. As expected, cadmium proved to be more toxic when tested in serum-free medium. With fish skin primary cultures, reduction of cell numbers was observed at concentrations as low as 5 mumol/L in serum-free medium compared to 100 mumol/L in serum-containing medium. This was found to be due to the reduction in the activity of free cadmium ions, caused by the presence of serum in the medium. It is concluded that serum-free formulations are probably preferable when testing pollutants in vitro. The results highlight the importance of establishing species-specific pollution tests and standardizing variables. In summary, the results show species and culture media differences in cadmium toxicity and suggest that caution is required when extrapolating results for pollutant effects from one in vitro system to another.

Animals↗
PubMed1996

Persistent expression of morphological abnormalities in the distant progeny of irradiated cells.

The phenomenon of delayed heritable lethal damage (often referred to as "lethal mutations") in the progeny of cells which survive irradiation is now well established, but little is known of the mechanism by which this cell death occurs. Current theories suggest a generalised genomic instability affecting all cells which leads to the production of some mutations which are lethal, or alternatively that a lethal mutation gene is activated, mutated or induced by radiation and leads to persistent and random cell death at high levels in the progeny. The aim of this study was to look at the morphology of progeny of irradiated cells at various times after irradiation to establish how widespread morphological abnormalities were in the population and whether there was any evidence that such abnormalities were clonal. Using two different cell lines, the results showed that morphological evidence possibly suggestive of apoptosis occurred in the cultures after all doses of radiation and up to 45 cell doublings after exposure. There was no evidence of a decrease in the numbers of damaged or dead cells in colonies with number of divisions after irradiation, or with decreasing original radiation dose. There was a significant dose-dependent increase in the number of cells with microvilli for both cell lines. The dose-dependency of this effect did not change with number of divisions after irradiation. It is clear that morphological evidence of cellular damage persists for several generations after the initial exposure. The effects are widespread in the cell population, and their constancy over time argues strongly for a general instability and against a clonal mechanism, since clonal descendants should die out and leave undamaged survivors. The lack of evidence for necrosis or senescence together with many morphological changes in the cultures suggestive of apoptosis could indicate an active mechanism of cell death. It is concluded that survivor populations of irradiated cells from two widely different mammalian cell lines demonstrate an altered phenotype including gross morphological changes. These result in a higher probability that cell division will fail to yield two healthy progeny.

Animals↗
PubMed1996

Expression of lethal mutations is suppressed in neoplastically transformed cells and after treatment of normal cells with carcinogens.

Recent evidence from our laboratory suggests that the fraction of cells with lethal mutations is lost from the population by apoptosis. The relationship of this process to genetic instability and carcinogenesis is unclear. To examine this, tumorigenic cell populations derived from spontaneously occurring, neoplastically transformed C3H 1OT1/2 foci and from radiation-induced foci were compared with wild-type C3H 10T1/2 cell populations to determine the frequency of induction of lethal mutations postirradiation. Lethal mutations did not occur in the progeny of cells from type 3 foci derived from cultures of spontaneously occurring or radiation-induced neoplastically transformed cells but were very frequent in the progeny of irradiated wild-type cells. Normal human cells (HPV-immortalized human keratinocytes and primary human normal uroepithelium) were then treated with carcinogens or transfected with the Ha-ras oncogene to see if these carcinogenic events affected the yield of lethal mutations postirradiation. In each case, cells which were exposed to a carcinogenic agent had reduced numbers of lethal mutations, elevated levels of stable p53 and Bcl-2 proteins and reduced evidence of apoptosis. It is suggested that lethal mutations may represent an active safety mechanism which may deal with radiation-induced genomic instability and which is disabled early in carcinogenesis.

Animals↗
PubMed1996

Effect of a tobacco-related nitrosamine on intercellular communication in human urothelial cells: a possible factor in smoking-related bladder carcinogenesis.

Bladder cancer is associated with smoking. Among the tobacco-derived carcinogens suspected of being involved in initiating the disease are nitrosamines found in urine. In this study a nitrosamine found in the urine of smokers was tested using a tissue culture model of normal human urothelium. Explant cultures were established from ureters and exposed to 5 ng/ml of the derivative. This level had been demonstrated previously to induce a variety of changes associated with initiation of carcinogenesis. Proliferation of the cultures was increased following exposure to the carcinogen, and the gap junction intercellular communication was reversibly inhibited. Examination of the connexin 43 protein and message status showed that the mRNA was unaffected, but the protein was not detectable using anti-connexin 43 antibody. The expression of the protein recovered within 24 h of removal of the carcinogen, indicating that the continued presence of the agent was necessary. Given the roles of cell proliferation and cell communication in carcinogenesis, the results may suggest a mechanism involving pre- or post-initiation deregulation of cell communication systems. Whether the enhanced growth is a separate effect or a consequence of reduced communication is an intriguing question.

Carcinogens↗
PubMed1995

Expression of p53 in urothelial cell cultures from tumour-bearing and tumour-free patients.

An explant culture technique was used to culture normal urothelium from patients with muscle-invasive bladder cancer (transitional cell carcinoma, TCC) (n = 11) and from non-tumour-bearing patients (n = 60). Cell cultures were examined for expression of p53 using the monoclonal antibody p53-240. There was a statistically significant increase in p53 expression in normal urothelial cell cultures from patients with TCC (P < 0.0005). Normal urothelial cultures from patients with TCC also showed more rapid proliferation in vitro when compared with non-tumour-bearing patients (P < 0.0005). A subgroup of non-tumour-bearing patients (n = 14) showed > 5% of cells expressing p53. p53 expression in this subgroup was found to correlate with cell proliferation in vitro (r2 = 0.766). None of these urothelial specimens was observed to express p53 when paraffin-embedded preparations were stained with p53-D07 antibody prior to culture. The rate of cellular proliferation in this subgroup did not differ from that of normal urothelium from TCC patients. Twenty-two paraffin-embedded, muscle-invasive TCC specimens were also evaluated for p53 expression using p53-D07. The expression of p53 in these tumours did not differ from that observed in normal urothelial cell cultures from patients with TCC (P = 0.26). This study identifies an overexpression of p53 in normal urothelial cells from patients with TCC and in proliferating cultures from a significant subgroup of patients without malignant disease. Increased p53 expression in normal cultured urothelial cells from patients with bladder cancer implies a global change in the mechanisms controlling urothelial cell division. This may represent an early step in the pathway to carcinogenesis.

Carcinoma, Transitional Cell↗
PubMed1995

Primary explants of human uroepithelium show an unusual response to low-dose irradiation with cobalt-60 gamma rays.

Recent results using very low doses of radiation have suggested that there is a hypersensitive region where cultures show an enhanced level of cell killing leading to a non-monotonic survival curve. This effect has been observed at doses below 2 Gy in mammalian systems and at much higher doses in insect cells. In this paper we report observation of the effect in primary human uroepithelial cell cultures. The effect was measured using a postirradiation proliferation assay where irradiated explants of standard size were allowed to proliferate for 14 days after exposure to 60Co gamma irradiation. By 14 days the majority of cultures derived from explants irradiated with 2-5 Gy showed little evidence of growth inhibition and cell numbers approached or even exceeded those obtained in the controls. There was, however, a significant reduction in cell number and growth rate in all cultures exposed to doses lower than 1 Gy. Oncoprotein (p53, c-myc, bcl-2, p21 ras) and EGFR expression were also measured in these cultures and were significantly increased. Morphological evidence of apoptosis was present in all irradiated cultures at 4 h after exposure, but this persisted for longer periods in cultures exposed to low doses.

Cell Survival↗
PubMed1995

Variation in the expression of p53, c-myc, and bcl-2 oncoproteins in individual patient cultures of normal urothelium exposed to cobalt 60 gamma-rays and N-nitrosodiethanolamine.

The controls determining the initial response of cells to DNA damage probably determine whether a cancer will ultimately occur. Efficient repair or apoptosis represents extremes of control mechanisms. Misrepair can lead to fixation of damage. The changes in oncoprotein expression of three genes involved in the regulation of repair of DNA damage and postdamage proliferation of cells were measured in cultures of normal urothelium from 55 patients without any malignancy. The aim was to obtain information on interperson variation in response to carcinogens in the human population. The group included 10 pediatric patients < 2 years old. Two different carcinogenic agents, ionizing radiation and N-nitrosodiethanolamine, which represent widely different DNA-damaging pathways, were used. Both of these cause bladder cancer in humans. Cells from explanted tissue were examined after carcinogen exposure for levels of p53, c-myc, and bcl-2 proteins. Both carcinogens led to increased levels of cytoplasmic p53 protein expression, although there was significant interpatient variation. bcl-2 showed a very significant increase in expression after radiation exposure. c-myc was high and variable pre- and postexposure. Individual patient culture changes in the expression of the three oncoproteins did not correlate significantly with each other or with cell growth, suggesting that the controls are complex. Pediatric samples had lower mean control values of p53 and bcl-2 than did adult samples. This was due to the absence in this group of high controls seen in some adult cultures. The result suggest that an early breakdown in control mechanisms of growth arrest and apoptosis may occur in urothelium after carcinogen exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Postirradiation expression of lethal mutations in an immortalized human keratinocyte cell line.

The quantification of the extent of delayed cell death and the rate and pattern of its occurrence in relation to the cell division cycle is important in radiotherapy and also in radiation transformation studies related to protection and dose limits. Here the numbers of lethal mutations occurring over 45 population doublings (clonal expansion to about 10(13) cells per cell originally surviving irradiation) was measured in an HPV 16 immortalized human keratinocyte cell lines used for transformation studies. The results showed that when postirradiation (dose range 1-6 Gy) growth curves were constructed, the difference in slopes could be accounted for entirely by correcting for the non-clonogenic fraction in the cell count, excluding a longer cell generation time as an explanation. When the cell loss was examined over the entire growth period of 6 weeks (about 45 doublings of the cell population), it was found to be dose dependent for the first two passages, but then to become more independent of dose. The mean loss for the population over the entire 45 cell doublings was about 15% at each cell division. There is some indication from the data that the numbers of lethal mutations actually begin to increase between passage 6 and 7 at every dose tested but this requires further investigation. The results allow a time/cell generation dependent factor to be derived for the cell line and used in survival curve equations where effects of radiation are being measured at times distant from the original exposure.

Cell Division↗
PubMed1994

High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation.

When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells were noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in "edge" cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed.

Cell Division↗
PubMed1994

Induction of stable p53 oncoprotein and of c-myc overexpression in cultured normal human uroepithelium by radiation and N-nitrosodiethanolamine.

Uroepithelium cultured from normal patients without cancer (60 individuals) was found to segregate into four subtypes based on the level of carcinogen treatment needed to induce abnormal p53 and c-myc. Twenty-two percent of patient cultures never showed abnormal p53 expression, even after chronic exposure to nitrosamines, in addition to irradiation. In these cultures, c-myc expression was confined to viable, normal-appearing cells at the growing edge of the culture and to apoptotic bodies. Twenty-eight percent of cultures were negative for abnormal p53 unless challenged with both radiation and chronic administration of nitrosamines, while a further 26% required only a single dose of radiation to induce the abnormal protein. The remaining patients had tissue which, while initially negative for stable p53, became positive when put into culture and stimulated to grow. The c-myc protein was overexpressed in all cultures with abnormal p53. It would appear that elevated expression of conformationally inactive p53 and of high levels of c-myc represents an early response of normal uroepithelial cells to carcinogen challenge. It also appears that a relatively high number of patients without cancer express these proteins when their cells are challenged to grow; a pre-exposure to environmental carcinogens such as nitrosamines in cigarette smoke is likely to be involved.

Carcinogens↗
PubMed1993

Recovery of the radiation survival-curve shoulder in CHO-KI, XRS-5 and revertant XRS-5 populations.

The response of wild-type CHO-KI, radiation-sensitive XRS-5 and radiation-resistant XRS-5 revertant populations was examined following single and fractionated doses of cobalt-60 gamma-radiation. The results show that it is possible to induce a shoulder on the fractionated dose-survival curve of XRS-5 radio-sensitive cells, which is the same size as the wild-type CHO-KI shoulder. This shoulder persists as does the wild-type CHO-KI shoulder even after correction of the curve for lethal damage occurring in the progeny. Since this mechanism involves induction of repair, CHO-KI cells, XRS-5 cells and revertant XRS-5 cells, which are repair-proficient, were exposed to 8-azacytidine--an agent which demethylates DNA and has been shown to recover repair proficiency in sensitive XRS-5 cells. The results confirmed that azacytidine had no effect on repair-proficient CHO-KI cells but it removed the shoulder and prevented split-dose repair in XRS-5 revertant populations. The absence of heritable, lethal defects in XRS-revertant cells exposed to single doses of radiation contrasts with the situation in the wild-type CHO-KI line where these defects occur in high numbers. These results suggest that the XRS revertant is not the same as the wild-type. They also suggest that the mechanisms involved in repair of damage and production of the survival-curve shoulder following single doses of radiation are different to those which occur following split doses of radiation.

Animals↗