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C S Potten

Publications and source records attributed to C S Potten.

At least 109 records · Page 6Linked to original sources
PubMed1997

The number of clonogenic cells in crypts in three regions of murine large intestine.

Data are presented for the kinetics of repair of sub-lethal damage in the large intestine of mice. The results are based on experiments using the crypt microcolony assay with two equal sized doses which were delivered with a variable interval of time between the doses. These show that this split-dose repair was largely complete after 5 h, and that there were no significant differences between three regions of the large intestine. Overall the half-time for the repair was 2.3 +/- 0.8 h, and the maximum split-dose repair ratio (the proportion of damage recovered by splitting the dose into two fractions) was 22 +/- 2% and the mean recovery factor (the ratio of the number of surviving crypts using long interfraction intervals to that at zero time) was 11 +/- 2. The split-dose approach (Hendry 1979) using a 5 h interval has been used to estimate the number of clonogenic cells in large intestinal crypts. A range of single and paired doses between 7 Gy and 10.5 Gy were used. There were significant differences between the three regions of the large intestine, the caecum, mid-colon and rectum. The estimate of the number of clonogens also depended in a significant way on the dose of radiation used to make the estimate. At low doses large intestinal crypts contain between 5 and 10 clonogenic cells while if high doses were used they contain an estimated 16 to 36 clonogenic cells. Considerable similarity exists between the small intestine and the large intestine for; (a) the repair kinetics (b), the clonogenic estimates, and (c) their dependence on dose.

Animals↗
PubMed1997

Differential survival of murine small and large intestinal crypts following ionizing radiation.

Radiation survival curves have been obtained for crypts in the small and large intestine of male BDF1 mice using the microcolony assay. Four regions of the small intestine and three regions of the large intestine were studied. One of the major objectives was to determine the optimal conditions for the microcolony assay in the large bowel. Various times post-irradiation were studied using different approaches and threshold criteria for surviving crypts. Small, but statistically significant, differences were observed along the length of the small intestine, but no differences were observed between different regions of the large intestine. There was a marked difference (p < 0.001) in the response in the large compared with the small intestine (D0 = 291 +/- 14 compared with 151 +/- 4 cGy respectively with corresponding extrapolation numbers of 18 +/- 4 and 377 +/- 65). The use of tritiated thymidine or vincristine helped in the identification of true survivors in the large bowel, but these approaches are not necessary in the small bowel. The longer the time after irradiation that the samples were fixed the easier it was to identify survivors in the colon. However, the longer the time the more animals died. The optimum compromise in these studies was to use partial-body irradiation (abdomen only) and to sample on the fifth day after irradiation. With these criteria the mid-colon survival curve had a D0 = 274 +/- 28 cGy and an extrapolation number of 22 +/- 10. The results can be considered in relation to published data on the levels of p53 and bcl-2 expression and apoptosis in murine small and large bowel.

Animals↗
PubMed1997

p53 deficiency sensitizes clonogenic cells to irradiation in the large but not the small intestine.

The role of p53 in the survival of irradiated crypts in the small intestine and three regions of the large intestine (cecum, mid-colon and rectum) was assessed by comparing the responses in p53 null, p53 heterozygous and wild-type mice. There was no difference in the levels of crypt survival in the small intestine between the three genotypes, although the rate of cell depletion and regeneration in the null mice appeared slower. In the large intestine, crypt survival was lowest in the null mice compared to the other genotypes, in particular after high doses. The levels of crypt survival in the heterozygotes were not significantly different from those in the wild-type mice. Hence the greater radioresistance of crypts in the colon than in the small intestine, reported previously by us and others using various other mouse strains, may be partly attributable to the presence of p53. This effect is not readily explained by current knowledge concerning the decreased p53 expression and the greater expression of the survival gene Bcl2 in the stem cell zone of crypts in the colon compared to those in the small intestine. Reduced repair associated with the lack of a G2-phase checkpoint delay, the predominant arrest point for intestinal cells, is a possible explanation for the decrease in survival.

Animals↗
PubMed1996

Protection of the small intestinal clonogenic stem cells from radiation-induced damage by pretreatment with interleukin 11 also increases murine survival time.

The effect of administering recombinant human interleukin 11 in conjunction with cytotoxic insults to the gastrointestinal tract has been studied using the crypt microcolony assay for stem cell function and whole-animal survival time studies. The cytotoxic regimens include single doses of gamma rays; single doses of 5-fluorouracil (5-FU) and multiple doses of 5-FU spaced 6 h apart. Interleukin 11 (IL-11) (100 micrograms/kg) delivered over a period of time prior to cytotoxic exposure afforded protection to the clonogenic cells in the crypts as seen with the microcolony assay and prolonged the animal survival time following radiation exposure. Continuing this dose of IL-11 after cytotoxic exposure afforded little additional protection. Three doses of 5-FU 6 h apart generated crypt survival curves similar to those obtained after a single dose of gamma rays. IL-11 given prior to two doses of 5-FU effectively abolished the cytotoxic effect of the second dose of 5-FU; i.e., 2.5-3.0 times more crypts survived if IL-11 was administered when the higher 5-FU doses are considered. IL-11 given before a dose of 12 Gy of gamma rays prolonged the survival time of animals by three to four days. This confirms earlier studies demonstrating that protecting clonogenic cells in the crypt survival assay can result in beneficial effects on whole-animal survival times.

Animals↗
PubMed1996

Changes in the cellularity of the cortex of human hairs as an indicator of radiation exposure.

Growing hair follicles with their rapid cell proliferation would be expected to be sensitive organs to cytotoxic agents such as radiation. Various abnormalities in the hair and hair follicles have been reported in the past. Changes in the number of cells in the newly forming hair cortex have been shown in the mouse to be one of the more sensitive assays for radiation effects, and this approach could provide a basis for a biological dosimeter. Here we show for the first time using hair cortex cell counts some preliminary data indicating that the number of cell nuclei in a unit of length (140 microns) of the cortex of human hairs from the chest and scalp of patients undergoing fractionated radiotherapy falls significantly (P = 0.005) by 5%-10% 3 days after the first dose in a fractionated sequence of irradiations. The first dose was delivered on a Friday, and no further exposures were delivered until after the hair sample was taken on the 3rd day (Monday). No significant effect of radiation dose could be detected over the available. limited range of doses studied (5-6.5 Gy with one exit dose sample at 2.6 Gy). Also, the width varies from hair to hair. If the width of the hair is taken into account and the cortical nuclei counts are normalised to the width of each hair, the effects seen at day 3 become slightly more significant (P = 0.002), and those at day 5 also become significant (P = 0.012). Samples taken on the 5th day after the first (Friday) exposure were also 2 days after the second exposure and 1 day after the third exposure. However, little expression of damage attributable to the 2nd and 3rd exposures was anticipated since their effects would take some time to be expressed in the cortical region examined, which is some distance from the proliferative region of the follicle.

Cell Count↗
PubMed1996

Evidence of reciprocity of bcl-2 and p53 expression in human colorectal adenomas and carcinomas.

Evidence of accumulating for the failure of apoptosis as an important factor in the evolution of colorectal cancer and its poor response to adjuvant therapy. The proto-oncogene bcl-2 suppresses apoptosis. Its expression could provide an important survival advantage permitting the development of colorectal cancer. The expression of bcl-2 and p53 was determined by immunohistochemistry in 47 samples of histologically normal colonic mucosa, 19 adenomas and 53 adenocarcinomas. Expression of bcl-2 in colonic crypts > 5 cm from the tumours was confined to crypt bases but was more extensive and intense in normal crypts < 5 mm from cancers. A higher proportion of adenomas (63.2%) than carcinomas (36.5%) expressed bcl-2 (P < 0.05). A lower proportion of adenomas (31.6%) than carcinomas (62.3%) expressed p53 (P < 0.02). A total of 26.3% of adenomas and 22% of carcinomas expressed both bcl-2 and p53. To determine whether these samples contained cells which expressed both proteins, a dual staining technique for bcl-2 and p53 was used. Only 1/19 adenomas and 2/53 carcinomas contained cells immunopositive for both bcl-2 and p53. Moreover there was evidence of reciprocity of expression of bcl-2 and p53 in these three double staining neoplasms. We suggest that bcl-2 provides a survival advantage in the proliferative compartment of normal crypts and colorectal neoplasms. However, its expression is lost during the evolution from adenoma to carcinoma, whereas p53 expression is increased, an event generally coincident with the expression of stabilised p53, which we presume to represent the mutant form.

Adenocarcinoma↗
PubMed1996

P53 deficiency produces fewer regenerating spermatogenic tubules after irradiation.

The survival of clonogenic spermatogonia was assessed by scoring regenerating tubules at 35 days after irradiation, in the p53 null, heterozygote and wild-type mouse. Survival levels in the p53 null mouse after doses between 6 and 16 Gy were reduced by a factor of 3-4 compared with the levels in the heterozygote or wild-type mouse, which responded similarly. However the radiosensitivity of the cells was similar in all three types of mice, and was characterised by a D0 = 1.7 Gy. A two-dose experimental protocol was used to show that the reduced level of survival in the null mouse at day 35 after irradiation was compatible with the interpretation that there were fewer functional radioresistant clonogenic spermatogonia in the testis of the unirradiated null mouse by about a factor of 3 compared with that in the testis of the wild-type. The lower cell number was similar to the number deduced in other mice (BDF1), where the cells were much more resistant (D0 = 3.2 +/- 0.2 Gy). It is concluded that the lack of p53 causes a reduced level of tubule regeneration at 35 days after irradiation. This is probably not due to cellular radiosensitization, but possibly to a change in the stem cell cycle phase distribution resulting in a smaller proportion of resistant stem cells, which are assayed after high doses.

Animals↗
PubMed1996

Cell kinetics in rat small intestine after exposure to 3 Gy of gamma-rays at different times of the day.

Qualitative and quantitative morphological changes in rat jejunum were studied after a whole-body exposure to 3 Gy of gamma rays. Four groups of animals were irradiated at different times of the day, namely midnight, 06.00, 12.00 and 18.00 hours. The number of epithelial cells, labelling and mitotic indices were evaluated in crypt sections and the spatial distribution of S-phase cells was determined. At 12 h after irradiation a marked reduction was observed in all parameters, but the proliferative activity was restored quickly and at 36 h after irradiation the values were significantly higher than the controls. The frequency distribution of labelled cells at different positions in the crypt was reduced at 12 h but a clear expansion of S phase cells to positions near to the crypt villus junction was observed during the recovery phase. The animals irradiated at different times of the day showed a similar general post-irradiation response in the number of cells along the side of the crypt, labelling and mitotic indices and in the distribution of S phase cells along the crypts. It is worth noting that the animals exposed at midnight had a distribution of S phase cells similar to controls at 72 h post-irradiation, i.e. earlier than the other groups.

Animals↗
PubMed1996

The in situ repair kinetics of epidermal thymine dimers and 6-4 photoproducts in human skin types I and II.

We assessed the in situ time-dependent loss of epidermal thymine dimers and 6-4 photoproducts in skin types I and II after exposure to two minimal erythema doses of solar-simulating radiation on previously unexposed buttock skin. Using quantitative image analysis, we evaluated biopsy sections stained with monoclonal antibodies. We then made comparisons, in the same volunteers, with unscheduled DNA synthesis, which is a direct marker of overall excision repair. Removal of thymine dimers was slow (half-life = 33.3 h), with high levels of lesions still present 24 h post-irradiation; some lesions were still present at 7 d. In contrast, removal of 6-4 photoproducts was rapid (half-life = 2.3 h), the decay kinetics of which correlated better with the decline in epidermal unscheduled DNA synthesis (half-life = 7.1 h). These data show that as in mouse, monkey, and in vitro models, the 6-4 photolesion is repaired preferentially in human epidermis in situ. They also raise the possibility that poor thymine dimer repair may be a feature of skin types I and II, who are more prone to skin cancer than are types III and IV. There was an inverse relationship between the onset of erythema and 6-4 photoproduct repair, suggesting that this repair process initiates erythema.

Adult↗
PubMed1996

Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme.

Xeroderma pigmentosum (XP) is a rare genetic disease in which patients are defective in DNA repair and are extremely sensitive to solar UV radiation exposure. A new treatment approach was tested in these patients, in which a prokaryotic DNA repair enzyme specific for UV-induced DNA damage was delivered into the skin by means of topically applied liposomes to supplement the deficient activity. Acute and chronic safety testing in both mice and humans showed neither adverse reactions nor significant changes in serum chemistry or in skin histology. The skin of XP patients treated with the DNA repair liposomes had fewer cyclobutylpyrimidine dimers in DNA and showed less erythema than did control sites. The results encourage further clinical testing of this new enzyme therapy approach.

Adolescent↗
PubMed1996

Gamma-ray-induced cell killing and chromosome abnormalities in the bone marrow of p53-deficient mice.

Resistance to the lethal effects of ionizing radiation has been demonstrated in a wide variety of cell types with defects in the p53 gene (thymocytes, splenic B and T cells, in vitro hemopoietic colony-forming cells and intestinal cells of the mouse, embryo cells of the rat, and human Burkitt's lymphoma cells). In contrast, Slichenmeyer et al. (Cancer Res. 53, 4164-4167, 1993) found no evidence of resistance in fibroblasts derived from p53 null mice. The aim of our study was to compare the radiation response of hemopoietic colony-forming cells (in vitro CFC) and of fibroblastoid colony-forming cells or units (CFU-F) within the same tissue (marrow) in p53 null mice (-/-), heterozygotes (+/-) and wild-type animals (+/+). We have also tested the hypothesis that, in proliferating cells, radiation-induced cell killing is mediated through chromosome damage by examining the relationship between these end points in hemopoietic cells of the three mouse types. Both in vitro CFC and CFU-F of -/- mice were resistant to cell killing compared with +/+ and +/- mice whose cellular sensitivities were indistinguishable. The resistance was characterized by a broader "shoulder" on the cell survival curve, i.e. a higher extrapolation number but similar D0 values using the multitarget model or a lower alpha coefficient using the linear-quadratic model. The frequency of chromosomally abnormal marrow cells after irradiation was similar for the three genotypes. However, marrow cells with aberrations carried more aberrations in -/- mice than in +/+ or +/- mice such that the total number of aberrations per 100 cells was higher in -/- mice. Since there were no differences in the yields of aberrations between genotypes in spleen lymphocytes or in CFU-F (both noncycling at the time of irradiation) and less mitotic inhibition in -/- marrow cells than in +/+ or +/- cells, the chromosomal radiosensitivity of -/- marrow hemopoietic cells might be related to reduced cell cycle delay allowing insufficient time for repair, but other explanations have been considered. We postulate that the radiation resistance of both hemopoietic CFC and CFU-F in -/- mice is a consequence of the failure of DNA/chromosome damage to trigger apoptosis or permanent cell cycle arrest to the same extent as in the +/+ or +/- mice: hence the lack of correlation between chromosome damage and cell death in the three mouse types.

Analysis of Variance↗
PubMed1996

A comparison of proliferation markers (BrdUrd, Ki-67, PCNA) determined at each cell position in the crypts of normal human colonic mucosa.

Samples of microscopically normal human sigmoid colon fixed in 70% ethanol from 15 patients who had received bromodeoxyuridine (BrdUrd) prior to surgery have been reanalyzed using a combination of proliferation markers. The specimens have been immunostained for proliferating cell nuclear antigen (PCNA) and after microwave treatment, they have been stained for BrdUrd and Ki-67. The 15 patients selected comprised 5 patients whose mucosa previously gave high BrdUrd labelling indices in the crypt, 5 that gave median values for BrdUrd labelling and 5 that gave low values for bromodeoxyuridine labelling on a previous analysis using tissue fixed in 70% ethanol and formal saline and using a different antibody (Potten et al., 1992). The relative levels of labelling at each cell position in the crypts has been compared using the 3 proliferation markers with the data being compared with the BrdUrd labelling as a standard labelling for S phase cells. One objective was to see whether all three proliferation markers discriminated equally well between the three groups of patient samples. The data show that the distinction between high, medium and low values seen with BrdUrd labelling was retained when Ki-67 immunostaining was analysed. PCNA immunostaining resulted in high levels of labelling and the different levels of labelling seen with BrdUrd and Ki-67 were largely lost.

Aged↗
PubMed1996

The quantitation and kinetics of unscheduled (repair) DNA synthesis in ultraviolet-irradiated human skin by automated image analysis.

Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing the concentration of tritiated thymidine and by increasing the incubation time.

Autoradiography↗
PubMed1995

Interleukin-11 protects the clonogenic stem cells in murine small-intestinal crypts from impairment of their reproductive capacity by radiation.

The rapidly proliferating gastrointestinal mucosa is one of the major limiting tissues in cancer therapy. Because of its short transit time and high sensitivity to radiation and chemotherapeutic drugs, damage is rapidly manifested. Protection of the important stem cells in the tissue could be achieved, in principle, by appropriate prior manipulation with cytokines or growth factors which might make them more resistant to the cytotoxic treatment, for example, by putting them out of cycle. Such strategies might reduce some of the adverse side-effects of cancer treatment and improve the quality of life of patients, while possibly allowing an escalation of therapeutic dose. The functional capacity of stem cells has been studied for many years using a microcolony assay technique which measures the regenerative capacity of the clonogenic stem cells in crypts. These cells determine the survival of crypts which themselves determine whether or not the mucosal integrity is maintained and ultimately whether the animal or patient survives. Here, we demonstrate that treatment with interleukin-11 for 2 days prior to radiation exposure can significantly increase the number of surviving crypts. Treatment with IL-11 both before (for 2 days) and after irradiation (for 3 days) produces a slightly enhanced protection. Up to about 4 times more crypts survive at the highest radiation dose after either of these treatment schedules. These studies may provide a radiobiological explanation for the increased survival of animals when IL-11 is administered during 5FU and radiation exposures.

Animals↗
PubMed1995

Apoptosis induced by high- and low-LET radiations.

Cell death after irradiation occurs by apoptosis in certain cell populations in tissues. The phenomenon also occurs after high linear energy transfer (LET) irradiation, and the relative biological effectiveness (RBE) is 3 to 4 (with respect to low-LET radiation and apoptosis in intestinal crypts) for neutrons with energies of 14 MeV and up to 600 MeV. It is thought that p53 plays a role in the phenomenon, as radiation-induced apoptosis is not observed in p53-null animals.

Animals↗
PubMed1995

Growth factor regulation of proliferation in primary cultures of small intestinal epithelium.

Although the intestinal epithelium is one of the most rapidly renewing tissues, little is known about the major growth factors that control the rate of cell replacement and migration. Recently, a primary culture model has been described for the developing rat small intestinal epithelium, which permits epithelial growth while maintaining interactions with associated stromal cells, thereby possessing several contextual advantages over established cell lines (Evans et al., 1992). We have used this model to begin to determine the factors that may be involved in controlling intestinal epithelial cell proliferation. Under the conditions examined, no single growth factor promoted exclusive proliferation of epithelial cells; stromal cell proliferation was also apparent. The most potent stimulators of epithelial proliferation were insulin and insulin-like growth factor 1 (IGF-1). These factors also appeared to inhibit migration of the epithelial cells. 5-10 ng/ml EGF, 5-20 ng/ml TGF alpha, and 10-20 ng/ml PDGF also slightly increased epithelial cell numbers. Cell proliferation was inhibited by 0.1 ng/ml TGF beta-1. In Dulbecco's modified Eagle's medium (DMEM) containing 0.25 IU/ml insulin, glucose levels of 2-3 g/liter permitted epithelial growth with limited expansion of the stromal cell population. Higher levels of glucose further stimulated the nonepithelial cell types. Transferrin was also a potent stimulator of both cell types.

Animals↗