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Biomedical subjects

C S Potten

Publications and source records attributed to C S Potten.

At least 127 records · Page 7Linked to original sources
PubMed1995

Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma.

Apoptosis is an important mechanism of cell death in tumours and it is seen both prior to and following radiotherapy. In this study patients with proven carcinoma of the cervix had measurement made of the percentage of apoptotic cells (apoptotic index or AI) in pre-therapy biopsies. Measurements of intrinsic radiosensitivity (SF2), already shown to be a predictor of outcome, had previously been made on the same pre-therapy biopsies. Mitotic index (MI) and Ki-67 antigen staining were also recorded as markers for proliferation. Patients were divided into those with an AI above or below the median and in general increasing apoptosis was associated with poor prognosis. The 5-year survival rate for tumours with an AI below the median was 79% and was significantly greater than the rate of 47% for those with an AI above the median (p = 0.003). There was also a significantly increased 5-year local recurrence-free rate for patients with an AI below the median compared with those with an AI above the median (79 versus 61%, p = 0.012). In addition, AI and SF2 acted as independent prognostic indicators. Patients with both an SF2 and AI value above the median did badly (25% 5-year survival, 46% local control) compared with those with an SF2 and AI below the median (80% 5-year survival, 100% local control). Apoptosis showed correlation with MI (n = 66, r = 0.34, p = 0.002) and cell staining for the Ki-67 antigen (n = 57, r = 0.25, p = 0.03), but neither MI nor Ki-67 were related to patient outcome. This suggests that while apoptosis may be a reflection of tumour proliferation this cannot in itself explain the ability of apoptosis to predict clinical outcome for this series of patients. The study raises the possibility of AI and SF2 being used together as predictors of tumour response to radiotherapy.

Adult↗
PubMed1995

The detection of cyclobutane thymine dimers, (6-4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects.

Ultraviolet irradiation of skin induces various DNA photolesions. Here we demonstrate that irradiation of human buttock skin with 300 nm UVR in situ induces thymine dimers and 6-4 photoproducts. Irradiation with 260 nm immediately followed by UVA (320 nm) induces the Dewar photoisomers of the 6-4 lesions. All three lesions can be detected in methanol-fixed paraffin sections using specific monoclonal antibodies. The sections have been analysed in an automated image analysis system (Discovery) and the level of immuno-DAB-peroxidase measured in individual epidermal cell nuclei as absorption at 460 nm (integrated optical density). The staining patterns with the antibodies showed no detectable change with epidermal depth by eye after 300 nm irradiation, however, the machine detected a fall off with depth of about 2.5% per cell layer. Following irradiation with a shorter wavelength (260 nm) there was a rapid fall off in staining with depth easily detectable by eye and machine (39% per cell layer).

Antibodies, Monoclonal↗
PubMed1995

Effects of IL-11 on the growth of intestinal epithelial cells in vitro.

The network of interacting factors that control proliferation in the intestinal epithelium is largely unknown. Recently, IL-11 was found to protect animals from lethal doses of cytotoxic agents. Part of this protective action was ascribed to a reduced level of damage in the intestinal epithelium. Whether this was due to a direct effect on epithelial cell cycle progression was unclear. We have addressed this question in vitro and found that IL-11 reversibly inhibited proliferation in untransformed small intestinal IEC18 cells. However, IL-11 did not inhibit transformed SW620 or HT29 colonic cell lines. IL-6 behaved in a similar manner to IL-11. Thus, these results suggest that IL-11 may be an ideal therapy adjuvant, protecting normal cells and further, these results suggest that IL-11 may be involved in the normal growth controls in the intestinal epithelium. The inhibitory response evoked by IL-11 is lost during carcinogenic transformation.

Animals↗
PubMed1995

The action spectrum for induction of chronic actinic dermatitis is similar to that for sunburn inflammation.

The action spectrum for induction of the abnormal cutaneous response at 24 h in the photosensitivity disorder chronic actinic dermatitis (CAD) was determined in 15 patients and found to be the same in shape as that for normal sunburn in fair-skinned individuals at 24 h, as determined for 47 control volunteers, although displaced in magnitude. This suggests that an endogenous chromophore(s), the same as or similar to that/those responsible for human sunburn, may be responsible for initiation of the abnormal reaction to irradiation in CAD, and that the putative antigen associated with the CAD reaction may be derived from that/those or associated molecules.

Aged↗
PubMed1995

Stimulation and inhibition of proliferation in the small intestinal crypts of the mouse after in vivo administration of growth factors.

The effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), insulin-like growth factor (IGF) I and II, acidic fibroblast growth factor (FGF), tumour necrosis factor alpha (TNF alpha), macrophage inhibitory protein 1 alpha (MIP-1 alpha) (LD78), and TGF beta-1 on cell proliferation in the crypts of the small intestine of mice were investigated. Various doses and dosing regimens were tested. Three in vivo assays were developed, in each case involving detailed cell positional analysis of methyl tritiated thymidine labelling and mitotic activity. These allowed deductions to be made about the regions of the crypt and hence regions of the proliferative hierarchy (stem cells versus dividing transit cells) that are affected by treatment with growth factors. The assays involved: (1) normal untreated mice (an assay most likely to be effective for detecting inhibitors); (2) mice shortly after whole body irradiation when compensatory proliferation has been endogenously triggered (another assay for inhibitory factors, possibly ones associated specifically with the regenerative process); and (3) mice at late times (96 hours) after irradiation in the regression phase after a proliferative overshoot (an assay designed to detect stimulators). Little effect was seen after treatment with acidic FGF, TNF alpha, or MIP-1 alpha but EGF, IGF-I and II, and TGF alpha can all be seen to exert some stimulatory effects on labelling or mitosis. EGF and IGF-I stimulate both unirradiated mice and 96 hour recipients, while TGF alpha had a greater effect on the 96 hour animals. In all cases, multiple doses were used. TGF beta-1 was an effective inhibitor of proliferation in unirradiated and early regenerating (18 hour) animals. EGF was the most effective of the stimulators, raising the levels of proliferation at all positions in the crypt, but particularly in the upper crypt. IGF-I also exerted its effect predominantly in the upper crypt, while TGF alpha raised proliferation at all cell positions. TGF beta-1 tended to have its strongest inhibitory effects in the lower (stem cell) regions of the crypt.

Animals↗
PubMed1995

The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but not progesterone.

In order to resolve the question of which ovarian steroid stimulates normal human mammary epithelial cell proliferation, we have implanted pieces of normal human breast tissue subcutaneously into athymic nude mice. These mice were then treated with slow-release pellets containing estradiol (E2) or progesterone (P) such that serum levels of E2 and P were increased to those seen in normal women. The proliferative activity of the tissue implants was assessed by uptake of tritiated thymidine and steroid receptor expression was measured immunocytochemically. Insertion of a 2 mg E2 pellet 14 days after tissue implantation increased the thymidine labeling index (TLI) from a median of 0.4% (n = 34) to a median of 2.1% after 7 days (n = 43; P < 0.001 by Mann Whitney U test). In contrast, treatment with a P pellet (4 mg) had no effect upon the TLI whereas P (4 mg) in combination with E2 (2 mg) had no effect over and above that of E2 alone. There was a significant correlation between the increase in TLI and either the E2 content of the pellets (P < 0.001 by linear regression) or the serum E2 levels achieved (P < 0.001). Expression of the P receptor was increased 15- to 20-fold by E2 treatment. We conclude that E2 is sufficient to stimulate human breast epithelial cell proliferation at physiologically relevant concentrations and that P does not affect proliferation either alone or after E2 priming.

Adult↗
PubMed1995

Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.

The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53.

Animals↗
PubMed1995

Deduction of the clonogen content of intestinal crypts: a direct comparison of two-dose and multiple-dose methodologies.

A microcolony assay was used in conjunction with fractionated gamma irradiation to determine the number of clonogens in murine intestinal crypts with varying doses of irradiation used in the determination. The experimental design allows direct comparison between two-dose methodologies, employing one and two (or two or four) equal dose fractions, and multiple-dose methodologies involving determination of the crypt survival curves for a number of fractionation regimens using equal doses per fraction. The two-dose methodology yielded estimates of clonogen number of between 3 and 4 at low delivered dose (single and double fractions each of 6.5-7.5 Gy), rising to around 40 at high biological doses (two and four fractions each of 5.75 or 6.5 Gy). The multifraction methodology yielded estimates of clonogen number which increased from 13 after a single fraction to values of 26 and 22 after three and four fractions. However, the latter values were reduced to 11 and 9, and showed little evidence of any dependence on fraction number, when data pertaining to high biologically effective doses were excluded. Hence it is concluded that the high values for clonogen number typically deduced from such multiple-dose protocols, compared with the generally lower (but dose-dependent) values obtained from two-dose protocols, may be explained at least partially by the higher biological doses generally employed in the multiple-dose protocols.

Animals↗
PubMed1995

The isolation and culture of adult mouse colonic epithelium.

A technique is described for the reproducible primary culture of colonic epithelium from adult mice. A collagenase-dispase digestion technique (adapted form Evans et al. 1992) is used to release the epithelium, followed by differential sedimentation to produce a high purity crypt preparation with maintained structural integrity and minimal mesenchymal contamination. The crypt units attach to collagen coated plastic within 24 h and the epithelial cells quickly begin to migrate outwards producing a monolayer surrounding the attached crypts. Electron microscopy revealed that the migrating epithelial cells possessed both desmosomes and microvilli. Proliferation in the colony supports the outward migration of cells until the migratory cells of adjacent colonies connect and a confluent monolayer begins to form. Proliferation is routinely maintained for 10 days (although cultures have now been maintained without subculturing for 35 days) and is demonstrated by increased cell numbers in spite of continuous cell loss into the culture media. Culture growth is enhanced by increasing concentrations of fetal calf and mouse serum and EGF but does not appear to respond significantly to added transferrin. Growth is also stimulated by a murine small intestinal extract thought to contain a potentially novel growth factor or cocktail of factors. This culture model has considerable potential for studies on growth factor control of this carcinoma susceptible tissue and its differentiated function as well as studies into the mechanisms of carinogenesis.

Animals↗
PubMed1994

Apoptosis and cancer chemotherapy.

The major disseminated cancers remain stubbornly resistant to systemic therapy. Drug-resistant tumours include both slow and fast growing types, with the carcinomas constituting the major problem. Strategies for drug discovery have, in the past, been focused on attempts to design antiproliferative agents, largely targeted to interfere with DNA integrity and replication. The malignant phenotype might be characterized by the emergence of cell populations with a greater survival potential: a lower proclivity to undergo apoptosis. This idea provides a possible explanation of the genesis and progression of cancer and of the inherent resistance of tumour cells to engage apoptosis. Work is described which identifies the molecular basis for differences in the survival potential of stem cells in the crypts of the colon and small intestine. The advantageous survival of colonic stem cells, provided by expression of bcl-2 and a muted p53 response to DNA damage, allows damaged cells to survive. Continued expression of bcl-2 renders tumour cells resistant to drug-induced DNA damage by a mechanism different from classical mechanisms of drug resistance. The attenuation of cell survival is described as a key component in strategies for the drug treatment of disseminated cancers.

Animals↗
PubMed1994

The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.

Three h after whole-body irradiation (8 Gy) of C57BL x DBA/2 F1 mice, p53 protein was expressed strongly in the stem cell compartment of the small intestine but at lower levels in the colon. At this time, apoptotic cells were also observed in the stem cell position of the small intestine, with fewer in the colon. In mice without copies of the p53 gene (nulls), the levels of spontaneous apoptosis, in both the small intestine and the colon, were not different from wild-type. Irradiation of the nulls with 8 Gy of gamma-rays failed to induce any further apoptosis: the loss of p53 essentially rendered the epithelial cells, from both the small intestine and the colon, radioresistant. The response of the epithelial stem cells of the small intestine suggests that p53 may play a role in the deletion of damaged cells with carcinogenic potential, whereas this process is limited in the colon.

Animals↗
PubMed1994

Characterization of radiation-induced apoptosis in the small intestine and its biological implications.

The small intestine with its high cell proliferation, well-accepted hierarchy, high radiation susceptibility and low cancer incidence is a useful model for studying the controls of cell replacement. Apoptosis, which represents part of the overall homeostatic process, occurs spontaneously at the stem cell position in the crypts, and very small doses of radiation elevate the levels of apoptosis rapidly in this region. Other cytotoxic agents also target cells in this region including several mutagenic chemicals. Yet other drugs target cells at higher positions in the crypt indicating that all crypt cells possess the programme for apoptosis, but this is normally suppressed in many of the cells. In contrast, high doses of radiation are required to reproductively sterilize the crypts and, using clonal regeneration techniques, the number of clonogenic cells is dependent on the levels of damage induced (dose), i.e. the more injury that is induced the greater number of cells that are recruited into the clonogenic compartment. All doses of radiation trigger rapid changes in proliferation in the stem cell region which suggests that the detection of the induced cell death (even small levels, such as one apoptotic cell per crypt) is efficient and has rapid consequences. p53 may be involved in this damage recognition and apoptosis initiation. The studies to date suggest that apoptosis plays an important role in this tissue in terms of its homeostasis and its protection against carcinogenesis by removal of potentially carcinogenic damaged cells.

Animals↗
PubMed1994

Clonogen content of intestinal crypts: its deduction using a microcolony assay on whole mount preparations and its dependence on radiation dose.

A new method for detecting surviving intestinal microcolonies in whole mount preparations is presented. This approach is not subject to the sampling problems encountered with microcolony assay using data from tissue sections, associated with the size of the microcolonies and the random nature of the sections in relation to these colonies. This new method has been used to determine the crypt survival after one or two fractions of gamma-irradiation, and hence to estimate the crypt clonogen number. The survival levels are similar using the two techniques and similar estimates for the number of clonogens per crypt are obtained. The results using this novel approach thus confirm the validity of the traditional approaches using sections. The estimates of initial clonogen number per crypt show a dependence on the size of dose used to estimate the number, and the estimates range from about eight clonogens per crypt for doses between 7 and 8 Gy, to about 40 clonogens per crypt for doses between 9 and 10 Gy.

Animals↗
PubMed1994

The time of onset and duration of 5-methoxypsoralen photochemoprotection from UVR-induced DNA damage in human skin.

Sites of previously unexposed buttock skin of eight human volunteers (skin type II) were treated daily for 3, 5, 8, or 10 days with suberythemogenic doses of solar-simulated radiation (SSR) in the presence of a UVB sunscreen containing 5-methoxypsoralen (5-MOP) at 30 p.p.m., or daily for 10 days with SSR+the same sunscreen without 5-MOP. One week after cessation of treatment, these sites, together with a control unexposed site, were challenged with 2 minimal erythema doses (2 MED) of SSR. Biopsy samples were taken within 15 min of the challenge dose, and were incubated for 1 h in tritiated thymidine. UV-induced DNA damage was measured indirectly by unscheduled DNA synthesis (UDS), and directly using a monoclonal antibody to thymine dimers, and automated image analysis. The level of pigmentation was assessed in sections in a semiquantiative fashion with Masson-Fontana staining, and the number of layers in the stratum corneum was used to assess changes in epidermal thickness. Using the UDS and dimer measurements, the level of photochemoprotection afforded by 5-MOP was determined from the reduction in the level of DNA damage observed. The photochemoprotection was expressed as a ratio of the 5-MOP-treated sites compared with the sites that did not receive 5-MOP treatment. The onset of 5-MOP photochemoprotection was shown to occur after three to five daily exposures, and became maximal after eight daily exposures. The onset of this protection coincided with increases in melanin and in stratum corneum thickness. In an extension of this study, it was found that following 10 daily exposures, 5-MOP photochemoprotection declined at a rate of about 5% per week, and in spite of several cycles of epidermal cell replacement, some protection still persisted up to 14 weeks after the end of the tanning protocol. The sites treated with sunscreen without 5-MOP was good correlation between the photochemoprotection endpoints of UDS and thymine dimer levels. The importance of 5-MOP photochemoprotection in the risk-benefit assessment of sunscreens used in suntanning is discussed.

5-Methoxypsoralen↗
PubMed1994

Slowly cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.

Slowly cycling label-retaining epidermal cells were identified by light microscopic autoradiography in the dorsal epidermis and hair follicles of adult mice 8-10 weeks after twice daily injection of [3H]dT on days three through five after birth. Pulse-labelled epidermal cells were identified in the epidermis and hair follicles of 7-8 week old mice 1 h after a single injection of [3H]dT at 8.00 a.m. For mice of both groups, epidermal cells including those from the hair follicles were harvested by trypsinization and were cultured from low density on feeder layers of irradiated Swiss mouse 3T3. On days 2, 4, 5, 7, 10 and 12, the cultures were fixed and processed for light microscopic autoradiography, and the distribution of labelled nuclei was quantified. On day 2 of culture, both label-retaining cells (LRC) and pulse labelled cells (PLC) were found primarily as single cells. After five days, LRC were found as pairs and clusters having silver grain counts consistent with their division. In contrast, PLC remained primarily as single cells. These results suggest that LRC may divide to form colonies (are clonogenic) whereas PLC are rarely clonogenic. The significance of this experiment is that it suggests that the LRC may not only be persistent in the epidermis, but that they may also be cells with relatively greater proliferative potential than the PLC and are thus likely to be stem cells.

Aging↗