Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.     

Ultrastructural site variations in mouse epidermal organization

Mouse dorsal, ear, tail and foot epidermis are compared according to their tissue architecture and cell kinetics. Cell proliferation is expected in terms of the daily volume of keratin replaced. The stratum corneum may be organized into vertical columns of squames, which may have minimal overlap as in dorsum and ear, or maximal overlap as in tail. Individual areas are adapted to their function both in squame fine structure and rate of cell replacement. The surface keratin loss/replacement rate is at its highest in foot and tail, and lowest in ear and dorsum. Observations on hairless mouse dorsum are also included.     

Postural motor programming in paraplegic patients during rehabilitation

One of the basic aims in the rehabilitation of thoracic spinal cord injured (SCI) patients concerns the regaining of sitting posture control. This implies the development of new postural strategies requiring the adjustment of motor programming processes. The aim of this study was to investigate the time course of postural reorganization during active, clinical rehabilitation of thoracic SCI patients with different SCI levels. Thus changes in motor programming in sitting balance control were investigated in two groups of complete low or high thoracic SCI patients. At several stages during the rehabilitation process an experiment was held in which sitting posture was perturbed systematically using submaximal reaching movements over four reaching distances. This bimanual reaching task was presented as a visual precue choice reaction time (RT) task in which reaching distance (i.e. grade of postural perturbation) was precued. Results indicated that in both high and low thoracic SCI patients RTs in movements involving postural perturbation became shorter during the course of the rehabilitation period. However, low thoracic SCI patients were generally slower in the programming of balance perturbing movements than high thoracic SCI patients, a phenomenon that did not change over time. Furthermore, initial differences in RTs as a function of grade of postural perturbation disappeared in both groups in the course of the rehabilitation phase. Precue benefit, equally large for both groups, did not change as a function of rehabilitation time. It is concluded that the observed phenomena signify the gradual development of new central postural control processes in both SCI groups during rehabilitation. Low thoracic SCI patients, having more residual sensorimotor functions, seem to adopt more complex strategies in maintaining and restoring sitting balance that take longer to specify and to programme. High thoracic SCI patients seem to rely on simpler strategies using more passive postural support.     

The intestinal epithelial stem cell: the mucosal governor

All epithelial cells in the small and large intestine are thought to originate from stem cells located towards the base of the crypts of Lieberkühn. To-date, there are no specific intestinal stem cell markers, hence stem cell properties can only be inferred. A range of experimental techniques have been employed including cell position mapping, radiation regeneration (clonogenic) assays, chimeric and transgenic mice. This review discusses the implications of experiments performed using these techniques in order to deduce the number, location and functional properties of stem cells. Stem cell homeostasis is maintained by cell proliferation and death 'through apoptosis'. The various growth and matrix factors and genes which may control these processes, and be important for stem cell function, are discussed along with their carcinogenic and clinical implications.     

Epithelial proliferation and hormone receptor status in the normal post-menopausal breast and the effects of hormone replacement therapy

The proliferation rate (as assessed by Ki67 expression) and expression of oestrogen-regulated progesterone receptor (PR) was studied in normal post-menopausal breast epithelium. Normal breast epithelium from patients receiving hormone replacement therapy (HRT) at the time of surgery containing either oestrogen alone (E2) or oestrogen and progesterone combined activities (E2 + P) was also studied, as HRT has been linked to an increased breast cancer risk. Samples of breast tissue, containing normal epithelium, from 185 patients undergoing surgery for benign or malignant disease were immunocytochemically stained for PR and Ki67. The percentage of labelled cells was expressed as the labelling index (LI). The median Ki67 LI in normal post-menopausal breast epithelium was 0.19 and median PR LI was 4.75, and both were unaffected by patient age, duration of menopause or if the tissue sample originated from a breast with benign or malignant disease. Proliferation did not alter significantly in patients taking HRT (P = 0.61); however, PR expression was up-regulated in both E2 and E2 + P users (P = 0.01). The dose and duration of HRT had no effect on either parameter. A possible attenuation of sensitivity to oestradiol-induced proliferation but not to PR expression occurs in the post-menopausal breast.     

The relationships between p53-dependent apoptosis, inhibition of proliferation, and 5-fluorouracil-induced histopathology in murine intestinal epithelia

The relationship between acute (<36 h) induction of apoptosis and longer-term (>72 h) intestinal histopathology was systematically investigated in vivo using p53 wild-type (+/+) and null (-/-) mice. Administration of the enterotoxin 5-fluorouracil (5-FU) at either 40 or 400 mg/kg to BDF1 mice induced an acute p53-dependent apoptosis in the crypts of both small intestine and midcolon. Although the amount of apoptosis was of the same order of magnitude at its peak (24 h) at both doses, only 400 mg/kg 5-FU brought about histopathological changes to the gut after 96 h, quantified as losses of crypt and villus cellularity. Only after the administration of 400 mg/kg 5-FU were mitotic index and DNA synthesis significantly suppressed in both small intestinal and midcolonic crypts at 24 h. This correlated with a prolonged, p53-dependent expression of p21waf-1/cip1. In p53 null (-/-) mice, significant reductions in both 5-FU-induced apoptosis and inhibition of cell cycle progression allowed retention of crypt integrity 96 h after 5-FU. These results show that quantitative measures of acute apoptosis in vivo may not accurately predict subsequent pathological changes in the gut. Rather, p53-dependent inhibition of cell cycle progression, together with cell loss by apoptosis, caused a loss of crypt integrity. Importantly, the tissue toxicity of 5-FU was genetically determined at a locus (p53) separate from that directly associated with drug action.     

Stem cells in gastrointestinal epithelium: numbers, characteristics and death

The mammalian intestinal mucosa, with its distinctive polarity, high rate of proliferation and rapid cell migration, is an excellent model system to study proliferative hierarchies and the regulation of cell division, differentiation and cell death. Each crypt contains a few lineage ancestral stem cells (the 'ultimate stem cells'). However, there are other potential stem cells within the early lineage, and many rapidly proliferating transit cells with no stem cell capabilities. Apoptosis under two circumstances has a specificity for the ultimate stem cells in the small intestine and this represents, in one case, part of the stem cell homeostatic process and, in another case, a protective mechanism against DNA damage. Apoptosis occurs with a lower frequency in the large intestine owing to the expression of the bcl-2 gene in this region, and this probably contributes to the causes for the low cancer risk in the small bowel and the high risk in the large bowel. Current studies are beginning to unravel the complex interaction of growth factors and regulatory genes that determine whether a cell divides, differentiates or dies.     

Modelling of the influence of HTdR on cell kinetics in mouse epidermis

Fluctuations occur in the labeling index (LI) of mouse epidermis over the first two days after application of tritiated thymidine (³HTdR) for which there has in the past been no explanation. Similarly, there has been no adequate explanation for the fluctuations in the percent labelled mitoses (PLM) data seen immediately after the first peak. Here we propose a model which provides an explanation for both these features. The hypothesis suggested is that some cells are killed by the ³HTdR either immediately when they are still in the S phase or later when they have reached G₁. In both cases the labelled precursor is released from the cell and may be incorporated into other S phase cells after an appropriate delay. The model has been formulated into a computer programme which can derive the appropriate LI or PLM curves and fit curves to the relevant data. The resulting curves fit the data well suggesting that such cytotoxic and reutilization processes may in fact occur.     

Circadian rhythms of presumptive stem cells in three different epithelia of the mouse

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with 3H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.