The radiosensitivity of cultured human and mouse keratinocytes

Clonogenic survival assays after gamma-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D0 when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.     

Measurement of the transit time for cells through the epidermis and stratum corneum of the mouse and guinea-pig

Abstract A new approach to determine the transit time through the epidermis is presented, involving a gentle washing of the skin surface to collect the loosely attached surface corneocytes. This, it is believed, will be less likely to stimulate the system than tape-stripping or scraping. Radioactively labelled thymidine and iododeoxyuridine have been used to label cells in the basal layer and various labelled amino acids (glycine, cystine and methionine) have been used to label the metabolically viable cell layers (up to and including the granular layer). The resulting changes in surface radioactivity levels have been interpreted to provide a basal to surface transit time of 8–9.5 days for hairless and haired mouse epidermis and about 13.5 days for guinea-pigs. The basal to granular layer transit time, which probably includes some basal layer residence time, is about 4.5 days in the mouse and 8 days in the guinea-pig. The granular to surface time in mice is about 5 days. The results also suggest that when nuclear and cytoplasmic organelles are degraded in the granular layer, material is released that can diffuse rapidly through the stratum corneum to the surface. Some of this can be shown by chromatography to be thymidine. Hence, the stratum corneum is pervious to molecules such as nucleosides. This rapid diffusion outwards through the skin can also be detected shortly after injecting [¹²⁵I]-iododeoxyuridine.     

The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

Intestinal Crypt Proliferation. Ii. Computer Modelling of Mitotic Index Data Provides Further Evidence For Lateral and Vertical Cell Migration In the Absence of Mitotic Activity

Abstract. The position-dependent mitotic index before, and 1, 2 and 3 h after vincristine was scored. the accumulation of cells in mitosis leads to an increase in the mitotic index from 0.06 to 0.34 at crypt positions 8-12. Surprisingly, the leading edge of the position-related mitotic index distribution moves to higher crypt positions although cell division was stopped. In addition, the vertical clustering of mitotic figures in sections was recorded. the data were examined using a previously described computer crypt model. We conclude: the average mitotic phase duration is about 0.7 h (40 min) and varies little with cell position; the geometrical correction factor for overscoring mitoses in crypt sections is about 0.6-0.7 and adjacent cell columns can merge. Lateral cell displacement after mitosis, as predicted in a previous model analysis, would be a mechanism to counteract other forces that tend to reduce the crypt circumference. In the normal steady state merging and expansion processes would just balance each other. This would not follow if one mechanism was blocked. Thus we propose a new concept in which the crypt geometry would be dynamically determined by cell proliferative activity in connection with lateral positioning of new cells on one hand and contracting forces on the other hand.     

Scoring mitotic activity in longitudinal sections of crypts of the small intestine

Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. The correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. The results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestine before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor fD for the mitotic index of 0.59 is obtained; (6) the correction factor fT derived from the shape and position of the mitotic figures measured in 3 microns longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor fmod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is fD; for mouse small intestine it is 0.59.     

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.     

The spatial organization of the hierarchical proliferative cells of the crypts of the small intestine into clusters of 'synchronized' cells

Abstract. A statistical analysis of the distribution of [³H]TdR-labelled cells in longitudinal and transverse sections of crypts from the ileum of the mouse, indicated that there was a strong tendency for labelled or unlabelled cells to be associated in short vertical runs and lateral clumps, suggesting the presence of clusters of labelled cells on the sides of the crypts. A model is discussed for the cellular spatial organization of the crypt that proposes a vertical alignment of the cells within branches of the proliferative cell lineage. The model would predict vertical alignment of partially synchronized cells as well as some lateral clumping.
In the present studies mitoses were not observed at higher levels in the crypt than labelled (S phase) cells. This observation would be predicted by the non-random spatial organization suggested by the model.
The model would also make certain predictions concerning cell migration. These are discussed in relation to cell migration studies which include evidence that migration continues in the absence of mitotic activity.     

INCORPORATION OF TRITIATED THYMIDINE INTO THE SKIN AND HAIR FOLLICLES

Diurnal fluctuations in incorporation of tritiated uridine and tritiated thymidine are reported for full thickness skin and its two proliferative populations, the epidermal basal layer and the hair follicle germ or matrix. Recently developed techniques whereby these two populations can be studied independently were utilized. Resting (unplucked) skin and skin in the middle of the hair growth cycle (10 days after plucking) was studied. the diurnal fluctuations differed at these two stages of the hair cycle. Generally a single minimum level of activity was observed shortly after the beginning of the dark period of the day, i.e. after 18.00 hours.     

Radiation, the ideal cytotoxic agent for studying the cell biology of tissues such as the small intestine

Epithelial tissues are highly polarized, with the proliferative compartment subdivided into units of proliferation in many instances. My interests have been in trying to understand how many cellular constituents exist, what their function is, and what the intercommunicants are that ensure appropriate steady-state cell replacement rates. Radiation has proven to be a valuable tool to induce cell death, reproductive sterilization, and regenerative proliferation in these systems, the responses to which can provide information on the number of regenerative cells (a function associated with stem cells). Such studies have helped define the epidermal proliferative units and the structurally similar units on the dorsal surface of the tongue. The radiation responses considered in conjunction with a wide range of cell kinetic, lineage tracking and somatic mutation studies together with complex mathematical modeling provide insights into the functioning of the proliferative units (crypts) of the small intestine. Comparative studies have then been undertaken with the crypts in the large bowel. In the small intestine, in which cancer rarely develops, various protective mechanisms have evolved to ensure the genetic integrity of the stem cell compartment. Stem cells in the small intestinal crypts are intolerant of genotoxic damage (including that induced by very low doses of radiation); they do not undergo cell cycle arrest and repair but commit an altruistic TP53-dependent cell suicide (apoptosis). This process is compromised in the large bowel by BCL2 expression. Recent studies have suggested a second genome protection mechanism operating in the stem cells of the small intestinal crypts that may also have a TP53 dependence. Such studies have allowed the cell lineages and genome protection mechanisms operating the small intestinal crypts to be defined.