Intestinal crypt clonogens: a new interpretation of radiation survival curve shape and clonogenic cell number

Estimates of the clonogen content (number of microcolony-forming cells) of murine intestinal crypts using microcolony assays show an apparent dependence on the radiation dose used in the assay of clonogen content. Crypt radiation survival curves often show increased curvature beyond that expected on the basis of the conventional linear-quadratic model. A novel form of crypt survival curve shape is proposed based on two contributory mechanisms of crypt killing. Six previously published sets of microcolony data were re-analysed using a dual-kill model, where target cells are killed by two contributory mechanisms, each described by a linear-quadratic function of dose. The data were analysed as two series--high-dose rate and low-dose rate irradiation. The data were fitted to the models using direct maximization of a quasi-likelihood, explicitly allowing for overdispersion. The dual-kill model can reproduce both the apparent dose-dependence of the clonogen estimates and the high-dose curvature of the dose-response curves. For both series of data the model was a significantly better fit to the data than the standard linear-quadratic model, with no evidence of any systematic lack of fit. The parameters of the clonogenic cell component of the model are consistent with other studies that suggest a low clonogen number (somewhat less than five) per crypt. The model implies that there is a secondary mechanism decreasing clonogen survival, and hence increasing clonogen number estimates, at high doses. The mechanisms underlying the modification of the dose-response are unclear, and the implied mechanisms of, for example, slow growth, induced either directly in the surviving cells or indirectly through stromal injury or bystander effects are only speculative. Nevertheless, the model fits the data well, demonstrating that there is greater kill at high doses in these experimental series than would be expected from the conventional linear-quadratic model. This alternative model, or another model with similar behaviour, needs to be considered when analysing in detail and interpreting microcolony data as a function of dose. The implied low number of < or = 5 of these regenerative and relatively radioresistant clonogenic cells is distinct from a similar number of much more radiosensitive precursor stem cells which undergo early apoptosis after doses around 1 Gy.     

The effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.

Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in an increased survival of animals exposed to a lethal dose of irradiation. The mechanisms underlying these effects are not clear. The present experiments were designed to investigate the nature of any proliferative changes induced in the crypts of the small intestine by protracted exposure to KGF. Tritiated thymidine or bromodeoxyuridine labeling showed statistically significant increases in labeling in the stem cell zone of the crypt, with a concomitant reduction in labeling in the upper regions of the crypt corresponding to the late-dividing transit population. The increase in labeling in the lower regions of the crypt was also observed with Ki-67 staining, but the reduction in the upper regions of the crypt seen with tritiated thymidine was not observed with Ki-67. Metaphase arrest data suggest that the rate of progression through the cell cycle is essentially the same in KGF-treated animals as in controls, but there is a statistically significant increase in the number of mitotic events per crypt. Double labeling studies suggest that, at certain times of the day, there is a greater influx into S phase than efflux. The data overall indicate that KGF induces some complex proliferative changes in the intestinal crypts and are consistent with the hypothesis that the radioprotection may be afforded, at least in part, by a KGF-induced increase in stem cell numbers and/or increases in the number of stem cells in the S phase of the cell cycle. This alteration in the homeostasis of the crypt is compensated for by a foreshortening of the dividing transit lineage.     

Cytotoxic effects of colcemid or high specific activity tritiated thymidine on clonogenic cell survival in B6CF1 mice

High specific activity tritiated thymidine (HSA-[3H]TdR) and colcemid were given in cytotoxic doses and regimens to B6CF1/Anl mice. The number of cells per intestinal crypt was reduced by the S-phase-specific (HSA-[3H]TdR and the metaphase blocking and cytotoxic effect of multiple injections of colcemid. In 50-day-old mice, the cytotoxic effect of multiple injections of colcemid reduced both the number of cells per crypt and the clonogenic cell survival. However, the number of surviving intestinal clonogenic or stem cells, assayed by the microcolony technique, did not change in 110--130-day old mice. These data suggest that most of the cells at risk from these cytotoxic agents are not clonogenic in adult 110--130-day old mice but are the cells in amplification division. However, since the stem cells of young mice are more susceptible to colcemid, they are apparently in a more rapid cell cycle than those of older mice. The clonogenic cell survival measured in 110--130-day old mice after a single radiation dose of 14 Gy (1400 rad) responded in a non-linear way to increasing time of continuous colcemid cytotoxicity. These data suggest that the intestinal stem cells can respond to amplification compartment cell death by a shortening of their cell cycle and thus, over time, the number of stem cells at risk to colcemid cytotoxicity increases.     

CYTOTOXIC EFFECTS OF COLCEMID OR HIGH SPECIFIC ACTIVITY TRITIATED THYMIDINE ON CLONOGENIC CELL SURVIVAL IN B6CF1 MICE

High specific activity tritiated thymidine (HSA-[³H]TdR) and colcemid were given in cytotoxic doses and regimens to B6CF₁/Anl mice. The number of cells per intestinal crypt was reduced by the S-phase-specific HSA-[³H]TdR and the metaphase blocking and cytotoxic effect of multiple injections of colcemid. In 50-day old mice, the cytotoxic effect of multiple injections of colcemid reduced both the number of cells per crypt and the clonogenic cell survival. However, the number of surviving intestinal clonogenic or stem cells, assayed by the micro-colony technique, did not change in 110–130-day old mice. These data suggest that most of the cells at risk from these cytotoxic agents are not clonogenic in adult 110–130-day old mice but are the cells in amplification division. However, since the stem cells of young mice are more susceptible to colcemid, they are apparently in a more rapid cell cycle than those of older mice. The clonogenic cell survival measured in 110–130-day old mice after a single radiation dose of 14 Gy (1400 rad) responded in a non-linear way to increasing time of continuous colcemid cytotoxicity. These data suggest that the intestinal stem cells can respond to amplification compartment cell death by a shortening of their cell cycle and thus, over time, the number of stem cells at risk to colcemid cytotoxicity increases.     

Radiation-induced mitotic delay: duration, dose and cell position dependence in the crypts of the small intestine in the mouse

The cells of the proliferative compartment in the crypt of the small intestine undergo a step by step differentiation and/or maturation from stem cells to the functional cells on the villi. The consequent hierarchical organization of the proliferative cell population can be related to the actual position of cells within the crypt. The stem cells are found near the bottom of the crypt with the more mature cells occurring at increasingly higher positions. The sensitivity of proliferative cells in the crypt of small intestine to radiation-induced mitotic delay was investigated at each position within the crypt. Using the stathmokinetic method (vincristine accumulation), the following were noted. The yield of mitotic figures 3 h immediately after irradiation showed a strong cell position dependence with the cells at the base of the crypt being most inhibited and those at the top of the proliferative compartment least affected. The mitotic yields were largely unaffected for the first 15 min suggesting that there is a transition point (Tp) for radiosensitivity which is located about 15 min before metaphase for all crypt cells. Cells located less than 15 min from metaphase are unaffected while those more than 15 min from metaphase are inhibited from further cell cycle progression. After this initial delay all proliferative cells were inhibited in their progression through G2 but some recovered more quickly than others. The ratio of the time of division delay (Td) in stem cells to that in cells at the top of the proliferative compartment was about 3:1. In absolute values Td after 1.0 Gy was about 1 h and 2.8 h, for cells at the top of the crypt and at the base, respectively. After 2.5 Gy the corresponding values were less than 3 h and between 5 and 6 h for the mid-crypt and crypt base respectively. There is thus a dependence on dose for the duration of the mitotic inhibition which for the cells at the top of the crypt is similar to the widely quoted average value 1 h per Gy, but the duration depends strongly on cell position. Thus not all proliferative cells respond in the same way. The duration is shorter the closer the proliferative cells are to their last cell division in the proliferative hierarchy in the crypt and longest for cells situated where the stem cells are to be expected.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

AN ESTIMATION OF PROLIFERATIVE POPULATION SIZE IN STOMACH, JEJUNUM AND COLON OF DBA-2 MICE

Liquid scintillation and autoradiographic techniques have been used to provide quantitative data on the proliferative units, the crypts, of stomach, jejunum and colon of DBA-2 mice. A slight modification of the crypt squash technique has provided data suggesting that about 50% of the cells of the jejunal crypt are at any given time in the proliferative state. This value is lower in the colon while in the stomach glands only 20% of the cells are involved in cell production. The data provide estimates for cell cycle times of 26·3, 16·0 and 23·2 hr for stomach, jejunum and colon respectively.
The size and number of proliferative units have been determined for three regions of the gastrointestinal tract of mice. A review of the literature suggests that considerable strain differences may exist.     

DIURNAL VARIATION IN PROLIFERATIVE COMPARTMENTS AND THEIR RELATION TO CRYPTOGENIC CELLS IN THE MOUSE COLON

The depth of the crypts in mouse descending colon varied diurnally, between twenty-six cells at 24.00 hours and thirty-eight cells at 12.00 hours. Cell loss from the colon was greatest immediately before the maximum faeces production, at the beginning of the dark period. The labelling index of the colon also changed, from 9% at 20.00 hours to 16% at 12.00 hours. The greatest variation in labelling index occurred at the top of the zone of proliferative cells, between the ninth and eighteenth cell position up the crypt. In this region a synchronized cohort of about forty cells apparently entered S phase once a day. Although the length of the proliferative zone doubled at 12.00 hours, that of the non-proliferative zone remained fairly constant all day. The number of cryptogenic cells per crypt was estimated by comparing single and split-dose X-ray survival curves. This gave a mean value of two cryptogenic cells per crypt. Crypts rarely regenerated from the base after irradiation. The cryptogenic cells probably lay between cell positions Nos 9 and 18 up the crypt and probably did not function as stem cells in the normal crypt.     

Radiobiology and stathmokinetics of intestinal crypts associated with patches of Peyer

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose-survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (approximately 35) but the final D0 for cells of the patch crypts was significantly higher (2.1 versus 1.7 Gy). A linear-quadratic fit gave a similar ratio of alpha/beta (approximately 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

The recruitability and cell-cycle state of intestinal stem cells

Evidence is presented which suggests that the crypts of the small intestine contain at least two discrete but interdependent classes of stem cells, some with discrete cell kinetic properties and some with discrete radiation responses or radiosensitivities. Very low doses of X rays or gamma rays, or neutrons, kill a few cells in the stem cell regions of the crypt in a sensitive dose-dependent manner. Similar doses generate several different cell kinetic responses within either the clonogenic fraction or the cells at the stem cell position within the crypt. The cell kinetic responses range from apparent recruitment of G0 clonogenic cells into cycle, to a marked shortening of the average cell cycle of the cells at the stem cell position. It is suggested that the cell kinetic changes may be the consequence of the cell destruction.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell kinetics were investigated in the descending colon of the rat. The number of cells per crypt was found to be approximately 625, with 33 cells per cell column and 19 cell columns per crypt circumference. The growth fraction of the colonic crypt was 0.42, and proliferating cells were situated largely in the lower half of the crypt. The cell cycle time was 50.5 h, with values for the G1, S and G2 phases of 40.0, 7.6 and 2.9 h respectively. Cell migration studies showed that it took 60-72 h for a cell to migrate from the upper border of the proliferative cell compartment in the crypt to the luminal surface of the colon. Data were also obtained from continuous labelling with tritiated thymidine and from studying the circadian rhythm of proliferative activity, which suggest that the cells in the bottom of the crypt may constitute a separate, more slowly cycling (stem)cell compartment.     

The proliferative status of intestinal epithelial clonogenic cells: sensitivity to S phase specific cytotoxic agents.

High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3 1/2 days after a range of radiation doses indicates that a majority of the clonogenic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.     

Circadian rhythms in the incidence of apoptotic cells and number of clonogenic cells in intestinal crypts after radiation using normal and reversed light conditions

Variations in the number of radiation-induced morphologically dead or dying cells (apoptotic cells) in the crypts in the small intestine of the mouse have been studied throughout a 24-h period under a normal light regimen (light on, 07.00-19.00 h; light off, 19.00-07.00 h). A clear circadian rhythm was displayed in the apoptotic incidence 3 or 6 h after irradiation for each gamma-ray dose studied (range 0.14-9.0 Gy). The most prominent circadian rhythm was obtained after 0.5 Gy. The peak time of day for inducing apoptosis was 06.00-09.00 h, and the trough occurred at 18.00-21.00 h. Some mice were also transferred to a room with the light cycle reversed, and were irradiated on different days after the transfer. The apoptosis induced by 0.5 Gy or 9.0 Gy, or the number of surviving crypts (microcolonies) after 11.0 Gy or 13.0 Gy was examined. The transition point for reversal (i.e. the switch time from the normal-light pattern to the reversed-light pattern) of the circadian rhythm in apoptosis (after 0.5 Gy) occurred 7 days after the transfer and the rhythm was reversed by 14 days. The rhythm for crypt survival (i.e. for clonogenic cell radiosensitivity) was disturbed on 1 day and the transition point for reversal occurred 3 days after the transfer. The rhythm became reversed by 7 days. These observations are discussed in relation to the identity of clonogenic cells, (functional) stem cells, proliferating transit cells and the cells sensitive to small doses of radiation (i.e. hypersensitive cells) in the crypt.     

Prosurvival and antiapoptotic effects of PGE2 in radiation injury are mediated by EP2 receptor in intestine

The biological activities of PGE(2) are mediated through EP receptors (EP(1)-EP(4)), plasma membrane G protein-coupled receptors that differ in ligand binding and signal-transduction pathways. We investigated gastrointestinal EP(2) receptor expression in adult mice before and after radiation injury and evaluated intestinal stem cell survival and crypt epithelial apoptosis after radiation injury in EP(2) null mice. EP(2) was expressed throughout the gut. Intestinal EP(2) mRNA increased fivefold after gamma-irradiation. Crypt survival was diminished in EP(2)-/- mice (4.06 crypts/cross section) compared with wild-type littermates (8.15 crypts/cross section). Radiation-induced apoptosis was significantly increased in EP(2)-/- mice compared with wild-type littermates. Apoptosis was 1.6-fold higher in EP(2) (-/-) mice (5.9 apoptotic cells/crypt) than in wild-type mice (3.5 apoptotic cells/crypt). The EP(2) receptor is expressed in mouse gastrointestinal epithelial cells and is upregulated following radiation injury. The effects of PGE(2) on both crypt epithelial apoptosis and intestinal crypt stem cell survival are mediated through the EP(2) receptor.     

A comprehensive model of the crypts of the small intestine of the mouse provides insight into the mechanisms of cell migration and the proliferation hierarchy

A comprehensive model has been formulated for the proliferative behaviour of the crypts of the small intestine based on individual cell to cell relationships rather than on the average effects of all cells. The model accommodates a wide range of cell kinetic data and provides an insight into the mechanisms involved in cell movement within the columnar sheet of cells and into the relationship between the stem cells and their progeny. The model permits the number of stem cells and transit generations to be estimated. The number of stem cells is predicted to be not less than 4 and not more than 16 per crypt with cell cycle times of between 12 and 32 h respectively. Certain conclusions can be drawn concerning the mechanisms involved in the initial cell displacements after cell division. The model also allows an estimation of parameters which cannot be measured directly such as the degree of cell generation disorder and the amount of dispersion of cells within a cell lineage.     

A stochastic branching model with formation of subunits applied to the growth of intestinal crypts

The intestinal epithelium is one of the most rapidly regenerating tissues in mammals. Cell production takes place in the intestinal crypts which contain about 250 cells. Only a minority of 1-60 proliferating cells are able to maintain a crypt over a long period of time. However, so far attempts to identify these stem cells were unsuccessful. Therefore, little is known about their cellular growth and selfmaintenance properties. On the other hand, the crypts appear to exhibit a life cycle which starts by fission of existing crypts and ends by fission or extinction. Data on these processes have recently become available. Here, we demonstrate how these data on the life cycle of the macroscopic crypt structure can be used to derive a quantitative model of the microscopic process of stem cell growth. The model assumptions are: (1) stem cells undergo a time independent supracritical Markovian branching process (Galton-Watson process); (2) a crypt divides if the number of stem cells exceeds a given threshold and the stem cells are distributed to both daughter crypts according to binomial statistics; (3) the size of the crypt is proportional to the stem cell number. This model combining two different stochastic branching processes describes a new class of processes whose stationary stability and asymptotic behavior are examined. This model should be applicable to various growth processes with formation of subunits (e.g. population growth with formation of colonies in biology, ecology and sociology). Comparison with crypt data shows that intestinal stem cells have a probability of over 0.8 of dividing asymmetrically and that the threshold number should be 8 or larger.     

The effect of estrogen on epithelial cell proliferation and differentiation in the crypts of the descending colon of the mouse: a radioautographic study

The regulatory role of estrogen on cell population kinetics in the descending colon was studied in intact female and ovariectomized mice. In the colonic crypts from intact mice, the crypt size (the number of epithelial cells per crypt column) and the proliferative activity of epithelial cells fluctuated slightly during the estrous cycle. Peak cellularity per crypt column was exhibted during estrus and early diestrus, whereas peaks in labeling index were seen during estrus and late metestrus. While the population size of mucous cells showed a minimal variation, the number of proliferative vacuolated cells per crypt column varied inversely with that of differentiated columnar cells during estrous cycle. The vacuolated cells were increased in number in the preovulatory phase and the columnar cells in the postovulatory phase. Three weeks after bilateral ovariectomy, the colonic crypt appeared to reach a new steady state, which was characterized by a small crypt size, a decrease in the number of differentiated cells, an increase in the relative number of proliferative cells and a relative increase in the proliferative activity of the crypt as compared to intact mice. When ovariectomized mice were treated with estrogen, the number of 3H-thymidine-labeled cells in the crypt was decreased as compared to untreated ovariectomized mice, the decrease being greater after a single injection than after multiple injections of estrogen, and the vacuolated-columnar cell line being affected more than mucous cell line. Meanwhile, the crypt size as well as the population size of differentiated cells in the crypt failed to return to normal after estrogen treatments. Thus, estrogen did not promote differentiation of epithelial cells in the crypt.     

Intestinal Cell Proliferation. I. A Comprehensive Model of Steady-State Proliferation In the Crypt

Abstract. Cell replacement in the crypt of the murine small intestine has been studied and modelled mathematically under steady-state conditions. A great deal of information is available for this system, e.g. cell cycle times, S phase durations, the rate of daily cell production, the Paneth cell distribution etc. the purpose of the present work was to consider simultaneously as much of these data as possible and to formulate a model based upon the behaviour of individual cells which adequately accounted for them. A simple mathematical representation of the crypt has been developed. This consists of sixteen stem cells per crypt (T_c= 16 hr, T_s= 9 hr), and four subsequent transit cell divisions (T_c= 11 to 12 hr, T_s= 8 hr) before maturation. Experimental data considered to test the modelling were LI and data on the number of vertical runs of similarly labelled cells. All data were obtained from the ileum after 25 μCi [³H]TdR given at 09.00 hours. A number of alternative assumptions have been considered and either accepted or rejected. Two alternative model concepts of cell displacement explain the data equally well. One is dependent upon strong local cell generation age determinance while the other could accommodate any weak local cell displacement process in conjunction with an environmental cut-off determinant at the middle of the crypt. Both models provide new interpretations of the data, e.g. certain rates of lateral cell exchange between neighbouring columns (250 to 350 per crypt per day out of a total of 420 cell divisions per day) can be concluded from run data, while LI data provide information about the mechanisms involved in maintaining a position-related age order in the crypt.     

Intestinal cell proliferation. I. A comprehensive model of steady-state proliferation in the crypt

Cell replacement in the crypt of the murine small intestine has been studied and modelled mathematically under steady-state conditions. A great deal of information is available for this system, e.g. cell cycle times, S phase durations, the rate of daily cell production, the Paneth cell distribution etc. The purpose of the present work was to consider simultaneously as much of these data as possible and to formulate a model based upon the behaviour of individual cells which adequately accounted for them. A simple mathematical representation of the crypt has been developed. This consists of sixteen stem cells per crypt (TC = 16 hr, TS = 9 hr), and four subsequent transit cell divisions (TC = 11 to 12 hr, TS = 8 hr) before maturation. Experimental data considered to test the modelling were LI and data on the number of vertical runs of similarly labelled cells. All data were obtained from the ileum after 25 microCi [3H]TdR given at 09:00 hours. A number of alternative assumptions have been considered and either accepted or rejected. Two alternative model concepts of cell displacement explain the data equally well. One is dependent upon strong local cell generation age determinance while the other could accommodate any weak local cell displacement process in conjunction with an environmental cut-off determinant at the middle of the crypt. Both models provide new interpretations of the data, e.g. certain rates of lateral cell exchange between neighbouring columns (250 to 350 per crypt per day out of a total of 420 cell divisions per day) can be concluded from run data, while LI data provide information about the mechanisms involved in maintaining a position-related age order in the crypt.     

Quantitation of intestinal clonogens by regeneration following cytotoxicity

Estimates of the number of colony-forming cells per intestinal crypt in the mouse are reviewed, together with their regeneration characteristics after cytotoxic doses of irradiation. The estimates are derived from the results of different designs of experiment, but all of them employ the scoring of sections of regenerating foci in prepared histological samples. It is shown that the estimates are dependent on the criterion used for scoring, and the necessary use of a correction factor to allow for the different sampling frequency of colonies of different sizes. Much of the data on this topic has emanated from the Paterson Institute for Cancer Research in Manchester, where various investigators have been involved over many years. Realistic estimates of the number of clonogens per crypt, derived using irradiation as the detection agent, are in the approximate range of 3 to 30. Higher values are obtained when a correction for sampling frequency is omitted. Lower values are obtained when injury is directed near the crypt base, using targeted irradiation or alternatively chemicals which possess some lineage-position-specific toxicity.