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Epithelial Biology Department, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 4BX, United Kingdom [C. S. P., J. A. O., C. B.]; EpiStem Limited, Manchester, M13 9XX, United Kingdom [C. S. P., C. B.]; and Amgen Inc., Thousand Oaks, California 91320-1789 [C. L. F., K. R.]
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Abstract |
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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.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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KGF has also been shown to alter the radiosensitivity of the clonogenic stem cells (8 , 10) in the small intestine, rendering them more resistant and, hence, protecting the intestine (and, as a consequence, the animal as a whole) from the symptoms of the gastrointestinal radiation syndrome (8 , 10) . The mechanism of action for KGF in such experiments remains obscure. The purpose of the present studies was to investigate whether repeated administration of KGF to mice resulted in any changes in proliferation along the crypt axis that could be interpreted in relation to cell lineages and the lineage ancestor stem cells, and thereby to provide a mechanistic explanation for the radioprotective effects reported above.
The small intestine is structurally organized into differentiated functional structures called villi and proliferative units called crypts. Within the crypts, it is believed that there are four–six cell lineages, each with a lineage ancestor stem cell. The lineages contain six–eight cell generations, the first two or three of which contain cells that are undifferentiated (uncommitted) and can repopulate the crypt with stem cells and their progenies; these are called potential regenerative or clonogenic stem cells, of which up to 30 exist in each crypt. The remaining 100–120 cells in the proliferative lineage have no stem cell capacity and are referred to as dividing transit cells. There are no markers for stem cells, but the small intestine is a highly polarized tissue, and the position of the cells on the crypt-villus axis in longitudinally sectioned crypts is closely related to their lineage status. Hence, by studying certain positions, the properties and responses of stem cells can be studied. In the mouse, the critical stem cell position is four cell positions from the base, immediately above the differentiated functional Paneth cells (11 , 12) .
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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The difference between the KGF and control LIs in each of the seven single-dosing protocols are represented as bar charts in Fig. 1
, with examples of the raw data shown below. The relative difference in labeling at each of these positional "windows" and in the crypt as a whole, can be seen in Fig. 1, A–C.
Those groups that show statistically significant differences using, first, the Mann-Whitney test (*) and, second, the difference plots and the modified median test (+) for the full distribution are identified on these bar diagrams. The cell positions over which a significant difference was detected in the median test are shown by the numbers adjacent to the bars.
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, as representative examples. The inserts show the difference plots used for the statistical test and the cell positions over which significant differences were detected and identified.
The results demonstrated that the time of day for KGF administration, the number of daily injections, or the number of KGF injections (one versus two) per day had little effect on the overall result. Hence, the data have been summarized by pooling all of the KGF-treated groups (11 groups in an initial run of the experiments; single labeling), and the corresponding saline-treated groups (Fig. 1D)
. This provided groups of 27 mice in each case (1 mouse died in one of the groups in each case), and 
120,000 crypt cells were scored in each pooled group.
Experiment to Determine Whether or not the Flux of Cells Entering S Phase Is Affected by KGF.
Fig. 2
shows the complementary data from the double-labeling experiments, the bar charts and LI curves demonstrating similar effects on the total number of either BrdUrd- or 3HdThd-labeled cells to those effects seen in Fig. 1
.
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Metaphase Accumulation Studies.
The results obtained from the crypt cell production rate at 24 h after KGF administration are presented in Fig. 3
. The analysis of the lines shows that the slopes are not different, which indicates that the rate of entry of crypt enterocytes into mitosis is the same in KGF and control animals at this time point, i.e., 24 h after injection of the last dose of KGF. The Y intercept of the line of KGF group is significantly higher than that of the control, which indicates that there are more cells in mitosis. Therefore, KGF is probably having some direct effect on cell cycle rather than on the rate of entry of cells into cycle. Unfortunately, no positional data are available from these studies.
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and 5
. The bar chart summaries indicate that the only consistent effects were in the lower, stem cell region of the crypt, in which an increase in labeling was seen (Fig. 4A)
. The pooled data from these experiments illustrate significant differences between the KGF and control groups at cell positions 3–5 and 9, which suggests increased cycling of cells in the stem cell zone, Fig. 5A
. Ki-67 labeling reached figures in excess of 90% in the central regions of the crypt, but there were no significant differences between the KGF and saline groups for the upper regions of the crypt. The pooled 3HdThd results on the same panel (Fig. 5A)
, demonstrate the higher levels of labeling achieved with Ki-67. These can be subtracted from the latter to show the distribution of cells that are expressing Ki-67 but are not undergoing DNA synthesis (Ki-67 minus 3HdThd), Fig. 5B
. Two representative examples for Ki-67 labeling in the individual groups (groups 4 and 5) are also shown to demonstrate the variability within these experiments (Fig. 5, C and D)
.
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and 7
summarize the data obtained from the double-labeling experiment for the influx and efflux for S phase in the KGF- and saline-treated groups. The results were rather variable and appeared to depend on the time of day. The data for the 9-a.m./6-day group show a clear and significant increase in influx into S phase, after KGF treatment in the lower regions of the crypt, i.e., associated with the early lineage, which is significant for cell positions 4–8. This was not seen for the 9-p.m./6-day group and was reversed for the 3-a.m./6-day group (i.e., reduced influx and increased efflux; data not shown). Efflux was not significantly affected by KGF pretreatment for the 9-a.m./6-day group. A small increase in efflux was seen for the 9-p.m./6-day group (not significant). Crypt length, as determined by the number of cells in the crypt column, was essentially constant (data not shown).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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). The mitotic accumulation data suggest more cells proliferating, a shorter cell cycle, or a longer duration of mitosis in the KGF-treated groups. The former is inconsistent with the overall labeling or Ki-67 data. A shorter cell cycle or more cells cycling could be consistent with the labeling data for the lower regions of the crypt and is, therefore, the favored interpretation.
The results of the double-labeling experiments suggested that KGF results in an increase in the influx of cells into S phase at the bottom of the crypt at some times of the day (e.g., at 9 a.m. for 6 days) but little sign of a corresponding increase of efflux at this time of day, which might suggest that the duration of S phase was increased. However, there was no indication that the number of labeled (S-phase) cells, or double-labeled cells was increased in the KGF-treated groups as would be expected if the S phase were extended. In fact, the number of double-labeled cells was reduced in the KGF-treated groups as was the simple S-phase LI. The fact that the influx was reduced and the efflux increased at 3 a.m. for 6 days suggests that the KGF effect depends on the circadian rhythm. The increase in the influx but no change in the efflux at the crypt base (Fig. 6)
could be interpreted to indicate that it takes 1–2 h for the KGF effects to be detectable. Overall, the data suggest that KGF does not result in increased cell production from the crypt. However, the similar trends at the crypt base seen in mitosis and more particularly in Ki-67 labeling suggest that there are more cells in cycle in the stem cell region.
Effects of KGF on the Transit Cells in the Crypt.
A more consistent and statistically significant effect was observed in the later transit compartments of the crypt. Here, no changes in influx or efflux were detected; however, there was a significant reduction in the number of cells replicating DNA in the upper regions of the dividing transit population (cell positions 9–15). The effect would suggest that, in the KGF-treated groups, there might be one less transit generation in the animals that have been repeatedly exposed to KGF. On first sight, this would appear to be in conflict with the observation that repeated exposure to KGF induces a hyperplastic state in the tissue, and is counterintuitive, bearing in mind the suggestion that more stem cells are cycling and, hence, producing more transit lineages, but it is consistent with the observation that the number of differentiated cells (e.g., goblet cells) is increased in KGF-exposed animals (7)
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Homeostatic Control on Crypt Size.
Overall, the data suggest that the stem cells are cycling faster and that there may be more stem cells present (which could account for the radioprotection). Each stem-cell-generated lineage appears to be shorter (less labeling in the upper crypt), resulting in a similar total cell production in the KGF and control groups. It appears that the overall homeostatic mechanisms in the crypt counteract the increased stem cell activity by reducing the transit lineage in an attempt to maintain a constant cell output or a stable crypt size.
The apparent stimulation of stem cell proliferation by KGF should result in increased numbers of stem cells, which may explain the radio- and chemo-protective effects (8 , 10 , 13) . Any new stem cells should produce entire transit lineages of 60–120 cells and, hence, crypt and proliferative compartment enlargement. However, stem cell numbers in the crypt are probably very tightly regulated with, under normal circumstances, homeostatic factors inducing any excess stem cells into apoptosis (14) . This process may be overridden in the KGF-treated animals and results in the activation of a secondary level of regulation: the reduction in cell proliferation, or number of transit generations, in the later transit cells.
KGF-induced Hyperplasia.
The hyperplasia after KGF exposure is particularly evident in the stratified epithelia of the mouth (6
, 13)
but is also observed in the esophagus and stomach and, to a lesser extent, in the intestine (7
, 8)
. However, in the intestine, the effect of increasing or decreasing the rate of cell production in the crypts is less easily detected by looking at the number of cells in the single-layered columnar epithelium in the crypts or the villus. Such measurements are dependent on other processes, such as cell migration along the basement membrane and cell extrusion and/or apoptosis at the villus tip, and on shrinkage or stretching of the tissue during processing and the amount of gut contents. If either migration or extrusion is affected independently, the cellularity and size of the crypt and/or the villus will be affected.
Ki-67 Labeling.
The somewhat surprising observation in the present experiments is the lack of any clear reduction in the Ki-67 LI for cells in the upper transit compartment of the crypt. This suggests that although KGF may have reduced the number of late transit cells cycling, or the number involved in cell production, they do not appear to have lost expression of the Ki-67 antigen. Ki-67 labeling index values are greatly increased at all of the cell positions relative to the S phase fraction. The 3HdThd or BrdUrd labeling both suggest a reduction in the number of cells in S phase in the upper regions of the crypt, i.e., fewer cells in cycle, or a longer cell cycle, either of which would result in fewer cells being produced after KGF, whereas the Ki-67 data suggest that the same number of cells are in cycle.
Conclusions.
In summary, the data suggest that KGF affects early lineage cells or stem cells, particularly when administered daily at 3 a.m. and 9 a.m. for 6 days. At the latter time, KGF treatment increases the influx into S phase over cell positions 4–8 but has no effect on the efflux. After most of the treatment protocols studied, KGF induces a fairly dramatic decrease in S phase cells in the late transit cell populations. The Ki-67 LI (cycling cells) is increased towards the base of the crypt, but the Ki-67 cell-positional distribution shows no foreshortening in the upper crypt. Thus, protracted exposure to KGF results in an increase in early lineage or stem cell cycling and a simultaneous induction of premature differentiation in late-transit cells, which continue to express the Ki-67 antigen for a time. It is also possible that KGF may regulate Ki-67 expression, which could provide another explanation for these results. The number of goblet cells was not measured in the studies but might be useful for clarifying any differentiation changes.
In terms of the radioprotective action of KGF in the intestine (8) , the present data suggest that the increased cycling of stem cells may produce a situation in which the crypts contain more steady-state and/or clonogenic stem cells, thus providing a larger target for the radiation. Because, for a given radiation dose, the same fraction of cells would be killed, a larger target pool in the KGF animals would result in a greater number of stem cells surviving per crypt and an increased animal surviving fraction and, hence, better gastrointestinal radiation outcome.
Although the present data suggest that one action of KGF is to increase the number of stem cells or early-lineage cells in S phase, this may not be the only explanation for the protective action on crypt clonogenic cells or animal survival (8) . The increase in S phase cells does not necessarily mean an increase in the number of stem cells. Other possibilities include: KGF acts as a survival factor that increases stem cell numbers by preventing radiation-induced apoptosis. Radiation induces apoptosis in the stem cell region of the crypt (15) ; KGF may also induce a change in the inherent radiosensitivity of the clonogenic cells, which may be related to changes in repair efficiency or to effects on antioxidants. In this context, the Do value, which is the reciprocal of the slope of the survival curve and a measure of radiosensitivity of the KGF-treated clonogenic cells, is increased from 1.43± 0.06 Gy to 1.91± 0.12 Gy (8) . Thus the clonogenic cells may be inherently more resistant after KGF treatment. However, the results are confusing because the survival curves also have a smaller shoulder, which suggests less repair capacity or a smaller intracellular radiation target. Changes in the rate at which KGF-treated clonogenic cells regenerate the crypt may also be a partial mechanistic explanation. The KGF-treated cells may be cycling faster or may be primed for regenerative cell cycles.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Various different injection protocols were tested to see which was the most effective and whether cell proliferation changes differed. Mice were given injections either once or twice daily for differing periods of time. The timing of these injections was also varied to determine whether the circadian rhythm influenced the magnitude of any KGF-induced crypt responses. Previous work has determined that peak proliferation in the mouse small intestine occurs at approximately 3 a.m., with a corresponding nadir at approximately 3 p.m. (17, 18, 19) .
Dosing protocols were as follows: (1) daily single injections of 125 µg of KGF at 9 a.m. for 6 days (0900/6-d); (2) 125 µg of KGF daily at 9 a.m. for 8 days (0900/8-d); (3) 125 µg of KGF at 3 a.m. for 6 days (0300/6-d). These animals were placed in a reverse light cycle room (lights on at 6 p.m., off at 6 a.m. for 2 weeks prior to the initiation of an experiment, i.e., the animals received injections at 3 p.m. human time, which was equivalent to 3 a.m. animal time); (4) 125 µg of KGF daily at 6 a.m. for 6 days (0600/6-d); (5) 125 µg of KGF daily at 9 p.m. for 6 days (2100/6-d); (6) 125 µg of KGF delivered twice daily at 9 a.m. and 9 p.m. for 6 days (0900, 2100/6-d); (7) 125 µg of KGF delivered twice daily at 9 a.m. and 9 p.m. for 3 days (0900, 2100/3-d); (8) 125 µg of KGF delivered at 12 midnight for 6 days (2400/6-d); and (9) 125 µg of KGF delivered at 3 a.m., 6 a.m., and 9 a.m. for 3 days (0300, 0600, 0900/3-d).
To measure cell proliferation, the S phase cells in the crypt were labeled with tritiated thymidine (3HdThd). One h after the last injection of KGF, the animals all received 25 µCi (0.925 MBq) of methyl 3HdThd i.p. (specific activity, 222 GBq/mM) (NEN Life Sciences, Hounslow, United Kingdom). For all of the above experimental groups and their respective controls, the animals were killed 40 min after the administration of tritiated thymidine.
To see whether the flux of cells moving into or out of the S phase varied, after KGF treatment, a double-labeling protocol was adopted (20 , 21) . Three additional groups and their respective controls, were double-labeled to determine the influx and efflux of the S-phase cells in the crypt (20 , 21) . These groups, numbered 10, 11, and 12, received KGF for 6 days at 9 a.m., 9 p.m., or 3 a.m. and then 25 µCi of tritiated thymidine 1 h after the last KGF administration. An additional dose of 10 mg of BrdUrd (Sigma Aldrich, Poole, United Kingdom) was then given i.p. in 0.5 ml 1 h after the 3HdThd. These animals were then killed 1 h later. These three experiments are abbreviated to: 9 a.m./6-day double-labeling; 9 p.m./6-day double-labeling; and 3 a.m./6-day double-labeling, respectively.
A vincristine metaphase arrest experiment was performed to test whether similar proliferative changes occurred in the rate of entry into the M phase (22) . In one experiment, 30 female BDF1 mice were randomized into two groups of 15 mice, housed five mice to a cage. The groups of mice were treated with a single s.c. dose of 125 µg per injection, or 5 mg/kg KGF, or the same volume of saline (vehicle control) at 8 a.m. for 3 consecutive days. The crypt cell production rate assay was performed 24 h after the last dose of KGF according to the method of Goodlad (22) . Briefly, mice were injected i.p. with 25 µg per injection or with 1 mg/kg of vincristine sulfate (VINCASAR PFS vincristine sulfate injection, USP; Pharmacia, Kalamazoo, MI) and then killed at timed intervals between 30 and 180 min after the injection. The small intestine was rapidly removed, rinsed, and divided into segments. Four cm of the jejunal segment was fixed in Carnoy’s fixative for 1 h, dehydrated in a series of ethanols, and hydrolyzed in 1 M hydrochloric acid for 10 min at 60°C. The tissue was then stained in Schiff’s reagent (American Master Tech Scientific Inc., Lodi, CA) for a minimum of 45 min, rinsed, and microdissected to separate crypts in 45% acetic acid. The crypts were counted on a slide in a drop of the acetic acid and coverslipped using gentle pressure to flatten the crypts. Metaphase-arrest figures were counted in 10 crypts/jejunal segment, and the counts were used to calculate an average number of crypt mitotic figures per mouse. These data were plotted versus time and were used to perform a regression analysis to obtain an equation for the line.
In other experiments, the small intestines were rapidly removed from the animals once killed, and were fixed for 30 min in ice-cold Carnoy’s solution (60% ethanol, 30% chloroform, and 10% propionic acid), after which they were stored in 70% ethanol prior to dehydration, embedding, and sectioning (3–5 µm). Autoradiographs were prepared using Ilford K5 emulsion (Ilford, Mobberly, United Kingdom) and standard procedures (23)
. For the double-labeling groups, immunohistochemistry was undertaken using a rat IgG monoclonal antibody for BrdUrd detection (MAS250b, clone BU1/7S; Harlan Sera Ltd., Loughborough, United Kingdom), again following standard procedures (20)
. Briefly, after blocking endogenous peroxidase activity with hydrogen peroxide, the slides were hydrolyzed in 1 M HCl at 60°C for 8 min followed by neutralization in boric acid buffer for 6 min. After washing in PBS, nonspecific binding was blocked by incubation in 5% normal rabbit serum (Sigma, Poole, Dorset, United Kingdom) for 30 min, and then a 1:5 dilution of the rat anti-BrdUrd (Sera Labs, Loughborough, United Kingdom) was applied for 1 h at room temperature. After further washing in PBS, rabbit antirat peroxidase (1:100; Dako, Cambridge, United Kingdom), diluted in 10% normal mouse serum, was applied for 1 h, and then, after further washing, the slides were developed using DAB. After the immunohistochemistry, the slides were washed for 24 h and then were dipped for autoradiography as above (see Fig. 8
).
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Briefly, after hydrating, the tissue was microwaved in citrate buffer to retrieve any cross-linked antigens, and then endogenous peroxidase activity was blocked with hydrogen peroxide. Nonspecific binding was blocked by incubation with 10% goat serum (Sigma, Poole, Dorset, United Kingdom) and then rat anti-Ki-67 was applied at a dilution of 1:500 for 1 h at room temperature. After washing in PBS, goat-biotinylated antirat antibody was applied, at 1:200 in 2% normal mouse serum for 30 min at room temperature. Tissue sections were then incubated in avidin-peroxidase solution (Elite ABC Vectorstain kit, Vector Labs, Peterborough, United Kingdom) for 30 min. After further washing, sections were developed with DAB. Some slides were then washed for 24 h and dipped for autoradiography as above (see Fig. 8
), others were simply dehydrated in 80% methanol and counterstained with 0.4% thionine, prior to transfer to 100% ethanol, clearing in xylene, and mounting in XAM (BDH Chemicals, Poole, Dorset, United Kingdom).
The gut-bundling procedure was used to ensure that each mouse provided 10 good transverse sections of the lower ileum, and that all four mice could be mounted together on a single slide (i.e., each mouse being identifiable) (25)
. These transverse sections were then used to determine the cell-positional LI in 50 complete longitudinally sectioned half-crypts from each mouse, the cell at the crypt base being designated cell position 1 as shown in Fig. 9
(26
, 27)
. Thus, each experimental group generated cell-positional frequency data for the LI (S-phase cells) from 200 half-crypts. From these cell-positional distributions, the fraction of cells in the S phase for cells of different hierarchical status, i.e., position along the crypt axis, could be determined with the presumptive stem cells being located around the 4th or 5th position from the base of the crypt (immediately above the crypt base Paneth cells) (11
, 28)
(see Fig. 9
).
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In addition, the cell-positional distribution for the 3HdThd-only (single) labeled cells was determined, i.e., the cells that were labeled only with 3HdThd but not labeled with BrdUrd. This cohort represents the efflux from S phase in the 1-h interval between the two injections of label. Similarly, the BrdUrd-only (single) LI was determined (i.e., BrdUrd-positive cells that contained no 3HdThd labeling). These represent the influx into S phase during the 1-h interval between injections (see Ref. 20 for additional details).
From a separate set of slides from each group, the Ki-67 LI was determined.
These labeling indices versus cell-position frequency plots can be analyzed and presented in a variety of ways. Here, for ease of presentation, the data are shown as smoothed graphs in which the running average over three adjacent cell positions has been determined (26) .
The frequency plots can be analyzed for statistical differences using a modified median test (26 , 29) that identifies those positions in the crypt over which statistically significant differences between the KGF-treated and the saline-BSA vehicle (control)-treated groups can be observed. The data can also be compared by looking at the average LI for the distributions as a whole for a group of mice, or for various selected windows along the cell-positional axis, e.g., cell positions 1–7.
The mean LI per mouse for the entire distribution, or for selected windows, was also calculated, and the group mean values were compared (treated versus control) using the Mann-Whitney test. Two windows were selected for analysis: first, cell positions 1–7, which include the area assumed to contain the stem cells for the crypt; and, second, cell positions 9–15, which encompass rapidly proliferating transit cells.
The LI cell-position plots (with difference plots showing areas of statistically significant difference) are accompanied by bar diagrams for the mean LI value, for the crypt as a whole, for cell positions 1–7, and cell positions 9–15.
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Footnotes |
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1 This work was supported by grants from the Cancer Research Campaign (United Kingdom) and Amgen Inc. 
2 To whom requests for reprints should be addressed, at Epithelial Biology Department, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, United Kingdom. Fax: 0044-(0)161-446-3181; E-mail: cpotten{at}picr.man.ac.uk
3 The abbreviations used are: KGF, keratinocyte growth factor; LI, labeling index; BrdUrd, bromodeoxyuridine; 3HdThd, [3H]thymidine; DAB, diaminobenzidine tetrahydrochloride.
Received for publication 10/23/00. Revision received 3/14/01. Accepted for publication 3/16/01.
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
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| Molecular Cancer Research | Cell Growth & Differentiation |
) or saline control (
). Simple, unweighted linear regression of the number of metaphases against time was analyzed. Standard F tests were used to test for differences between the slopes of the intercepts. Both groups show a very similar slope of close to 0.1 metaphase/h. The slopes are not significantly different (P = 0.91), whereas the intercepts are highly significantly different. Control, 8.8 (±3.3); KGF, 20.6 (±4.8).
