1Graduate Group in Molecular and Biochemical Nutrition, University of California, Berkeley 94720; and 2Division of Endocrinology and Metabolism, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94141
Submitted 9 April 2003 ; accepted in final form 22 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes
Long-term label-retaining cells (LRCs) have been identified as cells comprising or enriched with stem cells in a number of epithelial tissues, including colon (2, 7, 15, 1820, 25). LRCs are a subpopulation of cells characterized by having a longer cell-cycle time than transit cells. In addition, LRCs share several characteristics with stem cells (15, 18, 25, 28). LRCs in colon have been identified by the heterogeneity of labeling patterns after extensive delabeling (washout) of [3H]deoxythymidine ([3H]dT) following repeated injections of [3H]dT in rodents and humans (15, 25). Retention of [3H]dT label has not provided a means for physical isolation of LRCs, however. A technique for physically isolating LRCs would have substantial utility for assessing their physiological characteristics and for investigating their potential role in carcinogenesis.
We recently developed a method for measuring colon epithelial cell (CEC) proliferation in vivo that is well suited for cells with slow turnover rates (long-lived cells) (16, 22, 23). DNA replication, and thus cell proliferation, is measured on the basis of incorporation of deuterium from heavy water (2H2O) into the deoxyribose (dR) moiety of purine deoxyribonucleotides of dividing cells (Fig. 1). Previous methods for labeling DNA have used the nucleoside salvage pathway from labeled pyrimidine nucleosides {[3H]dT or bromodeoxyuridine (BrdU)}. In contrast, the stable isotope method with 2H2O, in addition to being safe for use in humans, utilizes the de novo nucleotide synthesis pathway, which introduces several technical advantages (discussed in Refs. 16, 22, 23).
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Labeling protocols. Fisher 344 rats (male, 4 wk old at the beginning of studies; Simonsen, San Jose, CA) were used. All procedures were approved by the University of California, Berkeley, Office of Laboratory Animal Care. Rats were housed three per cage and fed a diet of Purina rat chow, provided ad libitum. A 12:12-h light-dark cycle was maintained.
Animals were divided into three groups, receiving 2 (n = 9 rats), 4 (n = 12 rats), and 8 wk (n = 12 rats) of BrdU in drinking water, respectively. The animals in each group then stopped receiving BrdU and concurrently began receiving 2H2O (BrdU washout). Three animals from each BrdU group were not administered 2H2O but were killed 2, 4, and 8 wk after receiving BrdU. The other animals were killed 2, 4, and 8 wk after BrdU was stopped and 2H2O was started (Fig. 2).
|
The principle behind the use of BrdU to isolate nuclei of LRCs is as follows. During the BrdU delabeling period, cells with fast turnover are lost from the tissue within 8 days, given that their turnover time is 68 days (Refs. 12, 14; Kim SJ and Hellerstein MK, unpublished results). However, cells with slow turnover are retained in the tissue, and their BrdU content remains above the detection limit. The turnover time of stem cells has not previously been established but is estimated to be two to eight times longer than that of transit cells (24). The delabeling period of 28 wk was therefore chosen because it is longer than 8 days (to allow cells with fast turnover to be completely gone from the tissue) but not longer than 8 wk (to allow cells with slow turnover to retain BrdU).
Daily BrdU (Sigma) injection was given by the intraperitoneal route at 160 mg/kg body wt per day in dimethyl sulfoxide (final concentration 100 mg/ml) for up to 8 days. The 2H2O labeling protocol consisted of an initial intraperitoneal bolus injection of 100% 2H2O to achieve 4% enrichment of 2H2O in body water, followed by 8% 2H2O administration in drinking water ad libitum throughout the study (23). The bolus dose of 2H2O given to the rats was based on an estimated 60% of their body weight as water, i.e., for a 200-g rat with an estimated 120 ml of body water, 4% of which is 4.8 ml, 2.4 ml of 100% 2H2O was injected twice, 2 h apart. The 8% enrichment of 2H2O in drinking water was chosen because it produces relatively high enrichments in newly synthesized DNA and has no known toxicities. 2H2O was purchased from Cambridge Isotopes (Andover, MA). At each measurement time point, rats were killed by carbon dioxide asphyxiation.
Body Water Enrichment
Body water enrichments were measured using a gas chromatography-mass spectrometry (GC-MS) technique that we have described previously (23).
Isolation of CECs from Crypt Basal (Proliferative) Zones
The entire colon was excised fresh at necropsy. Feces were removed with cold 0.015 M NaCl solution with 0.001 M dithiothreitol (DTT), and the colon was placed in cold 1.6% Joklik's modified minimal essential medium (JMMEM; GIBCO, Grand Island, NY) for 120 min (4, 5, 29). CECs from different proliferative zones of the crypt were isolated sequentially according to the nonenzymatic, mechanical dissociation method (4, 5), rather than inverted or enzymatic methods, to increase the yield of cell collection. CECs from different crypt zones (top, middle, and basal) were collected by incubating the colon sac filled with three different solutions: 10% fetal bovine serum in 1.6% JMMEM (top zone isolation); citrate buffer with 0.027 M sodium citrate, 0.0015 M KCl, 0.096 M NaCl, 0.008 M KH2PO4, and 0.0056 M Na2HPO4, pH 7.3 (middle zone isolation); and 0.0015 M EDTA and 0.0005 M DTT in PBS, pH 7.2 (basal zone isolation). Antibiotics were added to each solution. The colon sac was clamped and immersed in an Erlenmyer flask filled with PBS. The tissue was shaken (75 oscillations/min) in a 37°C water bath for 20 min, then for 15 min, and then for 30 min to collect CECs from the top, middle, and basal zones, respectively. CECs from the middle zone (transitional cells) were discarded. The collected CECs from each zone were centrifuged at 500 g for 10 min to precipitate the cells. The collected cells were then separated from intraepithelial lymphocytes (IELs) and other contaminants by applying discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. Fifteen milliliters of 45% Percoll were overlaid on fifteen milliliters of 75% Percoll (29). The Percoll gradient was then centrifuged at 350 g for 30 min. CECs were collected from the supernatant at the 45% Percoll layer. CECs from the base of the crypts (proliferative zone) were collected (4, 5) and used for FACS and GC-MS analysis, because LRCs are known to be located at this region (24, 25). CECs from the top (mature) zone from rats that were given 2H2O for 23 wk were used as fully turned over CECs to represent the maximal 2H labeling in DNA (23). A part of the distal and proximal colon was used for immunohistochemistry.
Determination of IEL Contamination in CECs
CECs freshly collected after Percoll centrifugation were stained with 6A5 anti-RT6.2 (rat IgG1) antibodies specific for rat IELs (Bortell R, personal communication). Anti-RT6.2 antibodies were a generous gift from the laboratory of Dr. Rita Bortell (University of Massachusetts) and were derived from a hybridoma cell line secreting the antibodies. Controls were prepared as follows. Unstained control was prepared without the addition of any antibodies. Also, to detect nonspecific binding of primary antibody, 2 million cells were preincubated with isotype control for 6A5 anti-RT6.2, i.e., purified rat IgG (PP68; 5 µg/1 million cells in 100 µl; Chemicon) on ice for 30 min. The cells were then washed twice in 2.5 ml of magnetic cell sorting (MACS) buffer (Miltenyi Biotech) and stained with anti-RT6.2 (1 µg/100 µl) for 30 min at room temperature. The cells were washed twice and then stained with mouse anti-rat IgG1 monoclonal antibody (PharMingen) conjugated with fluorescein isothiocyanate (FITC; 2 µg/100 µl, as suggested by the manufacturer) for 30 min at room temperature. The cells were washed twice in 1 ml of MACS buffer and reconstituted in PBS before FACS analysis. IELs and spleen cells from rats were used as positive controls.
In addition, enriched CECs were stained with mouse anti-rat CD8a monoclonal antibody (BD-554856; 1 µg/1 million cells in 100 µl, reacts with CD8+ T cells), mouse anti-rat CD25 monoclonal antibody (BD-554865; 1 µg/1 million cells in 100 µl, reacts with the -chain of the IL-2 receptor on T cells and thymic and splenic dendritic cells), and mouse anti-rat pan-T cell monoclonal antibody (BD-554904; 1 µg/1 million cells in 100 µl, reacts with a 95- to 120-kDa antigen on T cells). Isotype control for each antibody was used to correct for possible background staining. We also stained IELs and splenocytes to confirm the specificity of the antibodies to IELs.
Flow Cytometric Analysis of BrdU-Labeled Nuclei
The staining protocol was described elsewhere (6), modified for CECs. The CECs were isolated from colon (as described) and filtered through a Falcon tube 2235. The cells were then fixed with ice-cold 95% ethanol while vortexing, centrifugated at 800 g for 3 min, and washed in cold PBS (without Ca2+ and Mg2+) once. Because intact CECs showed a tendency to aggregate under high-speed flow-sorting conditions, nuclei were extracted from the cells for isolation by flow cytometry (10). Two million cells were washed in 150 µl of PBS, incubated in 1% NP-40 (Sigma) solution in the refrigerator for 30 min, and centrifugated at 10,000 g, and the pellet was collected and washed in PBS. Two million CEC nuclei were added to each well in 96-well (round bottom) plates, and then 150 µl of 1% paraformaldehyde with 0.01% Tween 20 in PBS was added for additional fixation and permeabilization. The nuclei were kept at room temperature for 30 min and then at 4°C for 15 min before centrifugation at 800 g for 3 min and washing in cold PBS once. Next, 150 µl of DNase I (50 kU/ml) were added at room temperature for 15 min to allow denaturation of nuclear DNA. The nuclei were then washed with PBS once, and 50 µl of FITC-conjugated anti-BrdU monoclonal antibody (Becton Dickinson, Palo Alto, CA) were added with 20 µl of PBS containing 0.1% Tween 20. For negative controls, an equal concentration of mouse IgG1-FITC isotype control antibodies (Becton Dickinson, catalog no. 349041) were used to correct for background fluorescence. The nuclei were stained overnight at 4°C, washed two times with cold PBS the next day, and then filtered (using a Falcon tube 2235) before being analyzed. Nuclei were sorted with a Beckman-Coulter EPICS Elite cell sorter equipped with an argon ion 15-mW air-cooled laser, a helium-neon 15-mW air-cooled laser, and a water-cooled 5-W argon ion laser.
Measurement of Cell Proliferation
Isolation of deoxyadenosine from DNA and derivatization of dR. Genomic DNA was isolated from the collected nuclei of LRCs by using a Qiagen kit (Qiagen, Valencia, CA). DNA was hydrolyzed and deoxyadenosine (dA) was isolated as described previously (23). The isolated dA was cleaved of the base moiety to obtain dR (12, 23). The pentose-tetraacetate (PTA) derivative of dR was prepared for GC-MS analysis (23). GC-MS analysis was performed by methane chemical ionization with the use of a 30-m DB-225 column (0.25-mm inner diameter, 0.25-µm film thickness; J & W Scientific, Folsom, CA) with selected ion monitoring of m/z 245 and 246.
Calculations. Unlabeled dA standards (representing natural abundance) were analyzed simultaneously in each GC-MS run to establish the dependence of measured isotope ratios on the amount of sample injected [abundance sensitivity (23)]. This dependence can be characterized by plotting the abundance of the parent ion M+0 (m/z 245) against the ratio of M+1 to M+0 plus M+1 ions [246/(245 + 246)]. A linear regression of the ratio vs. M+0 abundance was calculated as described previously (23). The regression line was then used to calculate the natural abundance ratio at any particular M+0 abundance (23) for calculation of excess abundances in samples.
The fraction of new cells produced (fractional synthesis, f) was calculated using the precursor-product relationship. The isotopic enrichment of fully (or nearly fully) turned over CECs represents the true precursor enrichment (i.e., maximum enrichment) for CECs (23). Colonocytes are known to be fully replaced with new cells in 68 days in rodents (14). Rats were therefore maintained on 2H2O for 23 wk to ensure complete turnover of CECs, and the enrichments of those CECs were used as a comparison (denominator) or asymptotic value (16). The fractional replacement (f) of newly divided cells at each time point during 2H2O administration was determined as follows (16)
![]() |
![]() |
The replacement rate constant (k) was calculated as described previously (23)
![]() |
Percentage of LRCs Estimated from 2H2O Labeling In Utero
To confirm the percentage of LRC, as determined by the 2H2O labeling method, we also studied rats (n = 7) that had been labeled in utero (the mothers had been receiving 2H2O during their entire pregnancy). All seven pups were maintained on 4% 2H2O for 7 wk after they were born, and three of these were then killed (fully deuterated rats). The other four rats stopped receiving 2H2O at 7 wk of age, were transferred to natural abundance water for 2 wk (to allow 2H2O to wash out of their body water pool), and then were killed.
BrdU Immunohistochemistry
To locate LRCs, we cut and fixed the distal end of the colon (1 cm) in 10% PBS-buffered formalin overnight. The fixed tissue was embedded in paraffin and sliced into 5-µm sections for mounting onto slides (Histotec, Hayward, CA). Slides were warmed to 5658°C for 30 min and deparaffinized with Hemo-D clearing agent (Fisher Scientific, Pittsburgh, PA). Tissue was rehydrated using gradations of 100 to 70% ethanol. Slides were incubated at 40°C in 2 N HCl. Endogenous peroxidase activity was quenched with 3% H2O2 for 15 min. Nonspecific binding was blocked using a horse serum blocking solution (Vectastain ABC Elite kit; Vector, Burlingame, CA). Slides were then incubated with anti-BrdU monoclonal antibody (1:50 dilution in PBS; Becton-Dickinson, San Jose, CA) in a humid chamber at 4°C overnight, followed by a biotinylated secondary antibody (Vectastain ABC kit; Vector). Detection was performed using streptavidin (Vectastain ABC kit; Vector), and color was developed using 3,3'-diaminobenzidine tetrahydrochloride (Sigma) for 715 min (21). Cells were counterstained with Mayer's hematoxylin solution (Sigma), and slides were preserved with Permount (Fisher Scientific).
Chromogranin A Staining
Chromogranin A occurs in secretory granules of a wide variety of endocrine cells (27). The tissue sections were fixed, mounted on slides, and stained for chromogranin A (Histotec). Briefly, antigen was retrieved by heating in 10 mM citrate buffer, pH 6. The tissue section was stained with monoclonal mouse anti-human chromogranin A (1:100 dilution; DAKO), biotinylated secondary antibody, and streptavidin-horseradish peroxidase. Enteroendocrine cells appear brown on the background of hematoxylin and eosin staining.
Statistical Analysis
One-way ANOVA (statistical results were considered significant at P < 0.05) was followed by a Tukey's honestly significant difference test to determine differences among the treatment groups.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
There were no significant differences in the body weights of animals given BrdU in drinking water compared with control animals (Fig. 3). The amount of BrdU-labeled water consumed per day was 48 ± 12 ml per rat.
|
Body water 2H2O enrichments were measured serially from plasma obtained from rats killed at each time point. Body water enrichments reached a plateau level (3.94.3%) and were stable throughout the 2- to 8-wk study (Fig. 4).
|
There was <1% contamination from IELs in CECs that we isolated (n = 3, Fig. 5, AC): 0.10.3% by testing with anti-RT 6.2 antibody, 0.150.65% with anti-CD25 and anti-CD8a antibodies, and 0.250.79% from anti-pan-T antibody.
|
Histograms of CEC Nuclei from BrdU-Administered Rats
To determine the optimal administration route, we gave BrdU to separate groups of rats interperitoneally and per os (in drinking water) and compared their FACS histograms (Fig. 6). The CEC nuclei from repeatedly intraperitoneally injected rats (once per day for 8 days) resulted in more BrdU incorporation and thus higher fluorescence. However, intraperitoneally injected rats showed decreases in body weight, so we administered BrdU via the per os route.
|
Histograms of BrdU-Delabeled Nuclei from CECs
To determine the percentage of LRCs in the proliferative zone, we analyzed nuclei from CECs for BrdU. Figure 7A shows the FACS histograms of CEC nuclei from 2-, 4-, and 8-wk BrdU-delabeled rats. BrdU-positive nuclei were sorted and collected. Stringent criteria for selecting only the top 2040% of the brighter end of the BrdU-positive peaks were applied. We also reanalyzed the nuclei after they were collected and confirmed the absence of contamination of the collected BrdU-positive nuclei by BrdU-negative nuclei (Fig. 7B).
|
The percentage of BrdU-positive nuclei was determined during the BrdU delabeling period. On average, there were 77% (n = 9), 12% (n = 9), 7.0% (n = 9), and 3.8% (n = 4) of LRC after 0, 2, 4, and 8 wk of delabeling, respectively (Fig. 8). These fractions of LRCs are consistent with values reported by other researchers (1, 24, 25).
|
To measure the turnover rate of LRCs, nuclei from LRCs collected by FACS were further analyzed by GC-MS. Table 1 shows increases in 2H enrichments of nuclei from LRCs, reflecting LRC division during the 2H2O labeling period after BrdU intake was discontinued. The turnover rate of LRCs was between 0.33 and 0.90% per day. Assuming that the total number of cells in rat colon is 250 million (17) and 510% are LRCs, 0.10.25 million LRCs are newly produced per day. The t1/2 of LRCs was calculated to be between 77 and 210 days (Table 1). The apparent systematic lowering of replacement rate with prolonged duration of BrdU delabeling is discussed below.
|
To confirm the percent of LRCs by the 2H2O labeling method, we studied rats that had been labeled in utero. The 2H enrichments of the fully deuterated and the delabeled CECs were compared, with the 2H enrichments of CEC from animals labeled in utero considered to represent the maximum 2H enrichment possible. EM1 values were 10.5 ± 0.2 and 1.0 ± 0.18%, respectively, for fully labeled and 2-wk delabeled animals, or 9.9% LRCs (Fig. 9). This value is similar to the percentage of LRCs estimated from 2 wk of BrdU delabeling (Fig. 8).
|
We determined the location of LRC by BrdU immunohistochemistry on 2-wk BrdU-delabeled crypts (Fig. 10). Most of the CECs were fully BrdU labeled after 2-wk BrdU administration (Fig. 10A), whereas after 2-wk delabeling we could identify cells that retained BrdU and were located near and at the base of the crypt (representative pictures shown in Fig. 10B).
|
Enteroendocrine cells are a minor population of the total epithelial cells (27) but are believed to have a longer life span (35100 days) (8) than CECs with fast turnover. Accordingly, we investigated the presence of enteroendocrine cells in our samples by using chromogranin A staining (representative pictures shown in Fig. 11). Only 3 of 30 crypts had 11.5% of chromogranin A-positive enteroendocrine cells present in the base regions of the crypt. The rest of the crypts showed no chromogranin A-positive cells at the base regions of the crypt from which we isolated BrdU-retaining cells and where LRCs are known to reside and were identified in our study. Most of the positive chromogranin A staining was found in the upper parts of the crypt, which had a higher labeling index of 2.79.8% [from 9,250 cells counted (n = 4)]. Thus we could effectively rule out significant contamination by this type of long-lived cell in the LRCs that we collected from the basal (proliferative) zone of the colon crypt.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results from the FACS histograms confirmed the effective delivery of BrdU from drinking water to the colonocytes (Fig. 6A). Approximately 90% of the CEC nuclei from the proliferative zone were BrdU positive after 2 wk of BrdU administration. Interestingly, the percentage of BrdU-positive nuclei after 4 and 8 wk of BrdU administration decreased to 6577%. This reduction may be due to the induction of liver enzymes that dehalogenate BrdU as part of the adaptation to its extended administration (11), making BrdU less available to be incorporated into nuclear DNA.
The presence of BrdU-positive CEC nuclei after prolonged delabeling supports the existence of LRCs in colon crypts (Fig. 7A). The percentage of LRC after a delabeling period of 28 wk (Fig. 8) was in the range of values previously reported by others (24, 25).
Kinetic results confirmed the slow turnover rate of these putative stem cells (0.330.90% per day). Compared with the 17% replacement rate per day of CECs with fast turnover (23), LRCs have a >30 times slower turnover rate (Table 1).
Some technical issues deserve comment. Stable body water enrichments were maintained throughout the study (4.2 ± 0.2% of 2H2O in total body water, Fig. 4). Achievement of plateau 2H enrichments in body water during long-term 2H2O administration is important because it allows a stable precursor enrichment throughout the DNA-labeling period in slowly proliferating cells (23).
IELs were effectively removed from CEC preparation from rat colon, as confirmed by assays using rat IEL-specific antibodies (Fig. 5). Thus our results are representative of CECs free from IEL contamination.
The procedure for isolating LRCs from rat colon was labor intensive, because LRCs are rare (3.812%) and because we collected only the upper 2040% fraction of the BrdU-positive peak. Furthermore, CECs tend to aggregate, so we processed nuclei by FACS relatively slowly (at a data processing rate of 400500 events/s) compared with blood cells (5,000 events/s). Therefore, collecting 300,000500,000 nuclei, the minimum number of nuclei needed at the time for each GC-MS analysis, took 412 h. Recently, however, technical improvements in sensitivity of GC-MS measurement of dR have reduced the cell number required for a mass spectrometric signal by >2 orders of magnitude (Awada M, Neese RA, and Hellerstein MK, unpublished observations). Future isolation protocols for LRCs may therefore not require nuclei extraction. The nuclei extraction procedure removes cytoplasm and, hence, opportunities for further molecular, cellular, and biochemical characterization of the LRCs. In future experiments, technical improvements in GC-MS analyses may allow isolation of intact BrdU-retaining cells and analysis of their surface markers. It should be noted that cells can be stained for BrdU label and then collected and further analyzed for deuterium enrichments in their DNA. Although the current version of this method did not allow for the characterization of LRC nuclei for biochemical markers of "stemness," future iterations of this approach may also involve measurements on nuclei (e.g., expression or activity of transcription factors; DNA oxidation or other genetic damage). In any case, the results and technique shown here represent a successful proof of principle of this kinetic approach for isolating LRCs.
The BrdU concentration used (1 mg/ml) in drinking water in this study is commonly used for long-term administration (11, 30) and did not cause any detectable toxicity or weight loss. It has been reported that 0.8 mg/ml of BrdU-containing drinking water administered for up to 5 wk in B6 and BALB/C mice causes no thymic toxicity (11), and 1 mg/ml of BrdU-containing drinking water given in Lewis rats caused no apparent toxic effects on various tissues with fast turnover when given for up to 12 wk (30). There have been reports of inhibitory effects of BrdU on cell proliferation and differentiation in vitro and in vivo (9, 26) at various concentrations and in various cell types. In the current study, however, the LRCs continued to divide during the delabeling period, based on the measured 2H incorporation into DNA of BrdU-positive nuclei (Table 1). Moreover, the BrdU-treated rats showed the same 2H2O incorporation and proliferation rates in the bulk CECs, with fast turnover, as in the control rats in our study (data not shown). We conclude that any toxic effect of BrdU given at this dose on proliferation of CECs and LRCs is minimal.
In summary, nuclei from LRCs (putative stem cells) can now be physically isolated and their turnover rates routinely measured by application of a combined BrdU-FACS isolation/stable isotope-mass spectroscopic method. Many applications of this technique can be envisioned. For example, alterations in stem cell proliferation may be responsible for the abnormal phenotype seen in mice early in the pathogenesis of the adenomatous polyposis coli gene mutation (3, 33). The information obtained by this approach may be further applied to deciphering the roles of stem cells in settings relevant to epithelial cell differentiation and carcinogenesis.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bickenbach JR. Identification and behavior of label-retaining cells in oral mucosa and skin. J Dent Res 60, Spec No C: 16111620, 1981.[ISI][Medline]
3. Boman BM, Fields JZ, Bonham-Carter O, and Runquist OA. Computer modeling implicates stem cell overproduction in colon cancer initiation. Cancer Res 61: 84088411, 2001.
4. Brasitus TA. Isolation of proliferative epithelial cells from the rat cecum and proximal colon. Anal Biochem 123: 364372, 1982.[ISI][Medline]
5. Brasitus TA and Keresztes RS. Glycoprotein metabolism in rat colonic epithelial cell populations with different proliferative activities. Differentiation 24: 239244, 1983.[ISI][Medline]
6. Coles MC, and Raulet DH. NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J Immunol 164: 24122418, 2000.
7. Cotsarelis G, Sun TT, and Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61: 13291337, 1990.[ISI][Medline]
8. Deschner EE and Lipkin M. An autoradiographic study of the renewal of argentaffin cells in human rectal mucosa. Exp Cell Res 43: 661665, 1966.[ISI][Medline]
9. Goz B. The effects of incorporation of 5-halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol Rev 29: 249272, 1978.[ISI]
10. Hasbold J and Hodgkin PD. Flow cytometric cell division tracking using nuclei. Cytometry 40: 230237, 2000.[CrossRef][ISI][Medline]
11. Jecker P, Beuleke A, Dressendorfer I, Pabst R, and Westermann J. Long-term oral application of 5-bromo-2-deoxyuridine does not reliably label proliferating immune cells in the LEW rat. J Histochem Cytochem 45: 393401, 1997.
12. Kim J, Neese RA, Isnard P, McCune M, and Hellerstein MK. A method to isolate long-term label retaining cells (putative stem cells) from rat colon of rats (Abstract). Proc Am Assoc Cancer Res, 2002, p. 127.
13. Kim KM and Shibata D. Methylation reveals a niche: stem cell succession in human colon crypts. Oncogene 21: 54415449, 2002.[CrossRef][ISI][Medline]
14. Lipkin M. Growth and development of gastrointestinal cells. Annu Rev Physiol 47: 175197, 1985.[CrossRef][ISI][Medline]
15. Lipkin M, Bell B, and Sherlock P. Cell proliferation kinetics in the gastrointestinal tract of man. I. Cell renewal in colon and rectum. J Clin Invest 42: 767776, 1963.[ISI]
16. Macallan DC, Fullerton CA, Neese RA, Haddock K, Park SS, and Hellerstein MK. Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans. Proc Natl Acad Sci USA 95: 708713, 1998.
17. Maskens AP and Dujardin-Loits RM. Kinetics of tissue proliferation in colorectal mucosa during post-natal growth. Cell Tissue Kinet 14: 467477, 1981.[ISI][Medline]
18. Morris RJ. Keratinocyte stem cells: targets for cutaneous carcinogens. J Clin Invest 106: 38, 2000.
19. Morris RJ, Coulter K, Tryson K, and Steinberg SR. Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling. Cancer Res 57: 34363443, 1997.[Abstract]
20. Morris RJ, Fischer SM, and Slaga TJ. Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen. Cancer Res 46: 30613066, 1986.[Abstract]
21. Murrill WB, Brown NM, Zhang JX, Manzolillo PA, Barnes S, and Lamartiniere CA. Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 17: 14511457, 1996.[Abstract]
22. Neese RA, Misell LM, Turner S, Chu A, Kim J, Cesar D, Hoh R, Antelo F, Strawford A, McCune JM, Christiansen M, and Hellerstein MK. Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci USA 99: 1534515350, 2002.
23. Neese RA, Siler SQ, Cesar D, Antelo F, Lee D, Misell L, Patel K, Tehrani S, Shah P, and Hellerstein MK. Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation. Anal Biochem 298: 189195, 2001.[CrossRef][ISI][Medline]
24. Potten CS, Booth C, and Pritchard DM. The intestinal epithelial stem cell: the mucosal governor. Int J Exp Pathol 78: 219243, 1997.[ISI][Medline]
25. Pozharisski KM, Klimashevski VF, and Gushchin VA. Study of kinetics of epithelial cell populations in normal tissues of the rat's intestines and in carcinogenesis. I. A comparison of enterocyte population kinetics in different segments of the small intestine and colon. Exp Pathol (Jena) 18: 387406, 1980.[Medline]
26. Rocha B, Penit C, Baron C, Vasseur F, Dautigny N, and Freitas AA. Accumulation of bromodeoxyuridine-labeled cells in central and peripheral lymphoid organs: minimal estimates of production and turnover rates of mature lymphocytes. Eur J Immunol 20: 16971708, 1990.[ISI][Medline]
27. Roth KA and Gordon JI. Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice. Proc Natl Acad Sci USA 87: 64086412, 1990.[Abstract]
28. Sunter JP, Wright NA, and Appleton DR. Cell population kinetics in the epithelium of the colon of the male rat. Virchows Arch B Cell Pathol 26: 275287, 1978.[ISI]
29. Todd D, Singh AJ, Greiner DL, Mordes JP, Rossini AA, and Bortell R. A new isolation method for rat intraepithelial lymphocytes. J Immunol Methods 224: 111127, 1999.[CrossRef][ISI][Medline]
30. Tough DF and Sprent J. Turnover of naive- and memory-phenotype T cells. J Exp Med 179: 11271135, 1994.[Abstract]
31. Wong WM and Wright NA. Cell proliferation in gastrointestinal mucosa. J Clin Pathol 52: 321333, 1999.[Abstract]
32. Yatabe Y, Tavare S, and Shibata D. Investigating stem cells in human colon by using methylation patterns. Proc Natl Acad Sci USA 98: 1083910844, 2001.
33. Zhang T, Otevrel T, Gao Z, Ehrlich SM, Fields JZ, and Boman BM. Evidence that APC regulates survivin expression: a possible mechanism contributing to the stem cell origin of colon cancer. Cancer Res 61: 86648667, 2001.