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First published online November 29, 2007
Stem Cells Vol. 26 No. 3 March 2008, pp. 630 -637
doi:10.1634/stemcells.2007-0621; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Identification of a Novel Putative Gastrointestinal Stem Cell and Adenoma Stem Cell Marker, Doublecortin and CaM Kinase-Like-1, Following Radiation Injury and in Adenomatous Polyposis Coli/Multiple Intestinal Neoplasia Mice

Randal Maya, Terrence E. Riehlb, Clayton Huntc, Sripathi M. Surebana, Shrikant Ananta,d, Courtney W. Houchena

Departments of aMedicine and
dCell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA;
bDepartment of Internal Medicine, Division of Gastroenterology, and
cDepartment of Radiation Oncology, Radiation and Cancer Biology Division, Washington University School of Medicine, St. Louis, Missouri, USA

Key Words. Stem cell marker • Doublecortin and CaM kinase-like-1 • Adenoma stem cell marker • Gamma irradiation • Gastrointestinal cancer • Adenomatous polyposis coli/multiple intestinal neoplasia mice

Correspondence: Correspondence: Courtney W. Houchen, M.D., Department of Medicine, University of Oklahoma Health Sciences Center, 920 Stanton L. Young Boulevard, WP 1360, Oklahoma City, Oklahoma 73104, USA. Telephone: 405-271-2175; Fax: 405-271-5450; e-mail: Courtney-houchen{at}ouhsc.edu

Received on July 31, 2007; accepted for publication on November 19, 2007.

First published online in STEM CELLS EXPRESS  November 29, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase-like-1 (DCAMKL-1) to examine radiation-induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL-1 expression. Immunoreactive DCAMKL-1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL-1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL-1-positive cells. We also observed stem cell apoptosis and mitotic DCAMKL-1-expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL-1-expressing cells were negative for proliferating cell nuclear antigen and nuclear β-catenin in normal-appearing intestine. However, β-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of β-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Colorectal cancer is a major cause of cancer death in the Western world. Mutational activation of oncogenes joins with inactivation of tumor-suppressor genes to produce colorectal tumors [1]. The transformation of normal mucosal epithelial cells into invasive colorectal carcinoma occurs via a synchronized accumulation of mutations in a series of critical genes [2]. The long time span between initiation and gross development of tumors presents an enormous challenge in dissecting the critical molecular mechanisms that regulate neoplastic change.

Defining the mechanisms that regulate stem cell fate is critical in increasing our understanding of the neoplastic process. Tumorigenesis in the gut is thought to arise specifically in the stem cell [3, 4] population located at or near the base of the intestinal and colonic crypts. Transit cell populations originating from the stem cell zone become fully differentiated and are eventually sloughed into the lumen. Transit cells' short life span, whether they are mutated or not, limits their deleterious influence in the intestinal or colonic crypt [5]. Because no specific gut stem cell markers have been identified definitively [6, 7], recognizing and assaying resident intestinal stem cells is quite difficult and has raised contentious argument; however, the microcolony assay following {gamma}-irradiation (ionizing radiation [IR]) is by definition a functional evaluation of intestinal stem cell fate [8] and can potentially provide a mechanism for examining the early events of tumorigenesis. Because homeostatic mechanisms of stem cell proliferation are the same processes that become dysregulated in carcinogenesis [9], a complete examination of these proliferation mechanisms holds medical significance in targeting future cancer treatments; therefore, a more detailed understanding of the pathways that regulate stem cell behavior is essential.

As we work toward a complete understanding of these pathways that regulate stem cell behavior, one major obstacle in the study of gastrointestinal stem cell biology has been the lack of definitive markers to identify gastrointestinal stem cells. We have now confirmed that doublecortin and CaM kinase-like-1 (DCAMKL-1), a microtubule-associated kinase expressed in postmitotic neurons [10], is a putative intestinal stem cell marker. This discovery allows us to assay resident intestinal stem cells and their response to genotoxic injury. DCAMKL-1 was identified as a Gene Ontogeny-enriched transcript expressed in comparison with gastric epithelial progenitor and whole stomach libraries [11]. Immunohistochemical analysis using antibodies directed at DCAMKL-1 revealed single-cell staining in scattered intestinal crypt cross-sections at or near position 4 and in gastric isthmus cells in the putative stem cell location. We chose the radiation-injury model to investigate its effects on stem cell fate for several reasons: (a) the kinetics of radiation injury has been extensively characterized in the small intestine in mice [12, 13], (b) radiation injury can be induced uniformly throughout the gut at discrete points in time, and (c) the extent of radiation injury on crypt clonogenic survival can be varied with the dosage of radiation. In this study, we used immunohistochemical analysis to visualize crypt epithelial stem cells and to determine the cell-specific DCAMKL-1 expression at baseline and in response to radiation injury in adult mice.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Bright-Field Immunohistochemistry
Heat-induced epitope retrieval (HIER) was performed on 4-mm paraffin-embedded mouse small intestine and colon sections using a pressurized decloaking chamber (Biocare Medical, Concord, CA, http://www.biocare.net), and sections were incubated in citrate buffer (pH 6.0) at 99°C for 18 minutes. The sections were then washed three times with phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and endogenous biotin activity was blocked using the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and/or with DCAMKL-1 blocking peptide (Abgent, San Diego, http://www.abgent.com) wherever indicated, according to the manufacturer's instructions. Furthermore, endogenous peroxidase activity was quenched with 3% hydrogen peroxide. After washing, the slides were then incubated in horse normal serum (2%) and bovine serum albumin (BSA) (1%) at room temperature for 20 minutes to block nonspecific binding. The sections were then exposed to primary antibodies rabbit anti-DCAMKL-1 (Abgent), rabbit anti-Musashi-1 (Abcam, Cambridge, MA, http://www.abcam.com), rabbit proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), goat β-catenin (Santa Cruz Biotechnology), and rabbit anti-phospho-H2AX (Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) overnight at refrigerator temperature. Slides were then washed three times with PBS and incubated in the appropriate secondary antibody biotinylated donkey anti-rabbit and donkey anti-goat (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) for 30 minutes at room temperature. Slides were washed again and then incubated in streptavidin-horseradish peroxidase (Dako, Glostrup, Denmark, http://www.dako.com) at room temperature for 12 minutes. After final wash in PBS, chromogenic development was performed using 3,3'-Diaminobenzidine tetrahydrochloride (brown) and/or 3-amino, 9-ethyl cabazole (red) substrate (Sigma-Aldrich). All slides were counterstained with hematoxylin (Biocare Medical), dehydrated in graded alcohols, cleared in xylene, and permanently mounted with CryoSeal (Richard-Allan Scientific, Kalamazoo, MI, http://www.rallansci.com).

Fluorescence Immunohistochemistry
HIER was performed on 4-mm paraffin-embedded tissue sections using a pressurized decloaking chamber (Biocare Medical), and sections were incubated in citrate buffer (pH 6.0) at 99°C for 18 minutes. After washing three times with PBS, the slides were then incubated in horse normal serum (2%) and BSA (1%) at room temperature for 20 minutes to block nonspecific binding. Sections were then sequentially exposed to rabbit anti-DCAMKL-1 (Abgent) for 1 hour at 30°C and its appropriate secondary Cy3-conjugated donkey anti-rabbit (Jackson Immunoresearch Laboratories) for 30 minutes at room temperature. Finally, fluorescein-conjugated terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed using the In Situ Cell Death Kit (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's instructions. The slides were then wet-mounted and counterstained using Vectashield with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). For costaining of DCAMKL-1 with Musashi-1, the slides were incubated with normal goat serum after decloaking and exposed to rabbit anti-DCAMKL-1 (Abgent) for 1 hour at 30°C and its appropriate secondary goat anti-rabbit Alexa Fluor 568 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 30 minutes at room temperature. Furthermore, the slides were blocked with normal goat and normal donkey serum and exposed to rabbit anti-Musashi-1 (Abcam) for 1 hour at 30°C and its appropriate secondary donkey anti-rabbit Alexa Fluor 488 (Invitrogen) for 30 minutes at room temperature. Then, the slides were washed with Hoechst 33342 for staining of the nucleus.

Microscopic Examination Immunohistochemistry
Slides were examined using a Nikon 80i microscope base. For bright-field imaging, x60 digital images were taken with a PlanAPO objective and DXM1200C camera (Nikon, Tokyo, http://www.nikon.com). Fluorescent images were taken with a x60 PlanFluoro objective and x2 optical converter for a final magnification of x120, using a CoolSnap ES2 camera (Photometrics, Tucson, AZ, http://www.photomet.com). Filter sets with excitation ranges for Cy3, fluorescein isothiocyanate, and DAPI were used. All images were captured using NIS-Elements software (Nikon) and further processed using Adobe Photoshop 8.0 software.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Localization of DCAMKL-1, a Putative Intestinal Stem Cell Marker
In wild-type (WT) adult mouse intestine (Fig. 1A), we confirmed that immunoreactive DCAMKL-1 is expressed primarily in single cells in the putative stem cell zone in adult, conventionally housed C57 Bl/6 mice. In rare sections, villus staining was observed, particularly at the crypt villus junction (data not shown). Distinct cytoplasmic staining was observed at baseline, whereas DCAMKL-1 expression was a rare event. Staining was present in approximately one in six intestinal crypt cross-sections, on average. Immunostaining of the proposed columnar longitudinal epithelial cell interspersed between Paneth cells was also observed. These columnar longitudinal epithelial cells have previously been shown to be positive for the putative stem cell marker musashi-1 (MSI-1) [7, 14]. Preincubation with DCAMKL-1 blocking peptide (Abcam) completely abolished DCAMKL-1 immunoreactivity (Fig. 1B).


Figure 1
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  		Figure 1. Expression pattern of doublecortin and CaM kinase-like-1 (DCAMKL-1) in normal mouse small intestine. (A): Immunohistochemical staining of normal small intestine for DCAMKL-1. Arrows indicate the cells positive for DCAMKL-1 in the stem cell zone. (B): Preincubation with blocking peptide completely abolished DCAMKL-1 immunoreactivity. (C): Immunohistochemical staining of normal small intestine for DCAMKL-1. Brown indicates the cells positive for DCAMKL-1 (indicated by the arrows).

 
Colocalization of DCAMKL-1 and MSI-1
To determine whether DCAMKL-1 was expressed in the same cells that expressed the putative stem cell marker MSI-1, we performed immunostaining for MSI-1 using the intestines of adult WT uninjured mice. In the experiment shown in Figure 2A, we again observed distinct DCAMKL-1 staining in the crypt. In the experiment shown in Figure 2B, we observed crypt epithelial staining for MSI-1 in several cells at the base of the crypt, including cell position 4, just above the Paneth cell zone, consistent with its reported stem cell localization. Furthermore, we used immunofluorescence microscopy and double immunostaining for DCAMKL-1 and MSI-1. We again observed single-cell staining for DCAMKL-1 in the stem cell zone (Fig. 2C). We also observed MSI-1 staining in the crypts (Fig. 2D). We observed distinct colocalization, however, with DCAMKL-1 and MSI-1 (Fig. 2H, orange). These data suggest that DCAMKL-1 is expressed in the same cell as MSI-1 but likely represents a subset of MSI-1-expressing cells. Nuclei stained with Hoechst 33342 (blue) are shown in Figure 2F and 2G.


Figure 2
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  		Figure 2. Colocalization of Musashi-1 and doublecortin and CaM kinase-like-1 (DCAMKL-1) in mouse intestine. (A): Immunohistochemical staining of normal small intestine for DCAMKL-1 (brown, indicated by the arrow). (B): Immunohistochemical staining of normal small intestine for Musashi-1. Brown indicates the cells positive for Musashi-1 at the base of the crypts. (C): The cell positive for DCAMKL-1, stained red (indicated by the arrow), appears at the base of the crypt. (D): Intestinal section stained for Musashi-1 (green). (E): Colocalization of DCAMKL-1 and Musashi-1 (yellow, indicated by the arrow). The magnified inset image represents the single cell positive for both DCAMKL-1 and Musashi-1. (F): Costaining of DCMAKL-1 (red, indicated by the arrow) with nuclear Hoechst 33342 (blue) staining. (G): Costaining of Musashi-1 (green) with nuclear Hoechst 33342 (blue) staining. (H): Colocalization of DCAMKL-1 and Musashi-1 (yellow, indicated by the arrow), costained with nuclear Hoechst 33342 (blue) staining. The magnified inset image represents the single cell positive for both DCAMKL-1 and Musashi-1 (yellow).

 
Fate of DCAMKL-1-Positive Cells in Response to Radiation Injury
To investigate whether DCAMKl-1 expression was upregulated following IR, we treated adult mice with whole-body IR (6 Gy), at doses sufficient to induce epithelial apoptosis in the stem cell zone [15–17]. Initially, we chose the time point of 6 hours after 6 Gy of IR as this is the time when maximal p53-dependent apoptosis is observed in the intestinal crypt [16]. Here, we demonstrated DCAMKL-1 staining similar to that observed at baseline (Fig. 3A). Following 6 Gy of IR, we observed morphologically appearing apoptotic cells in the lower one-third of the intestinal crypt with a typical distribution following IR (Fig. 3A, arrow). Surprisingly, we did not observe apoptosis in DCAMKL-1-positive cells within the crypt in more than 100 crypt cross-sections counted. To confirm this finding, we performed a similar experiment and stained for DCAMKL-1 and TUNEL (a marker for apoptosis). Apoptotic cells within the crypt were identified by TUNEL staining (green), and DCAMKL-1 staining (red) at single cell positions in the intestinal crypt was again observed (Fig. 3B). There was no evidence of apoptosis in DCAMKL-1-expressing cells. Furthermore, radiation-induced DNA damage was observed in the crypt at 6 hours following IR, evidenced by the presence of phospho-H2AX-positive cells (Fig. 3D, magnified in inset), which was not observed in unirradiated mice (Fig. 3C, magnified in inset). The DCAMKL-1-positive cell was also positive for nuclear phospho-H2AX but did not undergo apoptosis at that time (Fig. 3E, magnified in inset). Indeed, this was not completely unexpected, as earlier reports suggest that two important waves of apoptosis exist following IR. The first wave occurs at 4.5–6 hours (p53-dependent), and the second is near 24 hours (p53-independent). The second wave of apoptosis is thought to affect stem cells primarily [16, 17]. To investigate this further, we examined animals 24 hours after IR and performed immunohistochemical analysis for DCAMKL-1 (Fig. 3F). In the experiment shown in Figure 3F, we demonstrated morphological evidence of apoptosis and immunoreactive DCAMKL-1 staining in the stem cell zone; however, at that time point, there was clear evidence of apoptosis in the DCAMKL-1-positive cell (arrow). In addition, we noted the appearance of mitotic figures, suggesting the release of these cells from radiation-induced cell cycle arrest (Fig. 3F, M). The mitotic figures were often DCAMKL-1 immunoreactive, but this staining pattern was not observed in all of the mitotic figures present throughout the intestine. It should be noted that many of the cells with morphologic features consistent with mitosis were on occasion immediately adjacent to cells in the process of apoptosis, and these exhibited striking expression of DCAMKL-1. Consequently, these data suggest that by 24 hours after low-dose IR (6 Gy), a few (one per cross-section) stem cell/progenitor cells are removed by apoptosis, and the potential descendants of these cells are able to divide and, at least transiently, express DCAMKL-1.


Figure 3
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  		Figure 3. Fate of doublecortin and CaM kinase-like-1 (DCAMKL-1)-positive cell following ionizing radiation (IR). (A): Six hours after whole-body IR (6 Gy), we observed morphologically appearing apoptotic cells in the lower one-third of the intestinal crypt, but we did not observe apoptosis in any of the DCAMKL-1-positive cells indicated by the arrow. (B): The small intestine stained for DCAMKL-1 (red) and by terminal deoxynucleotidyl transferase dUTP nick-end labeling (green) to demonstrate apoptosis in the crypts 6 hours following radiation. (C): Small intestine of unirradiated mice demonstrating no staining for phospho-H2AX. The crypt area is magnified in the inset. (D): Six hours post-IR; small intestine demonstrates DNA damage by positive phospho-H2AX staining (3,3'-Diaminobenzidine tetrahydrochloride brown). The crypt area is magnified in the inset. (E): Six hours post-IR; small intestine demonstrated DNA damage in the DCAMKL-1-positive cell indicated by the arrow. The magnified inset image represents the single cell positive for both DCAMKL-1 and phospho-H2AX. (F): After 24 hours after IR, we noted the appearance of multiple DCAMKL-1 immunoreactive M adjacent to morphologically appearing apoptotic cells, indicated by arrow, that were also expressing DCAMKL-1. Abbreviation: M, mitotic figures.

 
Expression Pattern of DCAMKL-1 in Regenerative Crypts
To determine whether or not DCAMKL-1 is expressed in regenerative crypts following radiation injury, we exposed adult mice to a lethal dose (12 Gy) of IR and examined DCAMKL-1 expression in regenerative crypt epithelial cells. We chose 12 Gy since this dosage has been demonstrated to induce crypt stem cell sterilization in a majority of intestinal crypts [18]. Regenerative crypts appear 3.5 days following radiation injury and represent the survival of at least one progenitor/stem cell per crypt. DCAMKL-1 staining was not observed in regenerative crypts following 12 Gy (Fig. 4A). These data suggest that DCAMKL-1 is not expressed at the protein level during the period of crypt regeneration when proliferation is at its peak. These data are consistent with the original report [11] and with our findings failing to demonstrate DCAMKL-1 staining in 5-bromo-2'-deoxyuridine (BrdUrd)-positive cells (data not shown). On the other hand, it is unclear why this marker is not expressed, as not every cell in the regenerative crypt is in a proliferative state. This may represent some form of loss of niche signaling in 3.5-day-postirradiation crypts lacking an intact crypt/villus axis or functional mesenchymal cells. Although it is interesting to speculate, more studies directed toward defining the regulatory mechanisms that control expression of DCAMKL-1 are required. Restoration of DCAMKL-1 expression within the crypts, however, was observed 7 days postirradiation, when the morphologic features of the crypts/villus axis were returning to baseline (Fig. 4B), yet the crypts appeared elongated, with heaping up of nuclei. In several cross-sections, DCAMKL-1-positive cells were not necessarily restricted to lower crypt region.


Figure 4
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  		Figure 4. Doublecortin and CaM kinase-like-1 (DCAMKL-1) expression in the regenerative crypts post-ionizing radiation (post-IR). (A): Eighty-four hours following IR, no DCAMKL-1 expression could be detected in regenerative crypts. (B): Staining at 144 hours after IR demonstrated restoration of DCAMKL-1 expression in the intestinal crypt indicated by arrows.

 
DCAMKL-1 as a Putative Adenoma Stem Cell Marker
To determine whether DCAMKL-1 could be used to label putative stem cells within tumors, we used immunohistochemical analysis to identify DCAMKL-1 in the intestines of adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice. These mice have a germline mutation in the APC gene and develop numerous intestinal polyps [19, 20]. APC mutations are among the earliest genetic alterations in epithelial tumor progression [21]. Indeed, more than 60% of human colorectal adenomas exhibit a mutation in APC [22]. In WT mice, we observed classic single-cell staining in scattered crypt epithelial cells. However, in APC/min mice, we observed a slightly different expression pattern compared with WT. Although we observed occasional single-cell staining in the crypts as before, there was a trend toward increased DCAMKL-1 expression on the villi (Fig. 5A, 5B) compared with WT mice. This was often particularly evident in villus epithelial tissues adjacent to or surrounding adenomas (Fig. 5B). Note the distinct cytoplasmic staining pattern in the villus epithelium (Fig. 5A, arrowhead). It is unclear whether this is a function of villus expression of stem cells or a loss of crypt niche restriction in DCAMKL-1-expressing cells. It should be noted that we occasionally observed villus epithelial DCAMKL-1 expression in WT mice as well. Additional studies following isolation of these cells are required to fully determine the functional significance of these villus DCAMKL-1-staining cells.


Figure 5
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  		Figure 5. Histological evaluation of small intestine of adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice. (A): Scattered single cells were immunoreactive for doublecortin and CaM kinase-like-1 (DCAMKL-1) in the intestinal crypts (arrows) and showed a trend toward increased expression on villi (arrowhead). (B): DCAMKL-1 staining within adenomas of APC/min mice, indicated by the arrows. DCAMKL-1 was also immunoreactive in the cells within the villus epithelium surrounding the adenoma (arrowheads). (C): APC/min intestinal adenoma immunostained with anti-proliferating cell nuclear antigen (anti-PCNA) (red) and costained with anti-DCAMKL-1 (brown). The cells immunoreactive for DCAMKL-1 are indicated by the arrows. (D): Portion of (A) magnified to demonstrate that the cell positive for DCAMKL-1 was not immunoreactive for PCNA. (E): Double staining of PCNA and DCAMKL-1 in putative stem cell zone of wild-type mouse demonstrated the quiescent state of the DCAMKL-1 expression cell indicated by the arrow.

 
DCAMKL-1-Positive Cells in Adenomas Are Quiescent
The potential stem cell origin of neoplastic tissues has become increasingly recognized [4, 17]. Accordingly, changes in the regulation of stem cells could potentially alter the risk of tumorigenesis. We used immunohistochemical analysis to assess DCAMKL-1 expression patterns in APC/min adenomas. We observed distinct staining (Fig. 5B) in a minority of cells within the adenoma. Given the limited expression pattern of DCAMKL-1 in adenomas, we wanted to determine whether DCAMKL-1 was expressed in proliferative cells within adenomas. We used double staining protocols for both DCAMKL-1 and PCNA in APC/min mice. As expected, the majority of the adenomas expressed the proliferation marker PCNA. Indeed, there were very few cells within the adenoma that did not express PCNA. As PCNA staining is primarily nuclear, we predicted that the cytoplasmic DCAMKL-1 would be identified in proliferating cells if costaining was present. DCAMKL-1 was expressed in cells within the adenoma that were not proliferating and therefore quiescent (Fig. 5C; magnified in Fig. 5D). This was confirmed in normal crypt epithelial cells in which DCAMKL-1-positive cells were negative for PCNA (Fig. 5E). This is consistent with the original report in FVB/N mice, where DCAMKL-1 cells were negative for BrdUrd [11]; however, this finding within adenomas has not previously been described.

Coexpression of β-Catenin and DCAMKL-1 in APC/min Tumors
To determine whether nuclear localization of β-catenin could be observed in DCAMKL-1-expressing cells, we sought to identify β-catenin in quiescent cells within adenomas. β-Catenin translocation to the nucleus is one of the earliest steps in neoplastic transformation and is readily observed in adenomas of APC/min mice. In the experiment shown in Figure 6, β-catenin and DCAMKL-1 coimmunostaining was demonstrated in normal-appearing intestinal crypts in APC/min mice and within a crypt adenoma. In normal-appearing crypts, DCAMKL-1 immunoreactive cells exhibited typical membrane β-catenin staining, without any evidence of nuclear translocation (Fig. 6A; magnified in Fig. 6B); however, within the adenoma, nuclear β-catenin was readily identified in the DCAMKL-1-expressing cell (Fig. 6C, arrow; magnified in Fig. 6D). These data, taken together, strongly suggest that the normal epithelial intestinal stem cell and the adenoma stem cell can be distinguished based on nuclear β-catenin and DCAMKL-1 immunostaining. Furthermore, the adenoma stem cell can be distinguished from the proliferative adenoma cells based on PCNA and DCAMKL-1 immunostaining.


Figure 6
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  		Figure 6. β-Catenin expression in the small intestine of adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice localized with doublecortin and CaM kinase-like-1 (DCAMKL-1). (A): Normal-appearing APC/min mouse intestine immunostained for membrane β-catenin (brown) and cytoplasmic DCAMKL-1 (red); coimmunostaining is indicated by arrow. (B): Magnified image of (A) showing the cell positive for DCAMKL-1 and β-catenin, indicated by arrow. (C): DCAMKL-1-expressing cell (arrow), along with other cells demonstrating nuclear translocation of β-catenin, within an APC/min adenoma, indicated by the arrow, just adjacent to normal membrane β-catenin staining epithelium. (D): Magnified image of (C), showing the DCAMKL-1-positive cell, indicated by the arrow.

 
Morphology of DCAMKL-1-Expressing Cells
Upon closer observation, the unique morphologic appearance of the DCAMKL-1-expressing cell resembles that of neural processes observed on gastric D cells [23] (Fig. 7A–7D). In the experiment shown in Figure 7A, we observed DCAMKL-1 expression in cells in the mid-crypt in the proximal colon. In the experiment shown in Figure 7B, we observed an expression at the crypt base in the distal colon. In addition, higher-power views in both colon (Fig. 7C) and distal jejunum (Fig. 7D) clearly illustrate the unique morphologic staining pattern resembling that of axonal-like processes.


Figure 7
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  		Figure 7. Colonic distribution of doublecortin and CaM kinase-like-1 (DCAMKL-1) and structure of cell positive for DCAMKL-1. (A): The cell positive for DCAMKL-1 appeared at the midpoint of the colonic crypt in the proximal colon. (B): In distal colon, the distribution of DCAMKL-1 expression appeared at the base of the colonic crypt. (C, D): Close views of DCAMKL-1-expressing cells within the colon (C) and distal jejunum (D) demonstrate the axonal-like process.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
We usually observed one crypt with definitive DCAMKL-1 staining per six crypts in a typical intestinal cross-section near cell position 4 in the crypt. Presumably, this is due to the three dimensional nature of the crypt and the low probability that every cross-section will contain a stem cell. Nevertheless, we consistently observed DCAMKL-1 immunoreactivity in the stem cell zone, as previously noted (Fig. 1). The response to acute radiation injury is the most extensively characterized model system for studying injury repair in the rodent gastrointestinal tract. The actively proliferating cell population in the intestinal crypt rapidly undergoes apoptotic cell death following sublethal doses of IR (<8 Gy) [24]. Because epithelial cells at the lower one-third of the small intestinal crypts are the first to undergo apoptosis following low-dose IR (1 Gy), it is postulated that these "true" or "ultimate" stem cells prefer to undergo apoptosis rather than repair even comparatively minor damage to their DNA [25]. This trait may serve to reduce the risk of propagating a mutated clone within the crypt. If all the so-called ultimate stem cells [25] are destroyed, then their more radio-resistant daughter cells will assume stem cell functions and maintain the crypt; however, the molecular mechanisms that regulate this transfer of clonogenic capacity are poorly understood. In the present study, we demonstrated that cells positive for DCAMKL-1 underwent DNA damage along with other cells in the crypt but did not undergo apoptosis, whereas 24 hours following IR, the putative stem cell or cells positive for DCAMKL-1 did undergo apoptosis. Following 12 Gy of IR, the DCAMKL-1 reactivity was lost in the regenerative crypts 3.5 days following IR. DCAMKL-1 expression was restored at day 7 postirradiation, when the morphologic features of the crypts/villus axis were returning to baseline. These data support the hypothesis that daughter cells are capable of taking on stem cell characteristics in response to radiation-induced deletion of the ultimate stem cell and also illustrate that this process occurs at some time beyond 6 hours and prior to 24 hours after low-dose radiation injury. These data may potentially explain why doses of IR <8 Gy do not result in crypt sterilization of stem cells and, as a result, have little effect on clonogenic survival [15].

These data, taken together, strongly support the use of DCAMKl-1 as a viable intestinal and possibly colonic stem cell marker, as do the data presented by Giannakis et al. [11]. Additional studies would be required to fully characterize the functional role of this microtubule-associated protein in stem cell function and to understand the regulatory factors that control its expression. Clearly, following a lethal dose of whole-body irradiation, expression is lost in regenerative crypts. This could represent a negative regulatory mechanism, indicating the recruitment of sufficient stem cells required to repopulate the crypt, thus maintaining normal crypt numbers. Nevertheless, identification of a potential adult stem cell marker that can be identified under both normal and stressed conditions represents a potentially major step forward in the study of gut stem cell biology.

Here, we report the identification of a novel intestinal stem cell marker that can be used to test the effects of DNA-damaging agents, chemotherapeutic agents, and radiation injury on stem cell deletion, both directly and in real time. The data presented here also support assessment of radiation-induced apoptosis of intestinal stem cells 24 hours after IR, as opposed to 6 hours in intestinal cross-sections. The demonstration of a more variable expression pattern of DCAMKL-1 in the normal epithelium of APC/min mice compared with WT mice suggests that APC/min mice may exhibit different mechanisms of stem cell niche regulation, particularly in the regions adjacent to adenoma. The small percentage of quiescent DCAMKL-1-expressing cells within a particular adenoma suggests that they may be the origin of the more proliferative neoplastic cells, but it remains unclear whether these cells by themselves have tumorigenic potential either outside of the adenoma or outside of the crypt niche (villi). In the normal-appearing crypts of APC/min mice, β-catenin was coexpressed in the cytoplasm along with DCAMKL-1, whereas in adenomas, DCAMKL-1-positive cells demonstrated nuclear localization of β-catenin. This finding potentially illustrates a fundamental difference between the normal and adenoma stem cells. Isolating these cells and injecting them into nude mice xenograft models are essential in addressing the tumorigenic potential of these cells. These experiments are currently under way in our laboratories.

Recently, a novel putative intestinal and colonic stem cell marker was identified [26]. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) is a Wnt target gene with restricted crypt expression. In that report, the investigators demonstrated the expression of Lgr5 in cycling columnar intestinal epithelial cells at the crypt base [26]. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments suggested that the Lgr5-positive crypt base columnar cell is capable of generating all epithelial lineages over a 60-day period. Their data provide another potential stem/progenitor cell marker in that Lgr5 is expressed in rapidly cycling mitotic cells under basal conditions [26]. In contrast, DCAMKL-1 expression is restricted to quiescent cells at baseline, and it is expressed in mitotic cells only after irradiation. This is a major difference between Lgr5 and DCAMKL-1. We also observed occasional expression of DCAMKL-1 in crypt base columnar epithelial cells, suggesting that these may very well be stem cells. It is tempting to speculate that the restrictive DCAMKL-1 expression pattern in crypt base columnar identifies a subset of the Lgr5 columnar epithelial cells that consists of quiescent stem cells. Nevertheless, DCAMKL-1 is usually expressed at position 4–5, which has long been considered to be the stem cell location. Coexpression of DCAMKL-1 and musashi-1 is also observed, but DCAMKL-1 is expressed in a much more restricted fashion and in only a few crypt sections per intestinal cross-section. Barker et al. [26] found that Lgr5 is expressed at the base of the gland in the stomach, but the stem cell zone in the stomach is considered to be at gastric isthmus and not the base [11, 27, 28]. We observed DCAMKL-1 cells at the mid-stomach location (data not shown), consistent with an earlier report of Giannakis et al. [11].


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
Taken together, the identification of novel intestinal and colonic stem cell markers represents an exciting advance in adult stem cell biology. Use of the marker DCAMKL-1 will allow us to study for the first time the effects of DNA-damaging agents, chemotherapeutic agents, and stem cell protective strategies on stem cell fate, in diverse models of injury and inflammation. Furthermore, development of strategies specifically designed to target stem cells may lead to novel approaches to gastrointestinal diseases and cancer and aid in the development of novel chemotherapeutic and/or chemopreventative agents.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by NIH Grants DK-066161 and DK-002822 (to C.W.H.) and Washington University Digestive Disease Research Cores Center P30 DK-52574.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

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