Original article
Immunolocalization of DCAMKL-1, a putative intestinal stem cell marker, in normal colonic tissue

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Abstract

Doublecortin and CaM kinase-like-1 (DCAMKL-1) is a microtubule-associated protein kinase which has been recently proposed as a gastrointestinal stem cell marker. The aim of our study was to characterize DCAMKL-1 expression in normal human colon by immunohistochemistry. DCAMKL-1 immunostaining was performed on histologically normal colorectal biopsies from 14 patients. Immunoreactivity was found in over 60% of colonic crypts and was represented by strong cytoplasmic staining, anti-luminal in distribution, and was reminiscent of cytoplasmic orientation of neuroendocrine cells. The highest number (48.5%) of DCAMKL-1 positive cells was found in the first 4 cells from the crypt base. Seventy percent of DCAMKL-1 positive cells were located in the lower third of the crypt, 26% in the middle third and 4% in the upper third. Therefore, in normal colonic mucosa, expression of DCAMKL-1 is not confined to the stem cell compartment. When we compared DCAMKL-1 immunostaining with that of the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5), an intestinal stem cell marker, and chromogranin-A (CgA), an enteroendocrine cell marker, we found that DCAMKL-1 positive cells co-stained with Lgr5 only at the crypt base, but co-stained with CgA throughout the crypt. Our findings suggest that DCAMKL-1 marks a subset of colorectal stem cells, as well as a subset of enteroendocrine cells.

Introduction

The intestinal epithelium is a highly dynamic tissue where cells are continuously replaced by a process of replications within the crypts of Lieberkuhn, migration toward the surface epithelium, differentiation and apoptosis. In the colon, there are four major terminally differentiated epithelial cell types: the colonocytes or absorptive cells, the mucus-secreting goblet cells, the enteroendocrine cells and the Paneth cells, which are sometimes present in the ascending colon [7]. All these cell types originate from a single multipotent stem that is also capable of self-renewal. However, self-renewing cells have to divide at a very low rate to prevent acquisition of DNA replication errors, while a higher rate of proliferation is necessary for rapid tissue turnover and repair [19]. It has therefore been suggested that two distinct types of stem cells perform these activities. In the adult intestine, two types of stem cells are described based on their location and cycling dynamics [1]. Fast-cycling stem cells express leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) and are located at the crypt base while slower-cycling stem cells express Bmi1 and represent a rarer cell population located at the +4 position relative to the crypt base [13]. Another possible marker of slow-cycling intestinal stem cell population is the serine/threonine protein kinase doublecortin and CaM kinase-like-1 (DCAMKL-1), a microtubule-associated Ca2+/calmodulin-dependent protein kinase found in post mitotic neurons [5], [8]. In mouse small intestine and colon, DCAMKL-1 positive cells have been located at the +4 position in the crypt, are apoptosis-resistant and retain bromo-deoxyuridine [9], [10], which are all characteristics of quiescent intestinal stem cells. Also, in mouse small intestine, co-localization studies show that DCAMKL-1 positive cells are a distinct population from rapidly cycling crypt base cells expressing Lgr5 [10]. Based on these findings DCAMKl-1 is increasingly used for assessing the progenitor cell compartment in the gastrointestinal tract. However, recent lineage tracing studies have shown that DCAMKL-1 positive cells may be derived from Lgr5-expressing intestinal stem cells and may represent terminally differentiated tuft cells [4] or enteroendocrine cells [3]. Therefore, the role of DCAMKL-1 as intestinal stem cell marker and its applicability to intestinal stem cell research is not clear. Moreover, expression of DCAMKL-1 in normal human colonic tissue has not yet been described. We sought to investigate the presence of DCAMKL-1 immunoreactivity in normal human colonic tissue. We also sought to compare the expression of DCAMKL-1 with Lgr5, a known intestinal stem cell marker, and with chromogranin-A (CgA), an enteroendocrine cell marker, to assess whether they identify the same population of cells in normal colonic tissue.

Section snippets

Tissue samples

The study was carried out on paraffin-embedded, formalin-fixed human tissue samples, obtained from endoscopic biopsy specimens. Patient medical records were reviewed, and gastrointestinal histopathology was correlated with each specimen to confirm the absence of abnormal pathologic findings. Fourteen specimens were selected and used in the study. The study was approved by the Tulane University Institutional Review Board.

Immunohistochemistry

Immunohistochemical staining was carried out on 5 mm sections of

Results

Immunostaining revealed expression of DCAMKL-1 predominantly in cells at the crypt base. It was represented by strong cytoplasmic immunostaining, anti-luminal in distribution and reminiscent of cytoplasmic orientation of neuroendocrine cells (Fig. 1). First we determined the number of DCAMKL-1 positive cells per crypt and their localization along the crypt axis. As shown in Table 1, the number of positive cells per crypt varied from a single cell to several, but many crypts showed no

Discussion

Although DCAMKL-1 has been shown to be upregulated in human colorectal cancer, the expression of DCAMKL-1 in human normal colon has not yet been fully investigated [16], [17]. In this study, we have employed immunohistochemical analysis to determine the expression of DCAMKL-1 in normal colon mucosa from endoscopic biopsy specimens. DCAMKL-1 was expressed in over 60% of normal human colonic crypts. The highest number of DCAMKL-1 positive cells was found in the first 4 cells from the crypt base.

Acknowledgment

Supported by a South Central Veterans Affairs Health Care Network Research Pilot Study Award.

References (20)

  • N. Barker et al.

    Tissue-resident adult stem cell populations of rapidly self-renewing organs

    Cell Stem Cell

    (2010)
  • M. Giannakis et al.

    Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches

    J. Biol. Chem.

    (2006)
  • C. Bezençon et al.

    Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells

    J. Comp. Neurol.

    (2008)
  • M. Bjerknes et al.

    Origin of the brush cell lineage in the mouse intestinal epithelium

    Dev. Biol.

    (2011)
  • F. Gerbe et al.

    Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

    J. Cell Biol.

    (2011)
  • A.R. Gunawardene et al.

    Classification and functions of enteroendocrine cells of the lower gastrointestinal tract

    Int. J. Exp. Pathol.

    (2011)
  • A. Humphries et al.

    Colonic crypt organization and tumorigenesis

    Nat. Rev. Cancer

    (2008)
  • P.T. Lin et al.

    DCAMKL1 encodes a protein kinase with homology to doublecortin that regulates microtubule polymerization

    J. Neurosci.

    (2000)
  • R. May et al.

    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

    Stem Cells

    (2008)
  • R. May et al.

    Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively

    Stem Cells

    (2009)
There are more references available in the full text version of this article.

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