Doublecortin And CaM Kinase-like-1 Research Articles

Correlation Between DCAMKL-1 Protein Expression and K-ras Gene Mutation in Colorectal Cancer.

To investigate the correlation between doublecortin and CaM kinase-like-1 (DCAMKL-1) protein expression, K-ras gene mutation, and their impact on patient prognosis in colorectal cancer (CRC). Immunohistochemistry was used to detect the expression of DCAMKL-1 protein in 60 cases of colorectal adenoma, 82 cases of CRC (including 65 cases of lymph node metastasis) and paraffin-embedded paracancerous intestinal mucosal tissue. K-ras gene mutations in primary CRC lesions were detected using an amplification-refractory mutation system and fluorescent polymerase chain reaction. The relationship between DCAMKL-1 protein expression and K-ras gene mutations with the clinicopathological characteristics of patients with CRC was analyzed. Univariate Kaplan‒Meier survival analysis and multivariate Cox regression analysis were performed using follow-up data. The mutation rate of the K-ras gene in 82 cases of CRC was 48.8% (40/82). The positivity rate for the presence of DCAMKL-1 protein in CRC was 70.7% (58/82), significantly higher than that for colorectal adenomas (53.3%; 32/60) and paracancerous intestinal mucosa (0%; 0/82) (P<0.05). The positive expression rate for the presence of DCAMKL-1 protein in 65 patients with lymph node metastasis was higher in the primary lesions (69.2%; 45/65) than in the lymph node metastases (52.3%; 34/65) (χ2=12.087, P=0.001). The K-ras gene mutation status was positively correlated with DCAMKL-1 protein expression (r=0.252, P=0.022). In this study, a potential positive correlation between K-ras gene mutation and DCAMKL-1 protein expression was identified in CRC tissues. The assessment of K-ras gene mutation status and DCAMKL-1 protein expression holds promise for augmenting early diagnosis and prognosis evaluation in CRC. This approach may improve the overall prognosis and survival outcomes for CRC patients.

DCAMKL1 is associated with the malignant status and poor outcome in bladder cancer.

DCAMKL1 (doublecortin and CaM kinase-like 1) has been found to be overexpressed and function as an oncogene in several types of cancer, but there are limited reports on the role of DCAMKL1 in bladder cancer. The messenger RNA and protein expression of DCAMKL1 in bladder cancer tissues and cell lines was measured by quantitative reverse transcription polymerase chain reaction, western blotting, or immunohistochemistry. The correlation between DCAMKL1 protein expression and clinicopathological characteristics was analyzed. Univariate and multivariate Cox regression models were adopted to evaluate prognostic significance of DCAMKL1 in bladder cancer patients. In our results, DCAMKL1 messenger RNA and protein were overexpressed in bladder cancer tissues compared with adjacent normal tissues. DCAMKL1 protein overexpression was positively associated with clinical stage, muscularis invasion, lymph node metastasis, and distant metastasis. The univariate and multivariate analyses suggested DCAMKL1 protein overexpression was an unfavorable prognostic factor in bladder cancer patients. In conclusion, DCAMKL1 is an independent poor prognostic factor for bladder cancer patients.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Hepatocellular carcinoma: from hepatocyte to liver cancer stem cell.

Cyclooxygenase 2 in gastric carcinoma is expressed in doublecortin- and CaM kinase-like-1-positive tuft cells.

Background/AimsDoublecortin and CaM kinase-like-1 (DCAMKL1) is a marker of stem cells expressed predominantly in the crypt base in the intestine. However, DCAMKL1-positive cells have been shown to be differentiated tuft cells rather than quiescent progenitors. Tuft cells are the only epithelial cells that express cyclooxygenase 2 (COX-2) in the normal intestinal epithelium. We previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia and gastric carcinoma. In the current study, we investigated the association between COX-2 and DCAMKL1 in gastric carcinoma.MethodsWe examined the association between COX-2 and DCAMKL1 expression in gastric carcinomas in clinical samples (early gastric well-differentiated adenocarcinoma) and Cdx2-transgenic mice; and the DCAMKL1-transgenic mouse stomach using immunohistochemistry and quantitative real-time polymerase chain reaction.ResultsThe COX-2-expressing cells were scattered, not diffusely expressed, in gastric carcinomas from humans and Cdx2-transgenic mice. DCAMKL1-positive cells were also scattered in the gastric carcinomas, indicating that tuft cells could still be present in gastric carcinoma. COX-2 was expressed in DCAMKL1-positive tuft cells in Cdx2- and DCAMKL1-transgenic mouse stomachs, whereas the Sox9 transcription factor was ubiquitously expressed in gastric carcinomas, including COX-2-positive cells.ConclusionsCOX-2 is expressed in DCAMKL1-expressing quiescent tuft cells in gastric carcinoma.

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

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.

Identification of the putative intestinal stem cell marker doublecortin and CaM kinase-like-1 in Barrett's esophagus and esophageal adenocarcinoma.

In Barrett's esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase-like-1 (DCAMKL-1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC. Human NEM, BE, EAC, and multitissue microarrays were analyzed for DCAMKL-1, and immunohistochemically scored based on staining intensity and tissue involvement, with epithelia and stroma scored separately. Total RNA isolated from BE and paired NEM was subjected to real-time reverse-transcription-polymerase chain reaction analysis for DCAMKL-1, leucine-rich repeat-containing G-protein-coupled receptor (LGR5), and Musashi-1 (Msi-1) mRNA expression. DCAMKL-1 was minimally expressed in squamous NEM, but increased in BE (with and without dysplasia) and EAC tissues. In EAC, we found increased stromal DCAMKL-1 staining compared to adjacent epithelia. Within the submucosa of dysplastic BE tissues, an increase in the endothelial cell expression of DCAMKL-1 was observed. Finally, an upregulation of DCAMKL-1, LGR5, and Msi-1 mRNA was seen in BE compared to squamous NEM. In the present study, we report the progressive increase of DCAMKL-1 expression in BE from dysplasia to EAC. Furthermore, there was an increase in putative stem cell markers DCAMKL-1, LGR5, and Msi-1 mRNA. Taken together, these data suggest that the regulation of resident stem cells might play an important role in the progression of BE to EAC.

Cancer Stem Cells and Pluripotency

Determining the functional roles of cancer stem cells (CSCs) and epithelial to mesenchymal transition (EMT) in carcinogenesis and tumorigenesis are pivotal topics in cancer biology research in general and pancreatic cancer in particular. Several publications, including ours, have demonstrated that CSCs and EMT function in tandem, ultimately leading to cancer progression and metastasis [1]. This has been demonstrated in breast, pancreas, and colorectal cancers and likely occurs in all types of cancers. A pressing issue in the field of CSC and EMT research is that of pancreatic adenocarcinoma (PDAC), as it has the worst prognosis of any major malignancy with a 3% 5-year survival rate [2]. Major obstacles in treating pancreatic cancer include delayed diagnosis, extensive local tumor invasion, and early metastasis. CSCs, or cancer initiating cells (CICs), represent approximately 1% to 5% of the tumor, and are capable of unlimited self-renewal, and are often resistant to chemotherapy and radiation therapy [3]. This may explain why these treatments do not cure or prevent recurrence of PDAC [4,5]. Improving survival for all types of cancer likely hinges on the identification and eradication of CSCs. The existence of CSCs was first demonstrated in acute myelogenous leukemia [6] and subsequently verified in breast [7], pancreatic [8], and brain tumors [9-11]. The CD133+ subpopulations from brain tumors can initiate clonally derived neurospheres in vitro showing self-renewal, differentiation, and proliferative characteristics similar to normal brain stem cells [9-11]. In a recent study, a subpopulation of CD44+ CD24+ ESA+ cells derived from primary human pancreatic adenocarcinoma CSCs [8] were implanted in immunocompromised mice and resulted in enhanced tumorigenic potential. We have recently demonstrated that doublecortin and CAM kinase-like-1 (DCAMKL-1) is a pancreatic stem cell marker that is upregulated in pancreatic cancer and may be a marker of CSCs [1,12].

Doublecortin and CaM Kinase-like-1 and Leucine-Rich-Repeat-Containing G-Protein-Coupled Receptor Mark Quiescent and Cycling Intestinal Stem Cells, Respectively

It is thought that small intestinal epithelia (IE) undergo continuous self-renewal primarily due to their population of undifferentiated stem cells. These stem cells give rise to transit amplifying (daughter/progenitor) cells, which can differentiate into all mature cell types required for normal gut function. Identification of stem cells in IE is paramount to fully understanding this renewal process. One major obstacle in gastrointestinal stem cell biology has been the lack of definitive markers that identify small intestinal stem cells (ISCs). Here we demonstrate that the novel putative ISC marker doublecortin and CaM kinase-like-1 (DCAMKL-1) is predominantly expressed in quiescent cells in the lower two-thirds of intestinal crypt epithelium and in occasional crypt-based columnar cells (CBCs). In contrast, the novel putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor (LGR5) is observed in rapidly cycling CBCs and in occasional crypt epithelial cells. Furthermore, functionally quiescent DCAMKL-1+ crypt epithelial cells retain bromo-deoxyuridine in a modified label retention assay. Moreover, we demonstrate that DCAMKL-1 is a cell surface expressing protein; DCAMKL-1+ cells, isolated from the adult mouse small intestine by fluorescence activated cell sorting, self-renew and ultimately form spheroids in suspension culture. These spheroids formed glandular epithelial structures in the flanks of athymic nude mice, which expressed multiple markers of gut epithelial lineage. Thus, DCAMKL-1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR5+). Moreover, DCAMKL-1 can be used to isolate normal small intestinal stem cells and represents a novel research tool for regenerative medicine and cancer therapy.

Inactivating cholecystokinin-2 receptor inhibits progastrin-dependent colonic crypt fission, proliferation, and colorectal cancer in mice

Hyperproliferation of the colonic epithelium, leading to expansion of colonic crypt progenitors, is a recognized risk factor for colorectal cancer. Overexpression of progastrin, a nonamidated and incompletely processed product of the gastrin gene, has been shown to induce colonic hyperproliferation and promote colorectal cancer in mice, but the mechanism of pathogenesis has not been defined. Cholecystokinin-2 receptor (CCK2R) is the primary receptor for cholecystokinin (CCK) and amidated gastrin. Here, we show that Cck2r was expressed in murine colonic crypts and upregulated in the transgenic mice that overexpress human progastrin. Murine deletion of Cck2r abrogated progastrin-dependent increases in colonic proliferation, mucosal thickness, and beta-catenin and CD44 expression in the colon tumor. In addition, either deletion or antagonism of Cck2r resulted in the inhibition of progastrin-dependent increases in progenitors expressing doublecortin and CaM kinase-like-1 (DCAMKL1), stem cells expressing leucine rich repeat-containing G protein-coupled receptor 5 (LgR5), and colonic crypt fission. Furthermore, in the azoxymethane mouse model of colorectal carcinogenesis, Cck2r deletion in human progastrin-overexpressing mice resulted in markedly decreased aberrant crypt foci formation and substantially reduced tumor size and multiplicity. Taken together, these observations indicate that progastrin induces proliferative effects, primarily in colonic progenitor cells, through a CCK2R-dependent pathway. Moreover, our data suggest that CCK2R may be a potential target in the treatment or prevention of colorectal cancer.

Tissue-Engineered Small Intestine and Stomach Form from Autologous Tissue in a Preclinical Large Animal Model

Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche. A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans.

Acid, Bile, and CDX: the ABCs of making Barrett's metaplasia

Barrett's esophagus, a squamous-to-columnar cell metaplasia that develops as a result of chronic gastroesophageal reflux disease (GERD), is a risk factor for esophageal adenocarcinoma. The molecular events underlying the pathogenesis of Barrett's metaplasia are poorly understood, but recent studies suggest that interactions among developmental signaling pathways, morphogenetic factors, and Caudal homeobox (Cdx) genes play key roles. Strong expression of Cdx genes normally is found in the intestine but not in the esophagus and stomach. When mice are genetically engineered so that their gastric cells express Cdx, the stomach develops a metaplastic, intestinal-type epithelium similar to that of Barrett's esophagus. Exposure to acid and bile has been shown to activate the Cdx promoter in certain esophageal cell lines, and Cdx expression has been found in inflamed esophageal squamous epithelium and in the specialized intestinal metaplasia of Barrett's esophagus. Barrett's metaplasia must be sustained by stem cells, which might be identified by putative, intestinal stem cell markers like leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and doublecortin and CaM kinase-like-1 (DCAMKL-1). Emerging concepts in tumor biology suggest that Barrett's cancers may develop from growth-promoting mutations in metaplastic stem cells or their progenitor cell progeny. This report reviews the roles of developmental signaling pathways and the Cdx genes in the development of normal gut epithelia and the potential mechanisms whereby GERD may induce the esophageal expression of Cdx genes and other morphogenetic factors that mediate the development of Barrett's metaplasia. The role of stem cells in the development of metaplasia and in carcinogenesis and the potential for therapies directed at those stem cells also is addressed.

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

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 beta-catenin in normal-appearing intestine. However, beta-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of beta-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents.

Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection

SummaryInsights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host‐ and virus‐mediated transcriptional responses. An oligo‐based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial‐related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real‐time reverse transcription polymerase chain reaction (RT‐PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two‐dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis‐associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase‐like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real‐time RT‐PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.