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Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Doublecortin-like kinase 1 protein (DCLK1) is a gastrointestinal tuft cell marker that has been proposed to identify quiescent and tumor growth--sustaining stem cells. [DCLK1.sup.+] tuft cells are increased in inflammation-induced carcinogenesis; however, the role of these cells within the gastrointestinal epithelium and their potential as cancer-initiating cells are poorly understood. Here, using a BAC-CreERT--dependent genetic lineage--tracing strategy, we determined that a subpopulation of [DCLK1.sup.+] cells is extremely long lived and possesses rare stem cell abilities. Moreover, genetic ablation of Dclk1 revealed that [DCLK1.sup.+] tuft cells contribute to recovery following intestinal and colonic injury. Surprisingly, conditional knockdown of the Wnt regulator APC in [DCLK1.sup.+] cells was not sufficient to drive colonic carcinogenesis under normal conditions; however, dextran sodium sulfate--induced (DSS-induced) colitis promoted the development of poorly differentiated colonic adenocarcinoma in mice lacking APC in [DCLK1.sup.+] cells. Importantly, colonic tumor formation occurred even when colitis onset was delayed for up to 3 months after induced APC loss in [DCLK1.sup.+] cells. Thus, our data define an intestinal [DCLK1.sup.+] tuft cell population that is long lived, quiescent, and important for intestinal homeostasis and regeneration. Long-lived [DCLK1.sup.+] cells maintain quiescence even following oncogenic mutation, but are activated by tissue injury and can serve to initiate colon cancer., Introduction Colorectal cancer arises as a result of a series of genetic changes that include activating mutations in oncogenes and inactivating mutations in tumor suppressor genes. The most common initial [...]

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-I antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer. stem/progenitor cell marker; DCAMKL-1; pancreas; neurogenin 3; nestin doi: 10.1152/ajpgi.00146.2010.

The microcolony assay following gamma irradiation (IR) is a functional assay of intestinal stem cell fate. The cyclin-dependent kinase (CDK) inhibitor [p21.sup.Waf1/Cip1/Sdi1] (p21) regulates cell cycle arrest following DNA damage. To explore the role of p21 on stem cell fate, we examined the effects of p21 deletion on intestinal crypt survival following IR and expression of the stem/progenitor cell marker Musashi-1 (Msi-1) and the antiapoptotic gene survivin. Intestinal stem cell survival in adult wild-type (WT) and [p21.sup.-/-] mice was measured using the microcolony assay. Msi-1, p21, and survivin mRNA were measured using real-time PCR and immunohistochemical analysis. Laser capture microdissection (LCM) was used to isolate mRNA from the crypt stem cell zone. No differences in radiation-induced apoptosis were observed between WT and [p21.sup.-/-] mice. However, increased crypt survival (3.0-fold) was observed in [p21.sup.-/-] compared with WT mice 3.5 days after 13 Gy. Msi-1 and survivin mRNA were elevated 12-and 7.5-fold, respectively, in LCM-dissected crypts of [p21.sup.-/-] compared with WT mice. In conclusion, deletion of p21 results in protection of crypt stem/progenitor cells from IR-induced cell death. Furthermore, the increase in crypt survival is associated with increased numbers of Msi-1- and survivin-expressing cells in regenerative crypts. Musashi-1; gamma irradiation; survivin; crypt survival

Prostaglandin [E.sub.2] (PG[E.sub.2]) is a potent inhibitor of ionizing radiation (IR)-induced cell death. Exposure of colon cancer cells to IR leads to increased CUGBP2 expression. Therefore, we tested the hypothesis that PG[E.sub.2] radioprotects colon cancer cells by inhibiting CUGBP2 expression. Exposure of HCT-116 cells to [gamma]-IR (0-12 Gy) resulted in a dose-dependent reduction in cell growth and an increase in the [G.sub.2]-M phase of the cell cycle. Western blot analyses demonstrated increased levels of activated caspase 9 and caspase 3. In addition, whereas Bax expression is increased, that of Bcl-2 and Bcl-[X.sub.L] was reduced. Further analyses demonstrated increased activation of Chk1 and Chk2 kinases, coupled with higher levels of nuclear cyclin B1 and Cdc2. Pretreatment with PG[E.sub.2] suppressed the activation of caspase 3 and caspase 7 and inhibited Bax expression. In addition, PG[E.sub.2] treatment restored growth and colony formation to control levels. IR significantly upregulated the expression of CUGBP2 in the cells, which was suppressed when cells were pretreated with PG[E.sub.2]. Ectopic overexpression of CUGBP2 also induced apoptosis. Furthermore, it reversed the PG[E.sub.2]-mediated protection from IR-induced mitotic catastrophe. Furthermore, there was an increase in nuclear localization of cyclin B1 and Cdc2 coupled with increased phosphorylation of p53, Chk1, Chk2, and Cdc25c proteins. Cell cycle analysis also demonstrated increased [G.sub.2]-M transition. In contrast, siRNA-mediated suppression of CUGBP2 expression restored normal cell cycle progression and decreased IR-induced apoptosis. Taken together, these data demonstrate that PG[E.sub.2] protects colon cancer cells from IR-induced mitotic catastrophe in part through suppression of CUGBP2 expression. cell cycle; apoptosis; caspase; check point kinases; RNA binding protein

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mc1-1, an antiapoptotic Bc1-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mc1-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the [G.sub.2]-M phase. Western blot analyses demonstrated that there was decreased Bc1-2 and Mc1-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for [alpha]-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the [G.sub.2]-M phase of the cell cycle. We next determined the mechanism of CUGBP2mediated reduction in Mc1-1 expression. Mc1-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mc1-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mc1-1 and luciferase mRNA containing the Mc1-1 3'-UTR. However, luciferase protein expression from the luciferase-Mc1-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mc1-1 expression by inhibiting Mc1-1 mRNA translation, resulting in driving the cells to apoptosis during the [G.sub.2] phase of the cell cycle. Bc1-2 family member; [G.sub.2]-M arrest; checkpoint kinases; RNA stability

CUG triplet repeat-binding protein 2 (CUGBP2) is a RNA-binding protein that regulates mRNA translation and modulates apoptosis. Here, we report the identification of two splice variants (termed variants 2 and 3) in cultured human intestinal epithelial cells and in mouse gastrointestinal tract. The variants are generated from alternative upstream promoters resulting in the inclusion of additional [NH.sub.2]-terminal residues. Although variant 2 is the predominant isoform in normal intestine, its expression is reduced, whereas variant 1 is overexpressed following -y-irradiation. All three variants bind cyclooxygenase-2 (COX-2) mRNA. However, only variant 1 inhibits the translation of the endogenous COX-2 mRNA and a chimeric luciferase mRNA containing the COX-2 3'untranslated region. Furthermore, whereas variant 1 is predominantly nuclear, variants 2 and 3 are predominantly cytoplasmic. These data imply that the additional amino acids affect CUGBP2 function. Previous studies have demonstrated that variant 1 induces intestinal epithelial cells to undergo apoptosis. However, in contrast to variant 1, the two novel variants do not affect proliferation or apoptosis of HCT116 cells. In addition, only variant 1 induced [G.sub.2]/M cell cycle arrest, which was overcome by prostaglandin E2. Moreover, variant 1 increased cellular levels of phosphorylated p53 and Bax and decreased Bc12. Caspase-3 and -9 were also activated, suggesting the initiation of the intrinsic apoptotic pathway. Furthermore, increased phosphorylation of checkpoint kinase (Chk)l and Chk2 kinases and increased nuclear localization of Cdc2 and cyclin B1 suggested that cells were in mitotic transition. Taken together, these data demonstrate that cells expressing CUGBP2 variant 1 undergo apoptosis during mitosis, suggesting mitotic catastrophe. cell cycle; apoptosis; [G.sub.2]/M arrest; checkpoint kinases

Azoxymethane (AOM) is a potent DNA-damaging agent and carcinogen that induces intestinal and colonic tumors in rodents. Evaluation of the stem cell population by colony formation assay reveals that, within 8 h after treatment, AOM (10 mg/kg) elicited a prosurvival response. In wild-type (WT) mice, AOM treatment induced a 2.5-fold increase in intestinal crypt stem cell survival. AOM treatment increased stem cell survival in cyclooxygenase (COX)-[2.sup.-/-] but not COX-[1.sup.-/-] mice, confirming a role of COX-1 in the AOM-induced increase in stem cell survival. COX-1 mRNA and protein expression as well as COX-1-derived [PGE.sub.2] synthesis were increased 8 h after AOM treatment. Immunohistochemical staining of COX-1 demonstrated expression of the enzyme in the crypt epithelial cells, especially in the columnar epithelial cells between the Paneth cells adjacent to the stem cell zone. WT mice receiving AOM exhibited increased intestinal apoptosis and a simultaneous reduction in crypt mitotic figures within 8 h of injection. There were no significant differences in baseline or AOM-induced intestinal epithelial apoptosis between WT and COX-[1.sup.-/-] mice, but there was a complete reversal of the AOM-mediated reduction in mitosis in COX-[1.sup.-/-] mice. This suggests that COX-[1.sup.-/-] derived [PGE.sub.2] may play a key role in the early phase of intestinal tumorigenesis in response to DNA damage and suggests that COX-1 may be a potential therapeutic target in this model of colon cancer. Paneth cells; apoptosis; tumorigenesis; colorectal cancer; crypt survival