Bottom Of Colonic Crypts Research Articles

Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer.

Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.

Unraveling the Transcriptional Heterogeneity in Human Colon Cancer: Potential New Insights regarding Pathobiology and Treatment

In a recent hallmark report in Nature Biotechnology , Dalerba and colleagues from Stephen Quake's and Michael Clarke's laboratories at Stanford demonstrated the power of single-cell expression analysis for the elucidation of human tissue function and pathology (1). They made a detailed molecular dissection of healthy and colon cancer tissue to address fundamental questions about tumor heterogeneity and clonality. This pioneering and impressive work shows that much is to be gained by resolving the cellular heterogeneity of tissues. This more detailed characterization of normal and diseased tissue can provide valuable insights into cancer pathobiology that can be used for improving diagnosis and stratification of patients, and it has the potential to lead to new insights regarding treatment. Dalerba and colleagues first made a single-cell suspension of healthy colon tissue. They used fluorescence-activated cell sorting with established differentiation markers to isolate and separate well-differentiated epithelial cells at the top of colon crypts from the more immature stem cell–like cells at the bottom of colon crypts. They then performed single-cell quantitative PCR analyses …

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

Deficiency of Smad3, an intracellular mediator of TGF-β, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3 ex8/ex8 C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3 −/− mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3 −/− mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear β-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3 −/− mice in accordance with nuclear β-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3 −/− mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3 −/− mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a “proliferative zone” at the bottom of colonic crypts in the normal colon.

Defective Claudin-7 Regulation by Tcf-4 and Sox-9 Disrupts the Polarity and Increases the Tumorigenicity of Colorectal Cancer Cells

Tight junctions have recently emerged as essential signaling regulators of proliferation and differentiation in epithelial tissues. Here, we aimed to identify the factors regulating claudin-7 expression in the colon, and analyzed the consequences of claudin-7 overexpression in colorectal carcinoma (CRC). In healthy human colonic crypts, claudin-7 expression was found to be low in the stem/progenitor cell compartment, where Tcf-4 activity is high, but strong in differentiated and postmitotic cells, where Tcf-4 is inactive. In contrast, claudin-7 was overexpressed in areas with high Tcf-4 target gene levels in CRC samples. In vitro, Tcf-4 was able to repress claudin-7 expression, and the high mobility group-box transcription factor Sox-9 was identified as an essential mediator of this effect. Claudin-7 was strongly expressed in the intestine of Sox-9-deficient mice and in CRC cells with low Sox transcriptional activity. Sox-9 overexpression in these cells reinstated claudin-7 repression, and residual claudin-7 was no longer localized along the basolateral membrane, but was instead restricted to tight junctions. Using HT-29Cl.16E CRC cell spheroids, we found that Sox-9-induced polarization was completely reversed after virus-mediated claudin-7 overexpression. Claudin-7 overexpression in this context increased Tcf-4 target gene expression, proliferation, and tumorigenicity after injection in nude mice. Our results indicate that Tcf-4 maintains low levels of claudin-7 at the bottom of colonic crypts, acting via Sox-9. This negative regulation seems to be defective in CRC, possibly due to decreased Sox-9 activity, and the resulting claudin-7 overexpression promotes a loss of tumor cell polarization and contributes to tumorigenesis.

Effect of a 12-Month Exercise Intervention on the Apoptotic Regulating Proteins Bax and Bcl-2 in Colon Crypts: A Randomized Controlled Trial

Cellular proliferation and apoptosis (cell death) are highly regulated in the colon as insufficient apoptosis may lead to polyps and cancer. Physical activity decreases risk of colon cancer in observational studies, but the biological basis is not well defined. The objective of this study is to examine the effects of a 12-month aerobic exercise program on expression of proteins that promote (Bax) or inhibit (Bcl-2) apoptosis in colon crypts. Two hundred two sedentary participants, 40 to 75 years, were randomly assigned to moderate-to-vigorous intensity exercise for 60 min per day, 6 days per week for 12 months, or usual lifestyle. Colon crypt samples were obtained at baseline and 12 months. Bcl-2 and Bax expression was measured by immunohistochemistry. Bax density at the bottom of crypts increased in male exercisers versus controls (+0.87 versus -0.18; P = 0.05), whereas the ratio of Bcl-2 to Bax at the bottom and middle of crypts decreased as aerobic fitness (VO(2)max) increased (P trend = 0.02 and 0.05, respectively). In female exercisers, Bax density in the middle of crypts decreased (-0.36 versus +0.69; P = 0.03) and Bcl-2 to Bax ratio at the top of crypts increased versus controls (+0.46 versus -0.85; P = 0.03). Bax density in the middle of crypts also decreased as minutes per week of exercise increased (P trend = 0.03). A 12-month exercise intervention resulted in greater expression of proteins that promote apoptosis at the bottom of colon crypts in men and decreased expression of proteins that promote apoptosis at the middle and top of colon crypts in women. The difference in effect by gender and location of observed changes warrants further study.

Bcl-2 expression in adult and embryonic non-haematopoietic tissues.

The B-cell leukaemia/lymphoma-2 (bcl-2) proto-oncogene is unusual as its product appears to provide survival advantage to B cells by blocking apoptosis. In this study, the expression of bcl-2 has been examined in normal non-haematopoietic tissues, embryos, and psoriatic skin by immunohistochemical staining. Bcl-2 protein expression is mainly observed in cell populations with a long life and/or proliferating ability such as duct cells in exocrine glands, basal keratinocytes, cells at the bottom of colon crypts, and neurons. In the skin of both adult and embryo and also embryonic kidney and cartilage, bcl-2 expression was observed in cells which were undergoing morphological transition from undifferentiated stem cells to committed precursor cells. The finding of bcl-2 expression in the terminal differentiated syncytial trophoblast, but not cytotrophoblast, and in some cells responsive to hormone stimulation such as in the endometrium and myometrium suggests that the gene expression may be related to hormone responsiveness. As no bcl-2 localization was seen in the benign hyperproliferative skin condition psoriasis, this does not suggest a straight-forward link to proliferation. These observations support the view that the bcl-2 gene may have an important role in cell development, maturation, and the path to terminal differentiation.

Lysozyme localization in normal and diseased human gastric and colonic mucosa. A correlative histochemical, immunohistochemical and immunoelectron microscopic investigation.

The distribution of lysozyme in normal and pathological human gastric and colonic mucosa was studied by light and electron microscopic immunocytochemical techniques and compared with histological and histochemical features. Lysozyme was localized in pyloric glandular epithelial cells, mucous neck cells of fundic glands, Paneth cells and some crypt cells of the mature colonic mucosa. In addition, lysozyme was detected in a large spectrum of "immature" or "regenerative" epithelium: neck cells of the gastric regenerative zone, undifferentiated columnar cells of surface and hyperplastic interfoveolar crests of the stomach, regenerative cells in a healed gastric ulcer, some goblet cells in incomplete intestinal metaplasia, cells of the regenerative zone at the bottom of colonic crypts and, finally, fetal intestinal epithelium. Electron microscopically, we localized lysozyme in the central core of mucous granules in the pyloric gastric glandular epithelium and in the dense mucous granules in gastric mucous neck cells. Lysozyme was also detected in some immature mucin-producing cells of the gastric regenerative zone and in the rough endoplasmic reticulum of surface hyperplastic columnar gastric cells. At the electron microscopic level, a peculiar correlation between the immunopattern of lysozyme and the morphology of mucous granules has been postulated. All our data support and extend the view that the presence of lysozyme may be related to cell immaturity as well as to a regenerative state of the cell. Finally, the lysozyme distribution and its relation to mucosubstances in gastric and colonic carcinoma suggest that lysozyme should not be considered an exclusive marker of cells of gastric derivation.