Previous work on somatic mtDNA mutations has con- centrated on the observations in postmitotic tissues, such as muscle and the CNS, since these tissues are pre- dominantly involved in patients with mtDNA disease. Even in these tissues, systematic studies of the nature and incidence of mutations is limited. A small number of reports cite the accumulation of mtDNA mutations in replicative cells (6, 10). An important question in mito- chondrial genetics is whether there is evidence of mtDNA mutations in stem cells, and if so, do these mutations accumulate to levels sufficient to result in a biochemical defect. If so, this could have profound impli- cations for our understanding of the potential impor- tance of these mutations in aging and cancer. To investi- gate the existence of mtDNA mutations in stem cells, we decided to study colonic crypt stem cells. Colonic crypt stem cells are present at the crypt base, and thus all the cells within the crypt must be derived from these stem cells. Thus, we would not have to isolate individual stem cells to look for mtDNA mutations, but could in theory study their progeny. While there is uncertainty about the number of stem cells within human colonic crypts, stud- ies in mice suggest that crypts are maintained by small numbers of stem cells (11). For example, in chimeric mouse strains a single crypt often shows evidence of clonality in adult mice, suggesting that the majority of cells within an individual crypt are the progeny of a sin- gle stem cell (12). Studies in humans are much more dif- ficult because strategies that directly mark cell fates, such as aggregation chimeras and mutagenesis, are impracti- cal in humans (13); however, current evidence would sup- port that the situation is similar to mice.
11 Read more
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 b-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.
12 Read more
In this study, similar channels were observed in 58/113 (51%) cell attached basola- teral membrane patches in the mid third of isolated human colonic crypts. With NaCl solution in the bath and KCl solution in the pipette, inward currents were seen at the rest- ing membrane potential (zero holding voltage) in cell attached basolateral membrane patches, and linear current-voltage relationships indicated a mean slope conductance and reversal potential of 27 (1) pS and 28 (6) mV, respectively (n=15), and channel activity was voltage independent between − 80 mV and − 20 mV. Curvilinear current- voltage relationships were seen after excision of the patches into the inside out configu- ration (n=11) from which the K +
transfected HEK-293 cells (40), indicating its proinﬂammatory activity. The decrease in the proliferative rate and the increase in the death rate were also observed after stimulation of colonic crypts with sonicates and puriﬁed LPSs from CSCM members. This was not observed in mice deﬁcient in TLR4, indicating the central role of LPS. These data are not consistent with a recent study showing that, in contrast to poly(I · C), LPS did not decrease colonoid viability (41). Other studies, however, showed that LPS reduced cell proliferation of the small intestinal epithelium (14, 42) and that TLR4 activation impaired enterocyte proliferation in the ilea of newborn mice (43). It was also shown that interferon (IFN) signaling could affect intestinal epithelial regeneration. Indeed, proliferative bromodeoxyuridine (BrdU)-positive cells increase in type I IFN receptor knockout mice, and it was demonstrated that this increase depends on microbiota composition (44). It was also shown that type I IFN controls the proliferation of the intestinal epithelium through its activation of ␤ -catenin (45). In contrast to what was found in the small intestine (14), we showed that LPS-stimulated crypt death was caused by TNF- ␣ -mediated necroptosis via the TNF receptor (TNFR)/RIPK1/RIPK3 path- way, rather than by apoptosis through the p53/PUMA pathway. Indeed, the deletion of RIPK3 improved the survival rate of colonic crypt cultures during stimulation with LPSs puriﬁed from CSCM members. Interestingly, it was recently shown that lung injury observed upon high-dose-LPS-induced acute respiratory distress syndrome in mice was due to RIPK3-mediated necroptosis (46). This could be linked to a study showing that sepsis affects quiescent muscle stem cells, causing a defect in muscle regeneration by inducing increased apoptosis of satellite cells (47). Necroptosis is induced by extracel- lular stimuli, ﬁrst by TNF, and is mediated by RIPK3 via TNF receptor. RIPK3 is a downstream target of RIPK1, and the complex containing RIPK1 and RIPK3 is involved in initiating necroptosis (48–50). Other molecules involved in necroptosis, such as IL-33 and damage-associated molecular patterns (DAMPs) such as S100A8, S100A9, and S100A14 were also increased in LPS-stimulated crypt cultures. Differences observed following stimulation with different LPSs puriﬁed from CSCM members are likely to respond to structural differences of their corresponding lipid A. For instance, D. tsuru- hatensis LPS that appeared the least active showed underacylation of its lipid A with very short fatty acid chains, a characteristic associated with a weak endotoxin activity (19, 20, 51). The association of endotoxins of different potencies may thus result in optimal endotoxic balance achieving the exact regulatory function on the crypt regen- erative functions by regulating the life-to-death balance in the stem cell population.
16 Read more
Although IK channels dominate the basolateral K þ conductance in human colon . basolateral membranes of mouse small intes- tinal and colonic crypt cells also contain small conductance K þ (SK or KCNQ1/KCNE3) channels [6 e 8]. However, the possible existence of KCNQ1/KCNE3 channels and their contribution to the basolateral K þ conductance of human colonic crypt cells, has never been explored. KCNQ1 is a K þ channel protein with six transmembrane domains, and interacts physically with the smaller b -subunit KCNE3 protein, which has a single transmembrane domain . KCNE3 changes the biophysical properties of KCNQ1, the KCNQ1/ KCNE3 complex forming a constitutively open basolateral K þ channel, which is present in mammalian small intestine and colon, is readily stimulated by cAMP, markedly inhibited by chromanol 293B, and is a prerequisite for cAMP-stimulated electrogenic Cl secretion, such as that triggered by a variety of intestinal pathogens . The role of KCNQ1/KCNE3 channels appears to be to maintain * Corresponding author. Clinical Sciences Building (Level 7), St James's University
displaying different levels of β-catenin accumulation and downstream gene expression (Fig. 6A), and tumour cells with nuclear β-catenin are dividing more slowly than surrounding tumour cells, suggesting that such cells are similar to cells at the crypt bottom of the normal colon. Thus, we propose that a hierarchical control of cell proliferation in the colonic crypt epithelium is retained to some extent in colonic neoplasms. Accordingly, we found that tumour cells with nuclear β-catenin are accompanied by high Notch signalling (Fig. 6D), as has been reported in crypt bottom cells (Kayahara et al., 2003). It is interesting to note that a γ-secretase inhibitor turned slow-cycling cells into actively proliferating cells (Fig. 4C-G; supplementary material Fig. S7A). A previous study showed that Notch inhibitors turn undifferentiated, proliferating cells into quiescent cells in colorectal neoplasias (van Es et al., 2005), indicating that the Notch inhibitor might be of therapeutic benefit in colorectal cancers. The discrepancy in the effects of Notch inhibitor could be explained by differences in states of the affected cells between proliferating progenitor cells and ISC- like cells. Although the previous study showed effects on the transition of proliferating cells into terminally differentiated quiescent cells, our data suggest that a Notch inhibitor may promote the transition of slow-cycling ISC-like cells into progenitor cells in the colon. Considering the chemoresistance of slow-cycling cancer stem cells, the results also suggest that Notch inhibitors combined with chemotherapeutic agents and/or irradiation might be effective as treatments targeting slow-cycling cancer stem cells in the colon. In summary, our results indicate that, although proliferating progenitor cells in colonic crypts physiologically express higher levels of β-catenin/Tcf transcriptions, a further activation of the canonical Wnt singalling leads to de novo crypt formation, consisting of relatively slow-cycling cells in the adult colon, which is accompanied by activation of Notch signalling with transactivation of Notch ligands and receptors. However, treatment with a Notch/γ-secretase inhibitor turns such slow-cycling cells into proliferating cells, although we cannot exclude the possibility that some of the observed phenotypes are the result of superphysiological b-catenin expression obtained with our transgenic system. These findings suggest that Wnt and Notch signalling act in a synergistic and hierarchical manner to control differentiation and proliferation of the colonic crypt epithelium in vivo.
10 Read more
While FAP is relatively uncommon (1 in 10,000 individuals), results reported here have wider implications for understanding mechanisms involved in development of commonly occurring sporadic adenomatous polyps (1 in 2 individuals) and sporadic CRC (1 in 20 individuals) . Thus, in both FAP and sporadic cases, mutations of APC genes and tumor formations are known initial and final events of CRC development . While mechanistic steps between initiation and CRC formation may be the same or different in FAP and sporadic cases, descriptions of logical mechanistic steps for FAP will augment and focus companion studies of sporadic CRC development, and simultaneously, promote opportunities for discovering new disease control strategies. Because a critical part of mechanism design requires testing proposed processes with quantitative data, a useful mechanistic process for CRC development must provide logical links between quantitative data and qualitative cellular information. Hence, to identify kinetic mechanisms involved in CRC development, we calculated latenzzeit of colonic crypt cells using data on crypt cell cycle times from FAP patients who carry APC mutations.
were sacrificed after receiving drinking water with or without 5% DSS for 7 days. The colons were collected and processed for histological studies as described in Methods. (A) The severity of epithelial injury was assessed by a grading system (22) described in Methods. Proximal colon, WT and KO, n = 12 and 9, respectively; *P < 0.01; distal colon, WT and KO, n = 11 and 11, respectively. Data are expressed as means ± SEM. (B). The percentages of fields involved with either grade I, II, III, or IV injury (23, 24) were quantified as described in Methods. WT, n = 9; KO, n = 9. Data are expressed as means ± SEM. # P < 0.005, ## P < 0.05. (C) Colonic crypt cell proliferation was assessed by quantification of 5-BrdU incorporation.
13 Read more
is that at least two stem cells — one COX-normal and the other COX-defi- cient — were involved in crypt forma- tion. Thus, all three patterns are con- sistent with the notion that a colonic crypt is indeed a clonal population derived from one or more stem cells. Upon sequencing the sectioned crypts, they found many crypts with a normal mtDNA genotype (no surprise here), but in many crypts they found numerous mtDNA mutations, both in COX-negative crypts (many, but not all, mutations were in COX or protein synthesis genes) and in some of the ostensibly normal COX-positive crypts (mainly neutral mutations or muta- tions in non-COX genes). Interesting- ly, there were some COX-negative crypts in which no mtDNA mutations were found at all — presumably there were nuclear mutations in these stem cells that affected COX function. Over- all, the amount of mutated mtDNA was extremely high (on average, one mtDNA point mutation per crypt), and, as has been known for a dozen years now (2), the mutational “load” increased with age.
Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (lCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded lCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by lCgN alone. Combined lCgN/MNU treatments induced greater MTCRII (Po0.01) as well as greater number (Po0.001) and crypt multiplicity (Po0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r ¼ 0.732; Po0.01). MTCRII are induced by lCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium- term murine bioassay relevant to early-stage colorectal tumorigenesis.
However, from day 7 first three cell parameters increased and dead cells decreased continuously upto day 30 in all experimental sets upto day 30, while this cell kinetic study was not possible in 8 and 10 Gy irradiation alone sets of animals due to their death. Employing a cell kinetic approach, earlier workers have also described similar alterations in a number of cell parameters of mouse intestinal epithelium after irradiation (Quastler and Sharman, 1959; Devik, 1968; Hagemann et al., 1970, 1971; Sigdestad et al., 1970; Tsubouchi and Matsuzawa, 1973; Uma Devi et al., 1979 and Delaney et al., 1994). Exposure to different doses (6,8 and 10 Gy) markedly damaged the stem cells and ceased the mitotic activity in jejunal crypts (fig. 1). Therefore, a significant and consistent decrease in mitotic figures, crypt epithelial cells / crypt section and villus height and increase in dead cell number was noticed from day 1- 3 (figs.1- 4 ). The changes in these cell parameters were found to be dose and time dependent. Similarly Devik (1971), Uma Devi (1979) and Samrath et al. (2002) have also reported the dose dependent reduction in mitotic activity and thereby a decrease in crypt epithelial cells and villus height. A constant flow of cells occurs from crypt to villus in intestine to maintain the steady state relationship between proliferative (crypt) and nonproliferative (villus) zone to ensure an unbroken mucosal covering of the villi.
12 Read more
collect this material as it is impractical, unpleasant and logistically challenging, although this has been done in previous studies, e.g. Jalanka et al. , using a different laxative (Moviprep). As the colon is a large and capacious tube, it can never be completely evacuated and inevitably some liquid remains in the bowel. The remaining liquid is therefore bathing the colonic mucosa and tends to shift naturally around the bowel with the aid of peristalsis and the constant changes in the movement and posture of the subject (e.g. supine vs recumbent or semi-recumbent posi- tions). This lavage fluid is often encountered at the time of colonoscopy and is very easy to aspirate through a suction channel in the colonoscope and directly into a collecting tube. Both biopsies and colonic lavage samples were collected simultaneously, lavage followed by biopsy, therefore observed relationships are representative of ac- curate comparisons between sample types and most likely reflect the scope of the majority of such samples available for collection for research studies.
15 Read more
mainly, to the lineage of traditional yoghurt bacteria combined with a specific bacillus, DanRegularis, which gives the product a proven capacity to intestinal regulation in humans. The aim of this study was to evaluate the antigenotoxic, antimutagenic, and anticarcinogenic proprieties of the Activia product, in response to damage caused by 1,2-dimethylhydrazine (DMH) in Swiss mice. Activia does not have shown antigenotoxic activity. However, the percent of DNA damage reduction, evaluated by the antimutagenicity assay, ranged from 69.23 to 96.15% indicating effective chemopreventive action. Activia reduced up to 79.82% the induction of aberrant crypt foci by DMH. Facing the results, it is inferred that Activia facilitates the weight loss, prevents DNA damage and pre-cancerous lesions in the intestinal mucosa.
12 Read more
within the surrounding water (Figure 2). In most teleosts, the shape of the OE is folded into lamellae whose numbers vary from a few to about 300 depending on the species (Hansen and Reutter 2004). Each lamella consists of a nonsensory region and a sensory region, containing nonsensory cells and olfactory sensory neurons, respectively. Depending on the species, the thickness of the olfactory epithelium ranges from about 15µm (zebrafish, Danio rerio) to 110µm (pike, Esox lucius) (Holl 1965). The nonsensory region is comprised of supporting cells, nonsensory ciliated cells and basal cells. Basal cells are mitotically active and differentiate into new OSNs, therefore they are competent for a life-long renewal of the OE. At least three types of olfactory sensory neurons (OSNs) have been identified in actinopterygian (ray-finned) fishes, which are intermingled within the sensory region of the teleost olfactory epithelium and show different morphological appearance as well as different expression patterns of olfactory receptors (explained in detail in 1.2.1). Ciliated (cOSNs) and microvillous (mOSNs) olfactory sensory neurons, also present in the rodent MOE and VNO, respectively, make up the most prominent populations of receptor neurons. A third OSN type, named crypt cells, represent a comparatively small population of OSNs and are absent in the mammalian olfactory system. Recently, a fourth OSN type, kappe neurons was identified in the zebrafish OE (Ahuja et al. 2014). Every OSN expresses one type of olfactory receptor at its apical dendritic protrusions, which might be cilia or microvilli, or in the case of crypt cells both (Table 1). The binding of an odorant will lead to a change of the OSNs membrane potential and therefore triggering action potentials which propagate the information along the axon. OSN axons, which express the same receptor, project into the olfactory bulb (OB) into the same defined neuropil structures, named glomeruli, and make synaptic contacts onto projection neurons which in turn process and mediate olfactory information to higher brain areas such as the telencephalon (explained in detail in 1.2.2.).
144 Read more
Each biopsy from contralateral wall (CO) and mid sig- moid (MS) area was scored in three categories: surface staining intensity, crypt staining intensity relative to surface staining, and extent of crypt staining. A maxi- mum of six crypts from six available sections on two slides were chosen for scoring from each biopsy. Means were calculated for each category scored. Inten- sity of surface staining was scored from 3 to 0, where 3 represented the best staining and 0 being minimal or no staining. Intensity of crypt staining was compared with intensity of surface staining and was scored 3 if better than surface staining, 2 if similar to surface staining and 1 if less than surface. Zero score was awarded if minimal or no staining observed in crypt. Score 3 was awarded if the whole extent of the crypt was stained. Score 2 being 2/3 and 1 being 1/3 of the crypt. If only few random cells were stained, that sec- tion was considered negative for scoring purposes. Immunohistochemical sections from cancer sites were scored from 0 to 3 on the basis of proportion of cells showing positive staining. Score 3 if more than 60% of cells were positively stained for K8; 2 if around half of the cells stained and 1 if less than 40% of the cells were stained with K8.
Results: A total of 17,456 subjects were enrolled. The prevalence of colonic diverticulosis and adenoma was 2.4% and 13.2%, respectively. With regard to distribution of diverticula, most (365/424, 86.1%) were right-sided. Multiple logistic regression analysis suggested that age and male gender were independent risk factors for adenoma and advanced adenoma. There was no relationship between diverticulosis or location of diverticulosis and presence of adenoma and advanced adenoma adjusting by age and gender. In a stratified analysis according to age and gender, similar results were also noted.
In spite of the fact that colonic lipomas have unusual symptoms, they should be taken into consideration for differential diagnosis of tumors of the large intestine. Surgical resection may be a feasible and safe therapy for most symptomatic lipomas. Occasionally, the colonic lipoma may separate from its root and be expelled from the rectum; most of these have a favorable prognosis. However, we recommend that surgeons perform re-colonoscopy inspec- tion after expulsion and strengthen their aware- ness of hemorrhagic focal ulcerations and presence of residual tissue. These steps would help in the timely detection and intervention of hemorrhages and perforations and aid in regu- lar follow up.
utilizing a continuous sucrose gradient. In the villus cells, Na:H exchange activity was found associated with both the brush border and basolateral membrane, whereas, in crypt cells, Na:H exchange activity was only found on the basolateral membrane. Cl:HCO3 exchange activity was found only on the brush border membrane, in both villus and crypt cells. These studies suggest functional heterogeneity in ion transport between […]
Colorectal cancers are the third most common type of cancer. They originate from intestinal crypts, glands that descend from the intestinal lumen into the underlying connective tissue. Normal crypts are thought to exist in a dynamic equilibrium where the rate of cell production at the base of a crypt is matched by that of loss at the top. Understanding how genetic alterations accumulate and proceed to disrupt this dynamic equi- librium is fundamental to understanding the origins of colorectal cancer. Colorectal cancer emerges from the interaction of biological processes that span several spatial scales, from mutations that cause inappropriate intra- cellular responses to changes at the cell/tissue level, such as uncontrolled proliferation and altered motility and adhesion. Multiscale mathematical modelling can provide insight into the spatiotemporal organisation of such a complex, highly regulated and dynamic system. Moreover, the afore- mentioned challenges are inherent to the multiscale modelling of biological tissue more generally. In this review we describe the mathematical ap- proaches that have been applied to investigate multiscale aspects of crypt behaviour, highlighting a number of model predictions that have since been validated experimentally. We also discuss some of the key mathe- matical and computational challenges associated with the multiscale mod- elling approach. We conclude by discussing recent efforts to derive coarse- grained descriptions of such models, which may offer one way of reducing the computational cost of simulation by leveraging well-established tools of mathematical analysis to address key problems in multiscale modelling.
27 Read more
Our study was developed in four steps by taking advantage of: (I) DNA Microarrays, (II) Taqman high density cards, (III) Taqman single assay qRT-PCR and (IV) in situ hybridization. To achieve more robust results, we used three different groups of patients (Table 1). The first group included 66 Italian patients (III Clinica Chirurgica, Universita’ di Roma ‘‘La Sapienza’’, Rome, Italy) with colonic adenocarcinoma: 18 patients did not have any metastasis, 33 had affected lymph nodes and 15 showed metastases at both lymph nodes and liver. Total RNAs from 18 colonic primary tumors (T) with no metastasis, 33 colonic primary tumors (T N ) with lymphonodal recurrences and the corresponding 33
11 Read more