Changes in the synthesis of ribosomal ribonucleic acid and of poly(A)-containing ribonucleic acid during the differentiation of intestinal epithelial cells in the rat and in the chick.

Epithelial cells were isolated from rat and chick small intestine by techniques which separated subpopulations of differentiating villus and upper crypt cells from each other and from populations of mitotically dividing lower crypt cells. Incorporation of precursors into epithelial-cell DNA, cytoplasmic rRNA and cytoplasmic poly(A)-containing RNA occurred in the lower crypt cells in vivo when precursor was supplied from the vascular system of the intestine. Incorporation of precursor into 28S and 18S rRNA continued in the upper crypt cells, but occurred to only a very slight extent (if at all) in villus cells, whereas incorporation into poly(A)-containing RNA continued (at a diminishing rate) as the differentiating cells migrated along the villi. When precursor was supplied from the intestinal lumen, its incorporation into DNA and into rRNA of crypt cells was not very different from that observed with the other mode of precursor administration, but incorporation into villus-cell poly(A)-containing RNA then occurred at essentially the same rate in all intestinal epithelial cells in vivo. Cytoplasmic poly(A)-containing RNA appeared to turn over in rat crypt cells with a half-life not exceeding 24 h; crypt-cell rRNA showed no turnover and no evidence could be found for the presence of 'metabolic DNA'.     

In vivo effects of epidermal and fibroblast growth factors on DNA replication in mouse skin

A method was developed for measuring in vivo DNA synthesis after exposure to epidermal growth factor (EGF) or fibroblast growth factor (FGF) in a local area of mouse skin using ring-shaped forceps in combination with autoradiography. The technique should be useful for analysing the effects of growth factors on individual cells of the skin in vivo. EGF induced semisynchronized DNA synthesis in basal cells of the epidermis dose-dependently, but FGF did not. Time course study showed that EGF-induced DNA synthesis in basal cells increased with time for 24 h, and then decreased rapidly. EGF-induced DNA synthesis in basal cells was proportional to the time exposed to EGF (0-60 min). FGF and EGF both had little effect on dermal fibroblastic cells. The discrepancy between in vivo observations and those with cultured mammalian cells is discussed.     

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Summary Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.Supported by a research grant from the Medical Research Council of Canada (J.H.)     

The crypt cycle. Crypt and villus production in the adult intestinal epithelium.

We propose a model for the growth of individual crypts that is able to account for the observed changes in the number of cells in crypts under normal conditions, after irradiation, and after 30% resection. Parameter values for this model are estimated both for mouse and man, and detailed predictions of crypt growth rates are made. This model does not predict a steady-state crypt size; rather it suggests that crypts grow until they bifurcate. We therefore propose a crypt cycle (analogous to the cell cycle) and present evidence that most if not all crypts in the adult mouse are cycling asynchronously and independently. This evidence consists of four experiments that indicate that branching crypts are randomly distributed over the intestinal epithelium, that the plane of bifurcation of branching crypts is randomly oriented with respect to the villus base, and that the size distribution of crypts is consistent with an expanding crypt population. We also report for the first time evidence of villus production in the adult mouse intestinal epithelium. We conclude that the crypt and villus populations in the adult mouse are not in a steady state.     

DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon

Colon epithelium is made up of two general classes of cells, surface cells which are post-mitotic and crypt cells which contain the proliferative population. Their relative vulnerability to environmental damage and ability to perform DNA repair are important issues in colon carcinogenesis. DNA damage and repair was studied by the nucleoid sedimentation method in freshly isolated crypt cells for comparison with previous studies of post-mitotic surface epithelial cells. Suspensions of crypt cells were isolated from preparations of mouse colon by a series of sequential incubations in buffer containing 1.5 mM EDTA. Treatment of crypt cells for 30 min with 1.2 X 10(-6) M methyl methane sulfonate (MMS), photoaffinity labeling with 1 X 10(-6) M ethidium monoazide, lithocholic acid (2.5 X 10(-4) M) treatment for 1 h or X-irradiation at 1500 rads resulted in single-strand breaks in the DNA, which were repaired after 2 h of additional incubation. Interestingly, X-rays at 1000 rads and lithocholic acid (LA) (2.5 X 10(-6) M) after 30 min incubation failed to produce the detectable shift in nucleoid sedimentation characteristic of single-strand breaks, perhaps due to rapid repair by these proliferative cells. UV-irradiation failed to provoke strand incision as was also observed for the superficial post-mitotic cells in the previous studies. These studies showed the feasibility of studying DNA damage and repair processes in these two classes of colon epithelial cells in response to specific carcinogenic insult.     

Circadian rhythm in the rate of cellular proliferation and in the size of the functional compartment of mouse jejunal epithelium

Circadian variation in the small bowel mucosa of male Balb/c mouse was studied. The labelling was studied at 2 hourly intervals throughout a 24 h period by using autoradiographic techniques with 3HTdR. A 12 h light-dark schedule was employed. Villus and crypt cell populations, together with the mitotic index, were studied using the micro-dissection technique. Growth fractions were determined from the labelling index distribution curves. The peaks in both villus and crypt cell population occurred during the day, with maximum villus population of 3,887 cells/villus at 13.00 h and maximum crypt population of 178 cells per crypt at 09.00 h. The peaks of labelling index (Is) and mitotic index (Im) occurred during the dark period. The peak value of Is 38% at 17.00 h-19.00 h, was about 6-8 h in advance of the peak value of Im (6%). The peak in growth fraction corresponded to that of the labelling index.     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

Overlapping DNA Methylation Dynamics in Mouse Intestinal Cell Differentiation and Early Stages of Malignant Progression

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2^high), differentiated cells (EphB2^negative), Apc^Min/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in Apc^Min/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.     

The effect of a single injection of cytosine arabinoside on cell population kinetics in the mouse jejunal crypt

The early effects of a single injection of cytosine arabinoside (ara-C) on cell population kinetics in the jejunal crypt of the mouse were studied using autoradiography with tritiated thymidine, and metaphase arrest with vincristine. Ara-C had three main effects on crypt cells: a block of cells near the transition from G1 to S, death of nearly all cells in S, and a temporary block of the survivors, which remained viable and were able to proceed through the cell cycle. Throughout the crypt there was a decrease in cell cycle time and an increase in growth fraction. Although changes in proliferative rate were highest in the lowest part of the crypt it was not possible to show that crypt repopulation originated only from basal crypt cells, and the data are consistent with repopulation from the faster cycling cells in the proliferative compartment.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

p53 and cell-cycle-regulated protein expression in small intestinal cells after fast-neutron irradiation in mice

The involvement of the p53 gene in apoptosis of many cell types towards -radiation is well established. However, little information is available on the relationship between p53 status and cells ability to undergo apoptosis following exposure to fast neutrons. The aim of this study was to characterize the apoptotic pathway traveled by neutrons in mouse intestinal crypt cells. Each mouse received whole body doses of 0.25–8 Gy fast neutrons and were sacrificed 0, 4, 6, 12, 24, 48, and 72 h, respectively, after irradiation. Apoptosis of crypt cells and expression of p53, cyclin A, cyclin B, cyclin D, and cyclin E were measured. The apoptosis in crypt cells was maximal at 4 and 6 h after irradiation, showing a gradual decline at 24 h. The highest frequency of apoptosis was seen at a 1 Gy dose and then declined gradually beyond a 2 Gy dose with high levels of damage. In immunoblot analysis, apoptosis was confirmed to be dependent on p53 function after fast-neutron irradiation. In addition, cyclin B1, cyclin D, and cyclin E were overexpressed in intestinal cells after fast-neutron irradiation and their immunoreactivities were increased strongly in round and oval cells of laminar propria in villi core and crypts. The results of the current study suggest that apoptosis in crypt cells shows a time- and dose-dependent increase after fast-neutron irradiation. In addition, fast-neutron-induced apoptosis in mouse intestinal crypt cells appears to be related to the increase in functional p53 proteins to a level sufficient to initiate apoptosis and up-regulation of cell-cycle-regulated proteins, which may lead to resistance to DNA damage through cell cycle arrest, is involved deeply in protection of gastrointestinal cells after low doses of fast-neutron irradiation. (Mol Cell Biochem 270: 21–28, 2005)     

DNA replication in asynchronous and synchronous ehrlich ascites cells in different conditions of growth

Ehrlich ascites tumour cells were labelled for DNA fibre autoradiography within the peritoneal cavity of a tumour-bearing mouse. The generation and the evaluation of the autoradiographic patterns is described and discussed. To study possible changes of the autoradiographic patterns during a natural S phase the labelling was performed in the mouse or in culture with asynchronous cells which were afterwards separated into synchronous subpopulations by zonal centrifugation. The subpopulations obtained were characterized by flow cytofluorometry in connection with the thymidine labelling index. We compared the DNA fibre autoradiographic patterns of several synchronous and asynchronous cell populations growing in the mouse or under different conditions in culture: The replicon size distributions of all populations examined were virtually the same. The fork movement rate was found to depend mainly on the metabolic condition of the cells. In culture it was significantly slower than in the mouse although a shortened S phase and therewith an increased DNA synthesis rate occurred. During a natural S phase it increased slightly, at most, while the DNA synthesis rate was considerably enhanced at the end of S. The changes in the rate of total DNA synthesis cannot account for the changes in the rate of chain growth. We conclude that the DNA synthesis rate is regulated almost exclusively by changing the replicon initiation frequency, while the fork movement rate is limited by the actual metabolic condition of the cells.     

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.     

DNA synthesis studies in pre-meiotic mouse oogenesis

DNA fiber autoradiography was used in mouse oogenesis to test Callan's hypothesis that a longer S phase results from a reduction in the number of active initiation sites. The data indicated that the premeiotic increase in the duration of S phase in mouse oogenesis was characterized by a rapid initial rate of chain growth and a larger replicon size when compared with replicating DNA of somatic mouse cells. These findings were at variance with those in mouse spermatogenesis and also did not support the Callan hypothesis of activation site repression.     

Double labelling with bromodeoxyuridine and [3H]-thymidine of proliferative cells in small intestinal epithelium in steady state and after irradiation

The simultaneous immunohistochemical detection of bromodeoxyuridine (BrdU) and [3H]-thymidine ([3H]TdR), by conventional autoradiography, was performed on the mouse small intestine (ileum). Proliferation was studied under normal conditions as well as after 3 Gy of gamma-rays. The BrdU method in conjunction with [3H]TdR autoradiography appears to be reliable and useful for the study of cell kinetics especially in disturbed states, on condition that [3H]TdR is delivered to the animals before BrdU. It has been found that cells in the crypt are delayed by irradiation in their progression through the cell cycle predominantly in late S phase. The cells at the bottom of the crypt are more affected than the more differentiated but proliferating cells in the upper part of the crypt.     

Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFP(SAT), demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues.     

Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase alpha activity

An approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2 alpha, insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca++-containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine-triphosphate for 1 hr at 37 degrees C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)-precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density-labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole-cell lysates prepared with a hypotonic buffer not containing Ca++. Using various templates, it was shown that the increase in activity of DNA polymerase alpha correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase beta activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.     

Analysis of DNA damage and repair accompanying differentiation in the intestinal crypt.

The ability to process damaged DNA may vary between cells depending on their differentiated status. However, there is little in vivo data available and it is not intuitively obvious how the activity of specific repair pathways may vary between different subpopulations (e.g. stem cells and proliferative, committed and differentiated cells) of a particular tissue. To obtain such information for the intestinal epithelium, we have developed an assay that detects differences in the way different regions of the crypt (stem, proliferative and maturation zones) respond to DNA damage. The assay is a variant of the ''comet'' assay, which detects DNA strand breaks by measuring the proportion of DNA migrating from individual cells, or in this case intact isolated crypts, in an electrophoretic field. The method is quantitative, with the amount of migrating DNA being proportional to the number of strand breaks. Isolated crypts are repair competent and spatial differences are apparent with some agents. The assay has the potential to characterize the repair properties of cells at different stages of differentiation within the crypt, determine the characteristics that might predispose them to damage and may help in understanding the route of stem cell mutation.     

Bringing target-matched PI3King from the bench to the clinic

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.     

Modeling Cell Dynamics in Colon and Intestinal Crypts: The Significance of Central Stem Cells in Tumorigenesis

Colon and intestinal crypts have been widely chosen to study cell dynamics because of their fairly simple structures. In the colon and intestinal crypts, stem cells (SCs) are located at very bottom of the crypt, fully differentiated cells (FDs) are located in the top of the crypt, and transit-amplifying cells (TAs) are in the middle of the crypt between FDs and SCs. Recently, it has been discovered that there are two types of stem cells in the intestinal crypts: central stem cells (CeSCs) and border stem cells. To investigate dynamics of mutants in colon and intestinal crypts, we develop a four-compartmental stochastic model, which includes two SC compartments, and TAs and FDs compartments. We calculate the probability of the progeny of marked or mutant cells located at each of these compartments taking over the entire crypt or being washed out from the crypt. We found that the progeny of CeSCs will take over the entire crypt with a probability close to one. Interestingly, the progeny of advantageous mutant TAs and FDs will be washed out faster than disadvantageous mutants. Saliently, the model predicts that the time that the progeny of wild-type central stem cells will take over the mouse intestinal crypt is around 60 days, which is in perfect agreement with an experimental observation.