Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo.

The gastrointestinal tract, with its rapid cell replacement, is sensitive to cytotoxic damage and can be a site of dose-limiting toxicity in cancer therapy. Here, we have investigated the use of one growth modulator to manipulate the cell cycle status of gastrointestinal stem cells before cytotoxic exposure to minimize damage to this normal tissue. Transforming growth factor beta-3 (TGF-beta3), a known inhibitor of cell cycle progression through G1, was used to alter intestinal crypt stem cell sensitivity before 12-16 Gy of gamma irradiation, which was used as a model cytotoxic agent. Using a crypt microcolony assay as a measure of functional competence of gastrointestinal stem cells, it was shown that the administration of TGF-beta3 over a 24-h period before irradiation increased the number of surviving crypts by four- to six-fold. To test whether changes in crypt survival are reflected in the well-being of the animal, survival time analyses were performed. After 14.5 Gy of radiation, only 35% of the animals survived within a period of about 12 days, while prior treatment with TGF-beta3 provided significant protection against this early gastrointestinal animal death, with 95% of the treated animals surviving for greater than 30 days.     

The initiation of colon cancer in a chronic inflammatory setting

Chronic inflammation predisposes to cancer. We used an inflammation-induced human model of tumorigenesis to explore how populations of mutated cells expand and initiate the earliest stages of cancer. Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with an increased risk of colorectal cancer mediated through a process of genomic instability. In order to characterize the process of clonal expansion, arbitrary primed (AR) and inter-simple sequence repeat (ISSR) PCR DNA fingerprint mutation profiles of single crypts were compared with the mutational profiles from clusters of crypts and whole biopsies within the same individual. To provide information at the earliest steps of neoplastic progression, we examined histologically negative crypts, as well as dysplastic crypts. Crypts from UC dysplasia/cancer show alterations in 10-20% of DNA fingerprint sites, regardless of (i) whether the crypts were dysplastic or non-dysplastic and (ii) whether the DNA came from one crypt or thousands of crypts. Of the mutational changes in single crypts, almost half are clonally expanded to adjacent crypts and/or to the thousands of crypts in a single biopsy. Using fluorescent in-situ hybridization to examine p53 alterations in individual crypt cells, we demonstrate that the mechanism of clonal expansion can occur through crypt fission. DNA alterations are initiated in colonic crypts and expand to adjacent crypts through crypt fission. Our data suggest that a continuous process of DNA mutations, clonal expansion through crypt fission and clonal succession initiates the development of inflammatory-associated colon cancer; this mutational process is moderated by crypt cell turn-over and cell death. This paradigm may apply to other inflammatory-induced cancers.     

How dysregulated colonic crypt dynamics cause stem cell overpopulation and initiate colon cancer

Based on investigation of the earliest colonic tissue alteration in familial adenomatous polyposis (FAP) patients, we present the hypothesis that initiation of colorectal cancer by adenomatous polyposis coli (APC) mutation is mediated by dysregulation of two cellular mechanisms. One involves differentiation, which normally decreases the proportion (proliferative fraction) of colonic crypt cells that can proliferate; the other is a cell cycle mechanism that simultaneously increases the probability that proliferative cells are in S phase. In normal crypts, stem cells (SC) at the crypt bottom generate rapidly proliferating cells, which undergo differentiation while migrating up the crypt. Our modeling of normal crypts suggests that these transitions are mediated by mechanisms that regulate proliferative fraction and S-phase probability. In FAP crypts, the population of rapidly proliferating cells is shifted upwards, as indicated by the labeling index (LI; i.e., crypt distribution of cells in S phase). Our analysis of FAP indicates that these transitions are delayed because the proliferative fraction and S-phase probability change more slowly as a function of crypt level. This leads to expansion of the proliferative cell population, including a subpopulation that has a low frequency of S-phase cells. We previously reported that crypt SC overpopulation explains the LI shift. Here, we determine that SCs (or cells having high stemness) are proliferative cells with a low probability of being in S phase. Thus, dysregulation of mechanisms that control proliferative fraction and S-phase probability explains how APC mutations induce SC overpopulation at the crypt bottom, shift the rapidly proliferating cell population upwards, and initiate colon tumorigenesis.     

Kinetics and possible regulation of crypt cell populations under normal and stress conditions.

The proliferative organisation of the crypts of the small intestine is considered with special reference to the existence, location and numbers of stem cells. It is concluded that the crypt contains a minority population of cells at its base that are the true stem cells. These cells provide an input of cells for the larger proliferative compartment higher up the crypt. The presumptive stem cells may be pluripotent and produce Paneth, goblet and columnar cells. They are probably also the cells which are capable of regenerating the crypt after X-ray depopulation. Radiobiological experiments indicate that the number of cryptogenic cells is less than 80, while the results of several experiments on the kinetics of the cell populations indicate that the number of stem cells is about 20. The stem cells are located in the Paneth cell zone of the crypt, and are apparently passing through the cell cycle at about half the speed of the proliferative cells. It is these vital stem cells that will determine the response of the mucosa to therapeutic agents, probably play a role in carcinogenesis and play a dominant role in mechanisms controlling cell proliferation.     

Progastrin Peptides Increase the Risk of Developing Colonic Tumors: Impact on Colonic Stem Cells

Preneoplastic lesions (aberrant crypt foci, hyperplastic/dysplastic polyps) are believed to be precursors of sporadic colorectal tumors (adenomas, adenocarcinomas). Aberrant crypt foci and hyperplastic/dysplastic polyps likely originate from abnormal growth of colonic crypts in response to aberrant queues in the microenvironment of colonic crypts. Thus, identifying factors which regulate homeostatic versus aberrant proliferation/apoptosis of colonocytes, especially stem/progenitor cells, may lead to effective preventative/treatment strategies. On the basis of this philosophy, the role of growth factors/peptide hormones potentially available in the circulation/microenvironment of colonic crypts is being examined extensively. Since the time gastrins were discovered as trophic (growth) factors for gastrointestinal cells, the effect of gastrins on the growth of normal/cancer cells has been investigated, leading to many discoveries. Seminal discoveries in the area of gastrins and colon cancer as it relates to molecular pathways associated with formation of colonic tumors are reviewed and the possible impact on diagnostic/preventative/treatment strategies is discussed.     

The suppression of aberrant crypt multiplicity in colonic tissue of 1,2-dimethylhydrazine-treated C57BL/6J mice by dietary flavone is associated with an increased expression of Krebs cycle enzymes

Colorectal cancer is the second leading cause of cancer deaths worldwide with diet playing a prominent role in disease initiation and progression. Flavonoids are secondary plant compounds that are suggested as protective ingredients of a diet rich in fruits and vegetables. We here tested whether flavone, a flavonoid that proved to be an effective apoptosis inducer in colon cancer cells in culture, can affect the development of aberrant crypt foci (ACFs) in C57BL/6J mice in vivo when preneoplastic lesions were induced by the carcinogen 1,2-dimethylhydrazine (DMH). Flavone applied at either a low dose (15 mg/kg body wt per day) or a high dose (400 mg/kg body wt per day) reduced the numbers of ACFs significantly, independent of whether it was supplied simultaneously with the carcinogen (blocking group) or subsequent to the tumor induction phase (suppressing group). Proteome analysis performed in colonic tissue samples revealed that flavone treatment increased the expression of a number of Krebs cycle enzymes in the suppressing group and this was associated with reduced crypt multiplicity. It suggests that mitochondrial substrate oxidation is increased by flavone in colonic cells in vivo as already observed in HT-29 cells in vitro as the prime mechanism underlying tumor cell apoptosis induction by flavone. In conclusion, flavone reduces the number of ACFs in DMH-treated mice at doses that can be achieved for flavonoids by a diet rich in fruits and vegetables. Moreover, reduction in crypt multiplicity by flavone is most probably due to the preservation of a normal oxidative metabolism.     

The relationship between ionizing radiation-induced apoptosis and stem cells in the small and large intestine.

Apoptosis is observed in the crypts of the small intestine of healthy animals and man (spontaneous apoptosis). The levels can be dramatically elevated 3-6 h following ionizing radiation exposure. Both the spontaneous and radiation-induced apoptosis in the small intestine crypts are most frequently observed at the positions in the crypt associated with stem cells (about four cell positions from the base of the crypt). The number of apoptotic deaths can be counted in routine histological preparations, but interpretation of the counts is complicated by numerous factors. However, recording the number of cells containing one or more apoptotic fragments in crypt sections provides a good estimate for the absolute number of cell deaths in crypts. Similarities are noted in the frequency and cell positional relationship of radiation-induced apoptosis in the small intestine of various strains of mice and one strain of rat. Apoptosis in the large intestine is generally lower in frequency than in the small intestine and, for the mid-colonic and rectal regions, has a different cell positional frequency distribution, with the highest apoptotic yield at the crypt base. The caecal colon has a pattern of apoptotic distribution more similar to that in the small intestine. After exposure to 1 Gy ionizing radiation, the maximum apoptotic yield occurs over a period of 3-6 h in the small intestine. There is some unexplained variability in the values between groups of mice and between different mouse strains. After 8 Gy, the yield remains elevated for several days, however a similar maximum yield is still observed at the early times. In mouse large intestine and rat small intestine, the yield continues to rise until about 6 Gy in mouse large intestine and until at least 10 Gy in rat small intestine. Spontaneous apoptosis is interpreted as part of the homeostatic mechanism regulating stem cell numbers. About 1.6 cells per crypt are dying at any one time. Following irradiation, there is an apparent relationship between mitotic and apoptotic levels, suggesting that these processes are linked. The dose-response relationship suggests that there are about six apoptosis-susceptible cells in crypts of the small intestine, with about 2-4 of these occurring at cell positions in which there are other more resistant clonogenic cells. In the large intestine, the position of these apoptosis-susceptible cells varies with region, but the numbers are similar.     

Tumoral mast cells exhibit a common spatial distribution

In the present study, we investigated whether human adipose tissue derived stem cells (hASCs) could enhance tumor invasion and whether these hASCs could be a potential source of CCL5. We observed a significant increase in the number of breast cancer cells that invaded the matrigel when Co-cultured with hASCs. We found that hASCs produce CCL5 in the Co-culture and cancer cell invasion was diminished by an antibody against CCL5. Furthermore, cancer cell invasion in the Co-culture was associated with an elevated level of MMP-9 activity. We conclude that CCL5 plays a crucial role for tumor invasion in the interplay of tissue resident stem cells from the fat tissue and breast cancer cells.     

Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.     

Death of intestinal crypts and of their constituent cells after treatment by chemotherapeutic drugs

The number and spatial distribution of necrotic cells in the jejunal crypts of mice, has been measured after treatment by each of 6 cytotoxic drugs. At the LD10/8 day dose of each drug, the majority of necrotic cells were found below position 9 and numbers per crypt were similar for all drugs (approximately 8). These findings resemble those for radiation. However, major differences between agents were found in the calculated numbers of the microcolony-forming units (MFU) that determine overall crypt survival or ablation after high doses of cytotoxic agent. Numbers of MFU as assayed by radiation were approximately 80 per crypt, but only 2 when assayed by mechlorethamine hydrochloride, adriamycin and 5-fluorouracil, and 7 using BCNU. No crypts were destroyed by either cyclophosphamide or actinomycin D, despite the appearance of numerous necrotic cells in the lower part of the crypt. We conclude that in drug-treated intestine, necrotic cells may arise from a non-MFU compartment and the incidence and distributions of such cells are likely to be poor indicators of the response of the MFU.     

P53 gene mutation increases progastrin dependent colonic proliferation and colon cancer formation in mice

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.     

Expression of ras oncogene mRNA and protein in aberrant crypt foci.

The expression of the ras oncogene in aberrant crypt foci was studied by both in situ hybridization and immunohistochemical approaches. Aberrant crypt foci are hypothesized to represent the earliest identifiable microscopic lesions of colon cancer in rodent colons. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg s.c.) once. Twelve weeks later, aberrant crypt foci were identified topographically, microdissected and processed for histology. In situ hybridization with an antisense oligomer of c-ras demonstrated increased expression of ras-specific RNA in aberrant crypts compared to normal crypts. A low amount of non-specific hybridization was obtained with the corresponding sense oligomer. The percentage of cells with grains (labeling index) was calculated in early and advanced aberrant crypt foci. This index was also calculated in normal appearing crypts. The labeling indices for the early and advanced aberrant crypt foci were significantly greater than that of normal crypts (18.0 and 25.0 versus 11.9). In the same tissue specimens, immunohistochemical staining for ras p21 with the monoclonal antibody (Y13-259) revealed strong staining intensity in early aberrant crypts (15/22) and advanced aberrant crypts (22/30) compared to normal crypts (3/50). The immunohistochemical results demonstrate the presence of elevated levels of ras p21 in the same tissue as increased levels of ras-specific message. This investigation provides the earliest demonstration of increased expression of the ras oncogene in precursor lesions of colon cancer possessing dysplastic features.     

Random Segregation of DNA Strands in Epidermal Basal Cells

According to the hypothesis proposed by Cairns, stem cells retain the older of the two parental DNA strands, whereas differentiating daughter cells receive the newly synthesized strand, so that a set of "immortal strands" persists in stem cells through successive cell divisions. To test this hypothesis, five successive divisions were induced in basal epidermal cells in vivo by two injections of cholera toxin into mouse skin and cells labeled with [³H]thymidine at the first cell cycle were chased for 50 days. If selective segregation occurs, the labeled strand should be transferred into a non-stem daughter cell after the second division and labeled cells would eventually be eliminated from the epidermis. However, the results suggest random segregation of DNA strands in epidermal basal cells. Labeled basal cells were persistently present throughout the whole epidermis for 50 days. Furthermore, labeled mitotic cells were found after the third division and their numbers of grains decreased exponentially through 5 cycles of divisions.     

Random segregation of DNA strands in epidermal basal cells

According to the hypothesis proposed by Cairns, stem cells retain the older of the two parental DNA strands, whereas differentiating daughter cells receive the newly synthesized strand, so that a set of "immortal strands" persists in stem cells through successive cell divisions. To test this hypothesis, five successive divisions were induced in basal epidermal cells in vivo by two injections of cholera toxin into mouse skin and cells labeled with [3H]thymidine at the first cell cycle were chased for 50 days. If selective segregation occurs, the labeled strand should be transferred into a non-stem daughter cell after the second division and labeled cells would eventually be eliminated from the epidermis. However, the results suggest random segregation of DNA strands in epidermal basal cells. Labeled basal cells were persistently present throughout the whole epidermis for 50 days. Furthermore, labeled mitotic cells were found after the third division and their numbers of grains decreased exponentially through 5 cycles of divisions.     

Are genetic determinants of asymmetric stem cell division active in hematopoietic stem cells?

Stem cells have acquired a golden glow in the past few years as they represent possible tools for reversing the damage wreak on organs. These cells are found not only in major regenerative tissues, such as the epithelia, blood and testes, but also in 'static tissues', such as the nervous system and liver, where they play a central role in tissue growth and maintenance. The mechanism by which stem cells maintain populations of highly differentiated, short-lived cells seems to involve a critical balance between alternate fates: daughter cells either maintain stem cell identity or initiate differentiation. Recent studies in lower organisms have unveiled the regulatory mechanisms of asymmetric stem cell divisions. In these models, the surrounding environment likely provides key instructive signals for the cells to choose one fate over another. Our understanding now extends to the intrinsic mechanisms of cell polarity that influence asymmetrical stem cell divisions. This article focuses on the genetic determinants of asymmetric stem cell divisions in lower organisms as a model for studying the process of self-renewal of mammalian hematopoietic stem cells.     

Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis.

Carcinogen-treated rats develop foci of aberrant crypts in the colon (ACFs) that have been interpreted as preneoplastic lesions. To characterise ACFs further, we studied in the unsectioned colon of rats the number, multiplicity, some morphological characteristics and the type of mucin production in ACFs. In ACFs observed 115 days after the administration of 50 mg kg-1 1,2-dimethylhydrazine (DMH), crypt multiplicity [number of aberrant crypts (AC) per focus] was positively correlated (P < 0.0001) with the reduction of goblet cells, and with luminal and nuclear alterations in the cells surrounding the lumen of the ACs. We studied mucin production in the unsectioned colon, demonstrating that ACFs producing sulphomucins (like the normal distal rat colon) were progressively reduced when ACF multiplicity increased, whereas ACFs containing sialomucins (correlated with an increased risk of colon cancer) or both sulphomucins and sialomucins increased with crypt multiplicity. We also studied ACFs in the colon and the occurrence of intestinal tumours in rats treated with azoxymethane (AOM; 64 mg kg-1). A significant association was found (P = 0.04) between tumours and the presence of ''large'' ACFs (AC/ACF > 14 crypts) and a borderline significant association (P = 0.057) between the presence of tumours and sialomucin-producing ACFs. We found no association between the number of ACFs, ACF multiplicity and the presence of tumours.     

The influence of age on colonic epithelial cell proliferation

Cancer of the large bowel is relatively rare in persons younger than 50 years of age, but its incidence increases sharply in persons older than 60 years of age. We thought that the evaluation of colonic cell proliferation, an accurate biomarker of predisposition to colorectal cancer, might help to elucidate the susceptibility of elderly persons to this common malignancy. Accordingly, 30 persons with normal lower endoscopy results were divided into three age groups (30 to 50,51 to 65, and 66 to 90 years of age; Groups 1, 2, and 3, respectively). Samples of rectal mucosa were taken at endoscopic examination, incubated with [3H]thymidine, and processed with standard autoradiographic techniques. At histologic examination, each intestinal hemicrypt was divided into five equal longitudinal compartments from the fundus (compartment 1) to the surface (compartment 5). The number and the position of labeled cells along the crypt were recorded. The total labeling index (LI) (the ratio of labeled cells to total cells) was significantly higher in Group 3 than in the two other groups. Similarly, the LI per crypt compartment in the most superficial portions of the crypts was consistently higher in persons older than 65 years of age (P less than 0.01 at least), indicating an expansion of the proliferative zone to the most superficial portion of the colonic glands. When the proliferative profiles of the three groups of subjects investigated were compared with those of patients with polyps, an almost complete overlap of values was observed between this population at increased risk for cancer and the subjects in Group 3. We conclude that aging is characterized by an overall increase of epithelial cell proliferation in colorectal mucosa and by an upwards expansion of the proliferative compartment, similar to that observed in a population at risk for cancer of the large bowel.     

Influence of diets containing high and low risk factors for colon cancer on early stages of carcinogenesis in human flora-associated (HFA) rats

Germ-free rats colonised with a human intestinal flora were fed diets containing high risk (HR) or low risk (LR) factors for colorectal cancer, and putative biomarkers were evaluated in the colonic mucosa; (i) proliferation, (ii) 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci and (iii) DMH-induced DNA damage. The HR diet was high in fat (45% of calories) and low in calcium and fibre, reflecting levels characteristic of typical western diets. The LR diet was low in fat (<5% of calories), and high in calcium and fibre. The nutrient/energy ratio of the two diets were similar. Mucosal crypt cell proliferation, assessed after microdissection, was higher on the LR diet (mean number of mitoses per crypt was 2.65 on the LR diet, and 1.62 on the HR diet; P < 0.05). Aberrant crypt foci (ACF) were assessed in the mucosa 12 weeks after DMH treatment. On the HR diet there were significantly more small ACF with 1 and 2 crypts per focus, but fewer ACF with 3, 5 and 7 or more crypts per focus. There was no significant difference in total ACF or the total number of crypts. The effect of diet on DNA damage in the colon was assessed in vivo by the comet assay. Animals were fed a HR or LR diet for 12 weeks before treatment with DMH or saline. For carcinogen-treated animals, DNA damage was significantly higher in colon cells from animals on the HR diet. On the LR diet both DNA damage and the induction of small ACF were reduced despite an increase in cell proliferation. The increase in large ACF on the LR diet may be attributable to elevated crypt cell proliferation possibly increasing crypt fission rates.      

Resistance of aberrant crypt foci to apoptosis induced by azoxymethane in rats chronically fed cholic acid

We have previously shown that chronic feeding of cholic acidto carcinogen treated rats reduces the number of putativepreneoplasticlesions of colonic cancer, aberrant crypt foci(ACF), but enhancesthe growth of remaining ACF and the incidence of colonic tumors.The following study was conducted to further explore the effectsof cholic acid on ACF growth by determining if ACF in cholicacid-fed animals display resistance to apoptotic cell death.ACF were induced in male Sprague- Dawley rats with two injectionsof azoxy-methane (20 mg/kg body wt). Rats were divided intotwo groups and fed either the control AIN-76 diet or the AIN-76diet containing 0.2% cholic acid. After 18 weeks, colonic apoptoticcelldeath was induced with an acute low dose of azoxymethane(10 mg/kg body wt). The number of cells, apoptotic bodies andbromodeoxyuridine (BUdR)-labeled cells were determined in coloniccrypts comprising ACF and surrounding normal crypts in ratsfrom each diet group. The number of apoptotic bodies per 100cells was lower in ACF crypts than in normal-appearing crypts(P = 0.0034). Both normal and ACF crypts from rats fed the cholicacid diet had fewer apoptotic bodies per 100 cells than cryptsfrom rats fed the control diet (P =0.0102). These data suggestthat ACF harbor resistanceto induction of apoptosis. Chronicfeeding of a diet containing 0.2% cholic acid results in thedevelopment of increased resistance to apoptosis. The lowerrate of cell death in ACF may contribute to the enhanced growthof ACF and higher tumor incidence previously observed in cholicacid-fed animals.