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Background: A high dietary calcium intake with adequate vitamin D status has been linked to lower colorectal cancer risk, but the mechanisms of these effects are poorly understood., Objective: The objective of this study was to elucidate the effects of a Western-style diet (WD) and supplemental calcium and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the colorectal mucosa., Design: We conducted 2 crossover trials to define molecular pathways in the human colorectum altered by 1) a 4-wk WD supplemented with and without 2 g calcium carbonate/d and 2) a 4-wk WD supplemented with 1,25(OH)2D3 (0.5 μg/d) with or without 2 g calcium carbonate/d. The primary study endpoint was genome-wide gene expression in biopsy specimens of the rectosigmoid colonic mucosa. Serum and urinary calcium concentrations were also measured., Results: Changes in urinary calcium accurately reflected calcium consumption. The WD induced modest upregulation of genes involved in inflammatory pathways, including interferon signaling, and calcium supplementation reversed these toward baseline. In contrast, supplementation of the WD with 1,25(OH)2D3 induced striking upregulation of genes involved in inflammation, immune response, extracellular matrix, and cell adhesion. Calcium supplementation largely abrogated these changes., Conclusions: Supplementing 1,25(OH)2D3 to a WD markedly upregulated genes in immune response and inflammation pathways, which were largely reversed by calcium supplementation. This study provides clinical trial evidence of global gene expression changes occurring in the human colorectum in response to calcium and 1,25(OH)2D3 intervention. One action of 1,25(OH)2D3 is to upregulate adaptive immunity. Calcium appears to modulate this effect, pointing to its biological interaction in the mucosa. This trial was registered at clinicaltrials.gov as NCT00298545 Trial protocol is available at http://clinicalstudies.rucares.org (protocol numbers PHO475 and PHO554)., (© 2016 American Society for Nutrition.)

Low folate status increases colorectal cancer risk. Paradoxically, overly abundant folate supplementation, which is not uncommon in the United States, may increase risk. The mechanisms of these effects are unknown. We conducted two translational studies to define molecular pathways in the human colon altered either by folate supplementation or by dietary folate depletion (followed by repletion). In the first study, 10 healthy, at-risk volunteers (with documented stable/normal folate intake) received supplemental folic acid (1 mg/d) for 8 weeks. In the second study, 10 similar subjects were admitted to a hospital as inpatients for 12 weeks to study folate depletion induced by a low folate diet. A repletion regimen of folic acid (1 mg/d) was provided for the last 4 of these weeks. Both studies included an 8-week run-in period to ensure stabilized folate levels prior to intervention. We obtained 12 rectosigmoid biopsies (from 4 quadrants of normal-appearing mucosa 10-15 cm from the anal verge) at baseline and at measured intervals in both studies for assessing the primary endpoints: genome-wide gene expression, genomic DNA methylation, promoter methylation (depletion/repletion study only), and p53 DNA strand breaks. Serum and rectosigmoid folate concentrations accurately tracked all changes in folate delivery (P < 0.05). In the first study, gene array analysis revealed that supplementation upregulated multiple inflammation- and immune-related pathways in addition to altering several 1-carbon-related enzymes (P < 0.001). In the second study, folate depletion downregulated genes involved in immune response, inflammation, the cell cycle, and mitochondrial/energy pathways; repletion reversed most of these changes. However, changes in gene expression after repletion in the second study (involving immune response and inflammation) did not reach the levels seen after supplementation in the first study. Neither genomic nor promoter-specific DNA methylation changed during the course of the depletion/repletion protocol, and genomic methylation did not change with supplementation in the first study. p53 DNA strand breaks increased with depletion after 12 weeks. In sum, depletion downregulates, whereas repletion or supplementation upregulates pathways related to inflammation and immune response. These findings provide novel support to the concept that excessive folate supplementation might promote colorectal carcinogenesis by enhancing proinflammatory and immune response pathways. These results indicate that modest changes in folate delivery create substantial changes in the molecular milieu of the human colon., (©2011 AACR.)

Dihydroxycholecalciferol bound to its receptor functions as a potent antimitotic, prodifferentiating, proapoptotic hormone in different cell types and tissues. Epidemiological studies have linked low human serum concentrations of the vitamin D precursor hydroxycholecalciferol to colorectal cancer incidence. We have demonstrated in human colorectal tissue and cells the conversion of the precursor to dihydroxycholecalciferol, as well as the existence of the vitamin D catabolic pathway. These findings suggest a role for the colonic vitamin D system in tumor prevention. Low calcium intake has been found to be associated with human colorectal cancer incidence. In mice fed calcium equivalent to a low human intake, the degradative vitamin D pathway was increased, mainly in the ascending colon. Refeeding the mice high levels of vitamin D and calcium lowered tissue 25-hydroxycholecalciferol 24-hydroxylase activity, but only replenishment of folic acid normalized expression of the degradative pathway completely. Normalization occurred also when mice consuming low calcium diets were fed soy or the phytoestrogen genistein. These results indicate that colonic vitamin D synthesis is not only under stringent control of nutritional calcium, but also of folate, a methyl donor, which suggests epigenetic control of vitamin D hydroxylases.

Epidemiological evidence suggests that dietary calcium and vitamin D intake are inversely related to incidence of colon cancer. Previous studies have demonstrated that supplementation of the diet with calcium in the form of calcium tablets or low-fat dairy foods alters colonic epithelial cell proliferation from a higher- to a lower-risk pattern. The present study compared relative effects of administration of calcium carbonate at approximately 900 mg/day (calcium) with those of a low-fat dairy food diet providing about the same amount of calcium (dairy) in a cross-over "head-to-head" study of 40 subjects at risk for colonic neoplasia. Dietary intake of macronutrients was similar in the two study periods, except for a slight increase in protein intake during dairy calcium supplementation. Rectal epithelial cell proliferation was studied in flat endoscopically normal-appearing mucosa at baseline and at the end of each of the two study periods and showed a significant reduction in epithelial crypt cell labeling index from 12.5% to 9.1% (calcium) or 9.3% (dairy) as well as in proliferating cells in the upper 40% of the crypt from 0.09 to 0.03 in the calcium- and low-fat dairy-supplemented intervention groups. No significant changes in two epithelial cell differentiation markers, cytokeratin AE1 and acidic mucins, were found. Furthermore, there were no differences in epithelial cell apoptosis or expression of the proapoptotic gene product BAK. These data indicate that increased dietary calcium given as supplements or in the diet in low-fat dairy foods lowers epithelial cell proliferation indexes from a higher- to a lower-risk pattern. Because supplemental calcium has been shown to reduce the recurrence of colonic adenomatous polyps in patients at increased risk for colonic neoplasia, our data suggest that supplemental low-fat dairy foods may also be effective.

Western-style diets (WDs) trigger and sustain the early phases of tumorigenesis in mouse colon, and when continued throughout the life span lead to the development of dysplastic crypts. In order to evaluate the roles both of cell proliferation and programmed cell death (PCD) in WD-induced tumorigenesis, immunohistochemical detection of proliferating nuclear antigen (PCNA), in situ end labeling (TUNEL) of DNA breaks, and p53 protein were carried out in mouse colonic mucosa during prolonged feeding of two WDs. PCNA Labeling Index of colonic crypts was significantly higher in WD-treated animals than in controls only at the beginning of the nutritional study, the gap rapidly bridged by increased cell proliferation spontaneously occurring in the colonic mucosa during aging. A transient early homeostatic activation of PCD at the base of the crypt also was observed in WD groups. No changes in PCD were seen in the upper third of the crypt or in surface epithelium throughout the study, indicating that PCD in that colonic crypt segment produces a constant flux of cell loss, uninfluenced by homeostatic fluctuations. A major finding was an irreversible, progressive, age-related decline of PCD at the crypt base in both control and treated animals that occurred during the second half of the rodents' life span. p53 protein was not immunohistochemically detected, suggesting that neither overexpression of wild-type nor mutated forms of the protein are involved in the above mentioned changes.

Several studies have recently reported the development of colonic epithelial cell hyperproliferation in rodents following the ingestion of Western-style diets. In this study, additional measurements related to differentiation and maturation of the colonic epithelial cells were made after feeding this type of diet. Two Western-style diets high in fat and phosphate content and low in calcium and vitamin D were fed to C57BL/6J mice for 12, 24, and 52 weeks. Diet A contained American Blend fat as a source of lipids, diet B contained corn oil, and control diet C was a standard AIN-76A semisynthetic diet which is lower in fat content and higher in calcium and vitamin D. Colonic epithelial cells were studied for three biomarkers: cytokeratin catalogue no. 18 (clone LE64) expression, soybean agglutinin carbohydrate lectin binding, and acidic mucins including sialo- and sulfomucins. Feeding of diets A and B revealed that colonic epithelial cells had increased expression of cytokeratin catalogue 18 and SBA carbohydrate lectin binding compared to controls (P = 0.0001 for diet A versus C and diet B versus C). Significant differences were found between diets B and C (P = 0.0001) and diets A and C (P = 0.0001) in total acidic mucins and in the ratio of sialomucin:sulfomucin (P = 0.0001). These findings demonstrate that both functional and structural modifications occurred in colonic epithelial cells under these dietary conditions, and further defined this rodent model for preclinical evaluation of nutritional and chemopreventive interventions.

A Western-style diet containing high-fat and phosphate, and low calcium and vitamin D was fed to mice for 20 weeks. Starting at week 8, subgroups of animals received the Western-style diet supplemented by two different calcium sources: tricalcium phosphate and calcium citrate malate. Hyperproliferation (increased [3H]thymidine-labelled cells/colonic crypt) and hyperplasia (increased total epithelial cells/crypt) developed in the sigmoid colon after 8 weeks of feeding the Western-style diet confirming previous results, and these were reversed at later periods by the addition of the two calcium sources to the Western-style diet. Findings indicate that the modified colonic epithelial cell hyperproliferation and hyperplasia which have been associated with subsequent development of colonic neoplasia, are induced in mice fed a Western-style diet, and the addition of calcium to the diet inhibited their development in the colonic mucosa.

In rodents and in humans, aging is associated with increased gastrointestinal epithelial cell proliferation and an expanded crypt proliferative compartment similar to that seen in the preneoplastic bowel. We have compared the distribution of a series of cytoskeletal antigens that are modified when colonic cancer cells differentiate in vitro in the colon of young (4-7 month) and aging (22-26 month) Fischer 344 rats. Two such proteins, p52 and p35, (that are increased in cultured senescent cells) differ in their position in the crypt axis and subcellular localization between young and aging rats. In young rats, immunoreactive p52 protein is present solely near the colonic crypt surface epithelium but in aging rats p52 expressing cells are found deeper in crypts. The intracellular localization of p35 also differs markedly in young and aging animals. The distribution of these proteins appears to be a reproducible biomarker of aging. Antigenic changes similar to those observed in aging colons also are seen in crypt cells of patients with ulcerative colitis and in the flat colonic mucosa of patients with adenomatous polyps and colon cancer. The combination of proliferative and differentiation changes suggest that the flat mucosa of the colon of aging rats has preneoplastic features.

New genetic, morphologic, proliferative and differentiation-associated biomarkers are available for study in Phase II chemoprevention clinical trials. These biomarkers have potential as surrogate intermediate endpoints for cancer incidence and eventual tumor inhibition. At this meeting, approaches to improve the standardization and quality control of biomarker assays were considered. Useful biomarkers should be amenable to quality control monitoring; sensitive, specific, and precise; sensitive to modulation by chemopreventive agents; and simple enough to be performed routinely in research or clinical laboratories. Human studies have more factors that contribute to biomarker assay variation than rodent studies. These include: (1) wider differences in genetic background of study subjects; (2) differing extent and duration of previous colonic diseases; (3) varying amounts of chronic irritation in the colonic mucosa of "normal" subjects induced by exposure to dietary and other factors; (4) differing initial levels of study nutrients ingested prior to the study; (5) administration of fluids prior to biomarker measurement which are followed by biopsies after hypermetabolic changes become quiescent; (6) failure to use strict morphologic criteria in counting fewer available human colonic crypts; (7) amplifying field variations in colonic mucosa by decreasing the number of colonic crypts counted; (8) colonoscopic miss-rate of adenomas; (9) a clinical trial window of observation of short duration which limits the stage of adenoma development that is measured; and (10) failure to measure the activity of a chemopreventive agent during the stage of adenoma development in which it is active. Some of these points are relevant to chemopreventive agents used in clinical trials involving other organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiological studies indicate that caloric intake and dietary fat content influence colonic carcinogenesis. In rodents, caloric restriction reduces, and some fats increase, carcinogen-induced colon cancer incidence. The present study was designed to investigate the effects of caloric restriction on colonic cell proliferation (CCP) in carcinogen-treated or control rats fed low- or high-fat diets. F344 rats were treated with azoxymethane (15 mg/kg x2) and then fed an isocaloric AIN 76A diet containing either 5 or 23% corn oil, ad libitum or calorie-restricted to 70 or 80% of the kilocalories consumed by ad libitum rats. Biopsies of the distal colon were taken at 10 and 20 weeks, and rats were sacrificed at 21 or 34 weeks on the experimental diets. Distal CCP was determined by microautoradiography after [3H]thymidine labeling in vitro or presacrifice administration in vivo. The labeling index and number of labeled cells per crypt column were significantly reduced by caloric restriction at all time points (10, 20, 21, 34 weeks). Caloric restriction reduced CCP in high fat- and low fat-fed rats and in azoxymethane-treated and control rats. High fat resulted in decreased CCP in the distal colon compared to low fat at 34 weeks but not earlier. The findings indicate that: (a) caloric restriction is effective in favorably modulating CCP, an intermediate biomarker of colon cancer risk; (b) a high fat ad libitum diet, which increased tumor yield, does not increase distal colon proliferation; (c) dietary fat intake alters proliferation in a manner differing from that induced by changing dietary caloric intake.

The effect of increasing amounts of wheat bran (0, 5, 10, 20%) in AIN-76 semisynthetic diet on colonic luminal short chain fatty acids, epithelial cell histone acetylation, and cytokinetics, was studied for 2 weeks in groups of 10 male Sprague-Dawley rats. Luminal contents were removed from the colon at sacrifice, quick frozen, and analyzed for short chain fatty acids by gas-liquid chromatography. Histone acetylation was assessed in cells isolated from the same animals. Cell proliferation was measured after a short pulse in vivo with [3H]thymidine. Colonic luminal butyrate levels were lower in the 0 and 20% fiber groups, and higher in the 5 and 10% fiber groups. In contrast, cell proliferation, as determined by labeling index, was higher in the 0 and 20% fiber groups, and lower in the 5 and 10% fiber groups. This resulted in a significant inverse correlation between luminal butyrate levels and colonic cell proliferation. In addition, there was a positive linear correlation between luminal butyric acid levels and colon epithelial cell histone acetylation. From these data it was concluded that colonic butyrate levels can be modulated by the addition of wheat bran to the diet and that butyrate can modulate DNA synthesis (calculated as labeling index) in the proliferative compartments of colonic crypts. The localization of dividing cells was unchanged and no induction of terminal differentiation was detectable (contrary to what has been observed for transformed cells in culture).

A biomarker of cell differentiation was analyzed in normal and abnormal colonic epithelial cells. Soybean agglutinin (SBA) lectin which binds to specific carbohydrate residues was studied in normal human colonic epithelial cells, in epithelial cells in transitional colonic mucosa adjacent to carcinomas, and in colonic carcinomas. Findings revealed that increased SBA binding occurred maximally in normal, well-differentiated colonic epithelial cells, and least in colonic carcinomas. Further quantitation of SBA lectin binding also was carried out before and after supplemental dietary calcium. Findings revealed that in subjects whose colonic crypt biopsies had normal SBA lectin binding before calcium supplementation, SBA remained unchanged after calcium supplementation. However, in subjects whose biopsies initially had reduced SBA binding, the SBA increased after calcium and became more characteristic of that observed in normal colonic epithelium. In subjects receiving calcium for less than 3 months, the increased SBA was not statistically significant; but when subjects received calcium for durations of 3 months or longer SBA lectin binding was significantly increased, changing towards that observed in normal mucosa containing greater numbers of well-differentiated colonic epithelial cells.

In a panel of eight cloned complementary DNA sequences whose level of expression characterize colon cells as transformed in vivo and in vitro, one which may also serve as a marker of risk in familial polyposis and familial colon cancer flat mucosa has been identified as mitochondrial cytochrome c oxidase subunit 3. Mean level of expression of cytochrome c oxidase subunit 3 decreases progressively in colon adenomas and carcinomas relative to normal mucosa in vivo, and returns to higher levels present in biopsies of normal mucosa when the HT29 human colonic adenocarcinoma cell line is induced to differentiate with sodium butyrate. Quantitation of cytochrome c oxidase subunit 3 DNA by dot blots indicated that these changes in expression were not associated with alterations in the number of mitochondrial genomes.

Biopsy specimens of human colonic mucosa taken from the rectosigmoid of 12 normal subjects were maintained in explant culture for 4 days. Histological, microautoradiographic and chemical measurements were carried out to evaluate cell replication, the effect of deoxycholic acid, and the incorporation of uridine and leucine into RNA and protein. Active cell replication was shown to be greatest during the first day of organ culture, and the number of cells that synthesized DNA also increased when deoxycholic acid was added to the culture medium. At later times, with morphological evidence of tissue viability, the synthesis of DNA, RNA, and protein within colonic epithelial cells decreased, and the total number of cells in the crypt columns declined. Findings indicate good maintenance of metabolic activities of colonic epithelial cells in short-term explant culture, and the utility of both cell-kinetic and morphological observations in assessing the status of colonic explants at early and late intervals.

Nine patients at high risk of developing colon cancer were placed on daily p.o. supplementation of 1500 mg of calcium for 4-8 weeks. The colonic epithelial cells in six of these patients showed a statistically significant decrease in their [3H]thymidine labeling indices in tissue culture so that they resembled those of patients at low risk of developing colon cancer. The three nonresponders had similar labeling indices before and after calcium supplementation. Biopsies from each of nine high-risk patients exhibited a decrease in proliferation when they were cultured in vitro with a high level of CaCl2 (2.2 mM compared with the 0.1 mM optimum value for proliferation). Two adenomas and two carcinomas showed a different pattern of response than normal cells, exhibiting no inhibition of growth at 2.2 mM CaCl2. These data indicate that the growth inhibition induced by high levels of extracellular calcium levels is lost at a stage in tumor development before cells become malignant.

The proliferation of epithelial cells in colonic mucosa was studied in humans at varying degrees of risk for colon cancer. Seventh-Day Adventist vegetarians, known to have significantly lower mortality from colon cancer than the general U.S. population, had the most quiescent proliferative activity of mucosal epithelial cells. Increased replication and expansion of the proliferative compartment accompanied increased colon cancer risk. The analytical methods of this study may be useful in assessing the influence of dietary components involved in the initiation, promotion or inhibition of colon cancer, and in developing strategies for nutritional intervention.

An analysis has been developed to improve the quantitation of abnormal patterns of tritiated thymidine [(3H]TdR) labelling of colonic epithelial cells, in biopsy specimens removed from human subjects at varying degrees of risk for colon cancer. After pulse incubation of specimens of colonic mucosa with [3H]TdR, each subject's microautoradiographic epithelial cell labelling distribution was segregated into eleven compartments over entire colonic crypts. The findings of each subject were then analysed to determine their relative degree of similarity to the findings for two reference populations of interest, i.e. a high-risk and a low-risk population; the individual was then classified as being closer to one or the other of the reference populations. The analysis developed is based upon a comparison of multinomial probabilities for the distributions of the labelled cells within the crypts, and permits the routine categorization of uneven distributions of labelled cells. For each subject, certain linear scores, a prognostic index based on them, and a related presumptive risk, were calculated. The sensitivity with which individuals known to be symptomatic for polyposis, and the specificity with which individuals known to be at lower risk were determined, were 73 and 93% respectively. The results suggest that this method of distinguishing among integer distributions of [3H]TdR- labelled cells in biopsies of colonic mucosa, may provide a useful basis for identifying individuals with familial polyposis, by separating their labelling patterns from those of low-risk subjects.

A computer-based scanning and image-processing system has been developed to quantitate the relative level of expression of each of 4000 cloned complementary DNA sequences in small biopsies routinely removed from the mucosa of normal and neoplastic human large intestine. Individuals have been studied from well-defined population groups in which colonic epithelial cells have progressed to increasingly advanced stages of neoplastic transformation. Comparison of normal colonic mucosa to colonic carcinomas demonstrated alterations in expression of approximately 7% of the cloned sequences; fewer changes were found between benign colonic adenomas and either normal colonic mucosa or carcinomas. A subset of the sequences which change in expression during progression from normal mucosa, to adenoma, to carcinoma showed complementary changes when colon carcinoma cells were induced to differentiate in vitro with sodium butyrate; quantitative correlations between in vivo and in vitro results were highly significant. Comparison of normal colonic mucosa with mucosa from patients with the autosomal dominant disease familial polyposis revealed more extensive alterations in gene expression involving approximately 25% of the clones screened. Flat colonic mucosa in familial polyposis is therefore markedly aberrant and may be highly dedifferentiated, suggesting several possible mechanisms for the very high incidence of cancer that develops in this epithelium.

Microautoradiographic measurements of TdR3H incorporation into epithelial cells of colonic biopsies were compared in normal and eight index cases with familial polyposis, an inherited trait characterized by the development of colonic polyps and carcinomas. Colonic epithelial cells normally synthesized DNA synthesis was proliferated in the deeper two-thirds of the crypts, while DNA synthesis was repressed in the upper one-third. Patients with familial polyposis and patients with isolated single polyps incorporated TdR into epithelial cells lining the surfaces of polyps and occasionally into surface cells of intervening flat mucosa. Symptom-free members of polyposis families also demonstrated TdR3H incorporation into surface epithelial cells in biopsies of morphologically flat mucosa. The findings indicate similar proliferative behavior of epithelial cells in the formation of all adenomas, be they single isolated polyps or multiple familial adenomatous polyps. A sequence of changes occurs involving a loss of the cells' ability to repress DNA synthesis and the appearance of cells with persistent DNA synthesis at the surface and along the upper portion of colonic crypts. These defects are focal and widespread in the colonic mucosa of those with familial polyposis and are expressed at early age.

Early lesions in the colonic mucosa of humans and rodents are characterized by similar proliferative changes within their epithelial cell population. Progressive phases of abnormal cell development appear during the evolution of neoplastic transformation in colonic cells of rodents exposed to chemical carcinogens and in humans highly susceptible to gastrointestinal cancer. Identification and classification by phenotype of cells of these individuals at increased risk for colon cancer are leading to new methods to improve the detection and diagnosis of neoplasia in high risk individuals and families. An analytical system of precise numerical definitions is aiding an approach to modify the evolution of advanced stages of neoplasia.

Supplemental dietary calcium decreased and normalized hyperproliferation of colonic epithelial cells in individuals in familial colon cancer kindreds, measured by rates and patterns of [3H]thymidine labeling of epithelial cells in colonic crypts. In whole colonic crypts hyperproliferation was decreased to lower levels in over one-half of the subjects individually studied during the course of the calcium supplementation regimen. The remaining familial colon cancer subjects did not show reductions in cell proliferation measured over the whole crypt. However, when their cell-labeling data were analyzed in regions of the colonic crypt, the size of the proliferative compartment decreased and contracted towards the crypt base after calcium, a pattern typical of individuals at decreased risk for colonic cancer. This contraction of the proliferative region of the crypts occurred through decreased cell labeling in the two crypt compartments closest to the luminal surface and increased cell labeling in the second crypt compartment nearest to the base of the crypt. Following in vitro exposure of colonic epithelial cells to increasing physiological amounts of calcium, cell proliferation in familial colon cancer subjects decreased uniformly and greater heterogeneity in responsiveness was observed in cells from individuals with familial polyposis.