Colonic aberrant crypt foci are associated with increased expression of c-fos: the possible role of modified c-fos expression in preneoplastic lesions in colon cancer.

Aberrant crypt foci represent the earliest detectable lesions of colon cancer. The expression of c-fos in these aberrant crypts was determined by in situ hybridization and immunohistochemistry. These lesions were induced in the colonic epithelium with azoxymethane using the rat model system. Expression of c-fos was markedly increased in the aberrant colonic crypts. Increases of approximately 60 and approximately 70% in the proportion of epithelial cells labelled were observed in the early and advanced aberrant crypts respectively. This was found to be statistically significant (P less than 0.001). Consistent with cell proliferation patterns in the colonic crypts, the epithelial cells of the lower crypt had greater levels of c-fos mRNA than those of the upper part of the crypts. In addition, immunohistochemical staining with c-fos polyclonal antibodies demonstrated increased levels of c-fos protein in aberrant crypts. This combined approach using in situ hybridization and immunohistochemistry has shown that increased c-fos expression at the RNA level in these lesions is associated with increased amounts of c-fos protein. Since c-fos has been implicated in the process of cell proliferation and differentiation, modifications in its expression may be significant to understanding the mechanisms whereby preneoplastic lesions transform to neoplastic lesions in colon cancer.     

Fatty acid synthase is over‐expressed in large aberrant crypt foci in rats treated with azoxymethane

Fatty acid synthase (FAS) is over‐expressed in many human cancers including colon. High levels of FAS expression have also been observed in a number of lesions that are precursors to invasive colorectal cancer. However, FAS expression in aberrant crypt foci (ACF), the earliest identifiable lesions in colon cancer development, has not been investigated. In this study, we treated Fisher rats with a single dose of the colon carcinogen azoxymethane then evaluated ACF 100 days later. We showed that large ACF (≥4 crypts/focus) have a significantly higher level of immunohistochemical staining for FAS than either small ACF (≤3 crypts/focus) or normal crypts. Furthermore, the severity of nuclear atypia in ACF was positively associated with increased expression of FAS. These findings suggest that the genes associated with FAS over‐expression are activated early in the stepwise development of colon cancer, though not until the ACF have reached a critical size with a level of nuclear atypia indicative of dysplasia. © 2008 Wiley‐Liss, Inc.     

Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague-Dawley rats: earliest recognizable precursor lesions of experimental colon cancer.

The main objective of the present investigation was to understand the molecular events involved in the genesis of aberrant crypt foci. Aberrant crypt foci were induced in Sprague-Dawley rats with a single injection of azoxymethane. Aberrant crypts have been identified topographically in the colon and are hypothesized to represent preneoplastic lesions. In order to understand the molecular events involved in the early stages of colon cancer, PCR-amplified DNA from aberrant crypts was hybridized with oligonucleotide probes specific for the detection of point mutations in codon 12 of K-ras. The mutation identified was a G to A transition resulting in the substitution of the amino acid aspartic acid (asp) for glycine (gly). This mutation was present in 6/19 (32%) of aberrant crypts examined. The identical mutation was also identified in adenomacarcinoma tissue while no mutation could be detected in normal intestinal mucosa. For further confirmation of these results, the presence of the mutated ras protein (rasAsp-12) was detected in aberrant crypts by immunohistochemistry. This investigation provides the first identification of a ras point mutation in aberrant crypt foci.     

Inhibition of aberrant crypt growth by non-steroidal anti-inflammatory agents and differentiation agents in the rat colon

Aberrant crypts are aggregates of single to multiple colonic crypts evidencing hallmarks of dysplasia and may be the earliest detectable pathological lesions for colon cancer. The aberrant crypt assay has been developed in 2 protocols. In one, putative chemoprevention agents are tested for inhibitory effects when administered concomitantly with a carcinogen. In the other, the objective of this study, aberrant crypts were induced in F344 rats by parenteral injection of the colon carcinogen azoxymeth-ane (AOM) and allowed to develop for 4 weeks, when an average of 90-100 aberrant crypt foci per colon were found in the methylene blue-stained colon. Then, during the second 4 weeks of the experiment, aberrant crypts were allowed to further develop to a frequency of > 150 foci per colon, a time when multi-crypt foci were observed. During this time we tested the inhibitory effects of 4 analgesic drugs and 2 differentiation agents for effects of aberrant crypt growth and development. We found the non-steroidal anti-inflammatory drugs piroxicam, aspirin and ibuprofen, but not acetaminophen, to be effective in suppressing aberrant crypt formation or the progression to foci of multiple aberrant crypts. Treatment with chemo-suppressing agents 13-cis-retinoic acid (13-cRA) and 4-hydroxy-phenretinamide (4-HPR), known differentiating agents, however, did suppress expansion of aberrant crypt foci, with 13-cRA being the much more potent agent.     

DNA alterations in human aberrant crypt foci and colon cancers by random primed polymerase chain reaction

Colon cancers are the result of the accumulation of multiple genetic alterations. To evaluate the role genomic instability plays during tumor development, we compared DNA fingerprints of 44 aberrant crypt foci (ACF; the earliest identified neoplastic lesion in the colon), 23 cancers, and normal crypts generated by random primers with PCR. The PCR products, separated by PAGE and viewed after silver staining, demonstrate altered fingerprints for 23.3% of the ACF and 95.7% of the cancers. In this first study of human ACF with this approach, the finding of altered DNA fingerprints in these microscopic lesions suggests that genomic instability can occur very early in human colon tumorigenesis.     

Aberrant crypts: putative preneoplastic foci in human colonic mucosa

Aberrant crypts were identified for the first time in whole-mount preparations of normal-appearing human colonic mucosa after staining with methylene blue. The foci of aberrant crypts varied from single altered glands to plaques of greater than 30 crypts. The mean proportion of colonic mucosa altered and the number of foci with aberrant crypts per cm2 of colonic mucosa were (a) higher in patients with colon cancer than in patients without colon cancer or predisposing conditions and (b) highest in our single case of Gardner's syndrome. Aberrant crypts are postulated to be the earliest identifiable potential precursors of colon cancer. Analysis of aberrant crypts may facilitate the study of the early pathological and molecular changes that precede colon cancer.     

Effects of all-trans retinoic acid as a potential chemopreventive agent on the formation of azoxymethane-induced aberrant crypt foci: Differential expression of c-myc and c-fos mrna and protein

The main objectives were to determine the modulating effects of all-trans retinoic acid on the number, size and multiplicity of aberrant crypt foci as well as the in vivo expression of the genes c-myc and c-fos. These foci, which are hypothesized to be the pre-malignant lesions of colon cancer, were induced in Sprague-Dawley rats with a single injection of azoxymethane. Rats were fed either a control diet (AIN-76) or the control diet to which had been added 75 mg/kg or 150 mg/kg all-trans retinoic acid. Within 4 weeks, we observed that the diets containing all-trans retinoic acid reduced the total number and multiplicity of aberrant crypt foci in the colon. However, all-trans retinoic acid increased the size of the lesions that persisted, possibly due to a greater proportion of lesions with dilated crypts. In situ hybridization and immunohistochemistry were performed on the colons for the in vivo analysis of gene expression in these lesions. The expression of myc-specific mRNA and protein in aberrant crypt foci significantly decreased with both levels of all-trans retinoic acid. In contrast, fos-specific mRNA and protein in aberrant crypt foci significantly increased when 150 mg/kg all-trans retinoic acid was added to the diet. The most important findings of this investigation are that intervention with all-trans retinoic acid in the pre-malignant stage of colon carcinogenesis is effective in decreasing the number and growth of aberrant crypt foci and altering the expression of the c-myc and c-fas genes.     

An immunohistochemical analysis of ras oncogene expression in epithelial neoplasms of the colon

Colonic epithelial tumors (101) including villoglandular adenomas, carcinomas in situ, adenocarcinomas, and neuroendocrine (NE) carcinomas were studied immunohistochemically with monoclonal antibodies (MoAb) RAP-5 and RAS-10 recognizing altered and unaltered ras oncogene products. In addition, 20 samples from multiple polyposis including adenomas with and without dysplasia, carcinomas in situ, and invasive carcinomas were studied. Using immunostaining techniques, normal mucosa was weakly stained, whereas the mucosa in the vicinity of tumors or inflammation showed enhanced staining. More tumors stained intensely with MoAb RAP-5 than with MoAb RAS-10. With MoAb RAP-5, most benign and malignant tumors showed enhanced staining. No significant differences in staining were noted in relation to superficial versus deeply invasive carcinomas or clinical staging. Immunostaining was also noted in some metastases. No significant differences in enhanced staining were found in carcinomas. Interestingly, the most extensive and enhanced immunostaining was noted in the villoglandular adenomas, dysplastic adenomas, and carcinomas in situ. The authors conclude that (1) ras protein expression is detectable in most benign, borderline, and malignant epithelial tumors of the colon as determined with MoAb RAP-5 and RAS-10, whereas enhanced expression is more often detected with RAP-5; (2) enhanced ras product expression in colon carcinomas does not seem to correlate with advanced tumor stages or with exocrine, NE, or phenotypically mixed tumors; and (3) the finding of the most intensely enhanced ras products expression in villoglandular polyps and carcinomas in situ suggests a possibly significant role for the oncogene in the early phases of transformation.     

Increased Cell Proliferation of Azoxymethane-induced Aberrant Crypt Foci of Rat Colon

Aberrant crypt foci (ACF) were induced in the colon of F344 rats by s.c. injection of azoxymethane (AOM) twice in a three day-interval and examined after 4 and 12 weeks. The number and crypt multiplicity of ACF in each section of rat colon increased during this period. Histologically, aberrant crypts consisted of proliferating atypical epithelial cells. Cell proliferation of ACF consisting of 4 aberrant crypts [ACF(4)] and 2 aberrant crypts [ACF(2)], and normal crypts in the colon of rats treated with AOM [normal crypts/AOM(+)] or saline [normal crypts/AOM(-)] was investigated by measurement of the mitotic index, proliferating cell nuclear antigen-labeling index (PCNA-LI), and 5-bromo-2'-deoxyuridine-labeling index (BrdU-LI). All three parameters of the cell proliferative activity of ACF(4) were higher than those of normal crypts/AOM(+) and normal crypts/AOM(-). The PCNA-LI and BrdU-LI in ACF(2) were the same as those in ACF(4). These findings suggest that ACF have increased cell proliferative activity. The correlation of these three parameters confirmed that the PCNA-LI is also a useful parameter for evaluating cell proliferative activity in ACF. The presence of many cells stained by PCNA in the upper portion of ACF suggested that ACF have more G₁ phase cells, which readily respond to mitogenic stimulation, than G₀ phase cells, which are predominant in normal crypts.     

Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity

Increased proliferative activity has been described frequently in the colons of animals treated with colon carcinogens and of patients at increased risk of colon cancer; it has been proposed as an intermediate biomarker of colon cancer. Aberrant crypt foci, microscopic lesions identified in whole-mount preparations of colons, are thought to be putative pre-neoplastic lesions. The present studies were carried out to evaluate the proliferative activity of aberrant crypt foci at several different time periods, and of tumors after a single dose of azoxymethane (AOM) in F344 rats. Rats were injected with 5-bromo-2′-deoxyuridine (BUdR) 1 hr before killing. Aberrant crypt foci and tumors were identified and marked in the whole-mount specimens, embedded in glycol methacrylate, and evaluated for histochemically demonstrable hexosaminidase activity. Hexosaminidase is known to be altered in over 95% of aberrant crypt foci. Serial sections were evaluated for BUdR incorporation immunohistochemically with a monoclonal antibody. The mean proliferative activity of aberrant crypt foci in the distal colons was found to be 'increased 3-to 4-fold over that of the adjacent normal crypts at every time period analyzed (4 to 36 weeks) and was comparable to that seen in benign and malignant colon tumors in the same animals. The observed increase in proliferative activity further supports the hypothesis that aberrant crypt foci are putative pre-neoplastic lesions. Similar aberrant crypt foci, identified in human colons at increased risk of colon cancer, may provide important biomarkers for this common human cancer.     

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.     

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.     

Increased expression of fatty acid synthase in human aberrant crypt foci: Possible target for colorectal cancer prevention

Aberrant crypt foci (ACF), the earliest identified monoclonal lesions in the colon, provide insights into changes that promote and/or accompany the transformation of normal colonic epithelial cells to colorectal cancer. Fatty acid synthase (FAS), the primary enzyme involved in de novo lipogenesis from carbohydrates, is expressed at low levels in most normal human tissues but is elevated in several human neoplasms including colorectal adenomas and carcinomas. To determine if this pathway is altered even earlier in colorectal tumorigenesis, 35 human ACF from 21 patients were evaluated for the immunohistochemical expression of FAS. Sections of colon cancer served as positive controls, and normal colonic mucosa distant from cancer or ACF served as negative controls. FAS expression was increased in 30 (86%) ACF compared with that in adjacent normal colonic mucosa. The expression of FAS in ACF was not related to the degree of dysplasia or to the number of crypts in the ACF. The over expression of FAS in a high proportion of ACF suggests that this enzyme plays an important role very early in colorectal tumorigenesis and may be a target for chemoprevention. © 2009 UICC     

CpG island methylation in aberrant crypt foci and cancers from the same patients

Aberrant methylation of CpG islands is a common alteration in human colon cancer. Methylation of only a limited number of loci has been studied in aberrant crypt foci (ACF), the earliest identified premalignant lesions in the colon, and in only a limited number of lesions from the same patients. Methylation-specific PCR was used to analyze 35 ACF, samples of normal crypts with the same number of crypts as were in each ACF and 22 cancers from the same patients. Aberrant methylation in cellular retinol-binding protein 1 (CRBP1), MINT31 or H-cadherin (CDH13) was identified in 19 of 35 (54%) ACF. Hypermethylation of CRBP1, MINT31 or CDH13 was more frequent (15 of 22, 68%) in cancers. DNA methylation of CRBP1, MINT31 or CDH13 was not correlated between cancers and ACF from the same patients. Aberrant methylation was not correlated with patient age, number of crypts in the ACF or cancer stage. These results suggest that hypermethylation in the promoter region of some genes is an independent event, occurs early in human colon tumorigenesis and may play an important role during the transformation and progression of some lesions to colon cancers.     

Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein.

BACKGROUND: We have previously shown that thermolyzed protein (casein) cooked with fat in the diet of the rat promotes the growth of aberrant crypt foci (putative precursors of colon cancer) assessed at 100 days. PURPOSE: To determine how thermolysis affects this promotion, we examined thermolysis conditions, quantity of thermolyzed protein in the diet, and duration of thermolysis. To determine whether the previous finding of promotion of aberrant crypt foci corresponds to promotion of cancers assessed much later, we carried out promotion studies until colon cancers appeared. METHODS: F344 rats were given an initiating dose of azoxymethane and were then randomly allocated to groups receiving diets differing in their quantity and quality of casein. The groups were examined for aberrant crypt foci and tumors in the colon. RESULTS: Aberrant crypt foci were promoted by diets containing thermolyzed casein (180 degrees C, 2 hours). Promotion increased with increasing level of thermolyzed casein in the diet (to 20%) and with increasing thermolysis time (to 4 hours). The number of animals with polyps and cancers was higher in the animals receiving thermolyzed protein (2 hours), 16/23 versus 9/26 (P less than .05) and 10/26 versus 3/27 (P less than .05), respectively. The number of aberrant crypts per focus and the number of large aberrant crypt foci were higher in the tumor-bearing animals. CONCLUSIONS: Thermolyzed casein promotes early colonic precursor lesions in a dose-dependent and thermolysis time-dependent manner; thermolyzed casein also promotes colon cancer. IMPLICATIONS: The promoter formed on thermolysis could be involved in colon cancers associated with diets cooked at elevated temperatures, such as can occur with high-fat diets.     

Ras, C-myc and C-erbB-2 oncoprotein expression in non-AIDS Mediterranean Kaposi's sarcoma

We have studied ras p21, c-myc p62 and c-erbB-2 oncogene expression in fourteen Greek patients with NON AIDS Mediterranean Kaposi's sarcoma using immunohistochemical analysis. Elevated expression of ras p21 expression was observed in all 14 cases studied (8 of which had intense levels of staining), whereas expression of c-myc and c-erbB-2 was less frequent, (6 out of 13 cases tested showed elevated c-myc expression and 6 out of 13 showed elevated c-erbB-2 expression). This report indicates that aberrant ras p21 oncogene expression is a feature of Kaposi's sarcoma and may be important in the early stages of this disease.     

Newly defined aberrant crypt foci as a marker for dysplasia in the rat colon

Dysplasia represents a preneoplastic status in multistep colon carcinogenesis. Whereas laborious preparation of thin sections is required for its diagnosis, we here show that newly defined aberrant crypt foci (ACF) simply mark the majority of the dysplasia on the whole colon. Specifically, decoloring of the azoxymethane‐treated rat colon after scoring classical ACF (cACF) resulted in visualization of a subset of aberrant crypts that remained densely stained. They were morphologically classified into three subtypes, of which two with compressed luminal openings proved highly correlated with dysplasia. Accordingly, we designated those foci harboring either of the two crypt subtypes as dysplasia‐associated ACF (dACF). By serially applying different detection methods for known preneoplastic lesions to the same colon, we showed that most dACF had already been identified as cACF, and a few newly identified dACF contained an entire population of more advanced lesions, such as flat ACF and mucin‐depleted foci. Consequently, integrative scoring of cACF and dACF enabled capture of all early lesions of the colon. Furthermore, 94% of the dACF showed dysplasia and 90% of the dysplastic lesions proved to be dACF. Thus, dACF is a promising marker for dysplasia, likely facilitating precise identification of the early stages of colon carcinogenesis.     

Diet Induced Obesity Increases the Risk of Colonic Tumorigenesis in Mice

A large body of epidemiological data indicates that obesity increases the risk of colon cancer in humans. There are limited studies using rodent models where the relationship between obesity and colon cancer has been studied. In this study, wild-type diet-induced obese (DIO) mice and lean wild-type controls were used to investigate the influence of obesity on the risk of colon cancer. We hypothesized that the obese phenotype would exhibit increased colonic tumorigenesis. Colon cancer was chemically induced by injecting the mice with azoxymethane (AOM) at levels that we experimentally determined to result in equivalent AOM concentrations in circulating blood. Risk of colon cancer was assessed via microscopic examination of entire colons for aberrant crypts, aberrant crypt foci and proliferation levels. The DIO mice were found to have significantly more aberrant crypts and aberrant crypt foci as well as increased proliferation of colonocytes per mouse compared to wild-type control mice, supporting the epidemiological data that obesity increases the risk of colonic tumorigenesis.     

Scanning electron microscopy of aberrant crypt foci in rat colon

The surface of the colon mucosa of 1,2-dimethylhydrazinetreatedF344 rats was examined with the scanning electron microscope.A detailed examination of the mucosal topography revealed fociwith one to several aberrant crypts. These were seen as structureselevated from the background mucosa. The shape of the luminalopenings of the aberrant crypts varied from elongated or tortuousto circular. However, we found no ultrastructural variationsbetween the different aberrant crypt foci (ACF) or between theACF and the background mucosa. There was no direct relationshipbetween the size of ACF and the number of aberrant crypts perfocus, which may be explained by the mechanism of crypt fission;in two aberrant crypts we discovered the formation of a transverseepithelial septum, dividing the large crypt into two smallercrypts. The gross morphology of the ACF observed by scanningelectron microscopy and light microscopy was in principle thesame.