Quantitative Image Analysis of Epithelial and Stromal Area in Histological Sections of Colorectal Cancer: An Emerging Diagnostic Tool.

In colorectal cancer (CRC), an increase in the stromal (S) area with the reduction of the epithelial (E) parts has been suggested as an indication of tumor progression. Therefore, an automated image method capable of discriminating E and S areas would allow an improved diagnosis. Immunofluorescence staining was performed on paraffin-embedded sections from colorectal tumors (16 samples from patients with liver metastasis and 18 without). Noncancerous tumor adjacent mucosa (n = 5) and normal mucosa (n = 4) were taken as controls. Epithelial cells were identified by an anti-keratin 8 (K8) antibody. Large tissue areas (5-63 mm(2)/slide) including tumor center, tumor front, and adjacent mucosa were scanned using an automated microscopy system (TissueFAXS). With our newly developed algorithms, we showed that there is more K8-immunoreactive E in the tumor center than in tumor adjacent and normal mucosa. Comparing patients with and without metastasis, the E/S ratio decreased by 20% in the tumor center and by 40% at tumor front in metastatic samples. The reduction of E might be due to a more aggressive phenotype in metastasis patients. The novel software allowed a detailed morphometric analysis of cancer tissue compartments as tools for objective quantitative measurements, reduced analysis time, and increased reproducibility of the data.

25-hydroxy-vitamin d metabolism in human colon cancer cells during tumor progression.

RT-PCR analysis showed elevated expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) and of 25-hydroxyvitamin D-24-hydroxylase (24-OHase) in well differentiated human colon carcinomas in comparison to normal mucosa. Further tumor progression is associated with a rise in 1alpha-OHase but with no significant change in 24-OHase mRNA expression. Accordingly, HPLC analysis of 25-hydroxy-vitamin D3 metabolism in freshly isolated tumor cells indicated that well to moderately differentiated colon cancers in situ are able to produce 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) and convert it through 24-OHase activity into side-chain modified metabolites, 1,24,25-(OH)3-D3 and 1,25-(OH)2- 24-oxo-D3. Likewise, 25-(OH)-D3 is metabolized into 24,25-(OH)2D3, 23,25-(OH)2D3, and 23,25-(OH)2-24-oxo-D3. Poorly-differentiated cancers expressed low levels of 1alpha-OHase mRNA, whereas 24-OHase was even over-expressed. RT-PCR and HPLC analysis of vitamin D metabolism in primary culture cell clones strongly suggested that the extent of endogenously produced 1alpha,25-(OH)2-D3 was inversely related to 24-OHase activity, which could thus limit the antimitotic efficacy of 1alpha,25-(OH)2-D3 particularly at late stages of colon cancer progression.

Establishment of primary cultures from human colonic tissue during tumor progression: vitamin-D responses and vitamin-D-receptor expression.

Primary cultures derived from pre-cancerous and cancerous human colon tissue are essential for understanding normal and abnormal growth function in the large intestine. Here presented are (i) the methodology for routine establishment of primary cultures of normal, adenoma- and carcinoma-derived cells, and (ii) data for the apparently protective role of vitamin-D compounds in colon carcinogenesis. The steroid hormone 1,25-dihydroxyvitamin D3 and some non-hypercalcemic analogs reduce the high mitotic rate of adenoma cells to that of normal colonocytes. After vitamin-D treatment, tumor cells are less proliferative and differentiation is enhanced. Primary-colon-cancer cultures display a mosaic pattern of vitamin-D-receptor expression, at the mRNA level and at the protein level, with varying intensity of expression in positive cells. This suggests that, in human colorectal tumors in vivo, a large fraction of cells will respond to genomic action of vitamin-D compounds.

Vitamin D receptor and cytokeratin expression may be progression indicators in human colon cancer.

Epidemiological data suggest the protective role of vitamin D against the development of colorectal carcinoma in man. This could be due to the anti-mitogenic effect of the steroid hormone on human colon carcinoma cells which is mediated by a specific nuclear vitamin D receptor (VDR). Western blot analysis showed that VDR expression increases during the transition from normal mucosa to polyps and later to pT3 tumors. In later stages, however, VDR is dramatically reduced. Cytokeratin 20, which was monitored as a differentiation marker, decreases in parallel with advancing proliferation and disappears from "normal" mucosa adjacent to later stage carcinoma. Interestingly, VDR density was conspicuously higher in all tumors tested when compared to adjacent "normal" tissue. This suggest that, up to a certain degree of dedifferentiation, malignant colonocytes can upregulate the VDR, probably as a counteractive measure in response to tumor cell growth, but that this ability is finally lost in highly undifferentiated carcinoma cells.

Human colonocytes in primary culture: a model to study epithelial growth, metabolism and differentiation.

The purpose of this work was to set up an in vitro model for the study of normal and pathological functions of the colonic epithelium. We have isolated colonic crypts by mild proteolytic digestion and mechanical dissociation of human biopsy material obtained during colonoscopy. The crypts, free of connective tissue, when placed in culture rapidly attached to the substrate and formed colonies containing over 95% of epithelial cells. Histochemical and ultrastructural characterization of the colonies showed the presence of both absorptive and secretory cells, exhibiting a high degree of differentiation. Proliferative activity occurred mostly during the first 24 h and progressively declined thereafter. The cells survived and maintained differentiated characteristics for at least three days in culture. This method can be used to study normal functions of the colonic epithelium. It may also be employed to investigate both noxious and protective factors in pathological conditions such as inflammatory bowel disease and colorectal neoplasia.

Expression of epithelial markers and retinoid-binding proteins in retinol- or retinoic acid-treated intestinal cells in vitro.

This study was undertaken with the aim of investigating the effects of retinoids on the expression of differentiated traits in intestinal cell models. The cell lines used included epithelial cells isolated from fetal rat intestines (FRIC), displaying a relatively undifferentiated phenotype, and the human colon adenocarcinoma cell lines Caco 2 and HT29, which express some of the traits of the mature enterocytes under defined culture conditions. The effects of retinoids were also studied in organ cultures of fetal rat intestine, where the epithelial-mesenchymal interactions are preserved. All-trans-retinol and all-trans-retinoic acid treatments were compared in their ability to regulate the expression of genes coding for proteins involved in retinoid metabolism and for cytoskeletal proteins. The results have shown that the effects of the two retinoids were qualitatively similar. A specific induction of the cellular retinol-binding protein CRBP I mRNA was observed following retinoid treatment in one of the two FRIC lines examined (FRIC B) and in organ culture. The expression of the retinoic acid receptors RAR alpha and gamma was not affected by treatment in any of the cultures examined, while RAR beta was expressed only by the organ cultures and was transcriptionally induced by retinoic acid treatment. The retinoids also induced a reorganization of the actin cytoskeleton in the FRIC B cell line, accompanied by a decrease in the expression of two components of the microvillar cytoskeleton, ezrin and villin. The results obtained in both cell and organ cultures suggest that retinoids alone are not able to trigger the differentiation program in the intestinal epithelial cell, irrespective of the level of differentiation already achieved at the time of treatment.

Experimental atherosclerosis in rabbits fed cholesterol-free diets.

Rabbits were fed a semipurified, cholesterol-free atherogenic diet containing 40% sucrose, 25% casein, 14% fat, 15% fiber, 5% salt mix and 1% vitamin mix. The fats were corn oil (CO), palm kernel oil (PO), cocoa butter (CB), and coconut oil (CNO). The rabbits were bled at 3, 6, and 9 months and killed at 9 months. Serum lipids of rabbits fed CO were unaffected. Serum cholesterol levels (mg/dl) at 9 months were: CO -- 64; PO -- 436; CB -- 220; and CNO -- 474. HDL-cholesterol (%) was: CO -- 37; PO -- 8.6; CB -- 25.1; and CNO -- 7.0. Average atherosclerosis (arch + thoracic/2) was: CO -- 0.15; PO -- 1.28; CB -- 0.53; and CNO -- 1.60. Cocoa butter (iodine value 33) is significantly less cholesterolemic and atherogenic than palm oil (iodine value 17) or coconut oil (iodine value 6). The difference between the atherogenic effects of cocoa butter and palm oil may lie in the fact that about half of the fatty acids of palm oil are C 16 or shorter, whereas 76% of the fatty acids of cocoa butter are C 18 or longer.

Evaluation of protein quality: comparison of single-point and multi-point assays.

The quality of different protein sources having a wide spectrum of potential biological values has been assessed with multi-point and singly-point assays on growing Sprague-Dawley rats weighing 60 g. Values obtained with multi-point assays, expressed goth as absolute and relative values, were well correlated irrespective of the response parameter, i.e. as absolute and relative values, were well correlated irrespective of the response parameter, i.e. body weight change, body water or body nitrogen (r = 0.980). Single-point assay values based on body nitrogen content were also well correlated with the multi-point assays, but some discrepancies were noted for what assays based on body weight change was concerned. The modification of body protein concentration, in particular of rats fed the protein-free diet, was the main cause of these discrepancies. The problem of reference protein, when protein quality was expressed as relative value, was also discussed.