The delay of anoikis due to the inhibition of protein tyrosine dephosphorylation enables the maintenance of normal rat colonocyte primary culture.

Apoptosis induced by detachment of cells from the extracellular matrix (anoikis) appears to be one of the main obstacles in attempts to establish long-term primary culture of normal colonocytes. In the present study, the dynamics of molecular events related to apoptosis of isolated normal rat colonocytes was investigated. The whole colonic crypts were isolated using collagenase/dispase digestion technique. DNA fragmentation typical for the apoptosis and the apoptotic morphology of cells were observed already at the end of their isolation. Considerable increase in caspase-3 activity was noted during the first two hours of cell cultivation. Delaying of apoptosis by treatment of cells with sodium orthovanadate, the specific protein tyrosine phosphatase inhibitor, was found to be possible. It may facilitate long-term culture of intestinal epithelial cells.

Biological activity of Desulfovibrio desulfuricans lipopolysaccharides evaluated via interleukin-8 secretion by Caco-2 cells.

BACKGROUND: Although Desulfovibrio desulfuricans species, besides existing in the natural environment, is also found in the human digestive tract, no information is currently available on its role in the intestinal ecosystem and its activity in regard to the intestinal mucosa. Bacterial products (lipopolysaccharides, LPSs) are generally known for their ability to trigger inflammatory response by stimulating cytokine expression, such as interleukin-8 (IL-8). METHODS: Colonic Caco-2 cells were exposed to LPSs isolated from the soil type and intestinal wild strains of D. desulfuricans bacteria. The amount of IL-8 secreted was measured by ELISA. The effects of sodium butyrate and cell preincubation with sodium butyrate on the IL-8 secretion in response to LPSs were also analysed. RESULTS: LPSs from D. desulfuricans down-regulated IL-8 secretion by the cells. Incubation of these cells with butyrate alone resulted in a dose-dependent stimulation of IL-8 release. Butyrate also modulated IL-8 secretion by cells stimulated with LPSs. CONCLUSIONS: Our findings suggest the lack of inflammatory response of intestinal mucosa in the presence of LPSs of D. desulfuricans. This response can be conditioned by the natural bacterial product, butyrate, which exerts a stimulatory effect on the IL-8 secretion and modulates its release in response to LPSs.

Quantification of P21 mRNA in biopsy specimens of colon tissue at different stages of neoplastic transformation.

Induction of the P21 protein is an integral part of cell growth arrest associated with the cellular response to senescence or damage. Changes in the P21 protein level are often observed in tumour cells and it is suggested, that they correlate with malignancy. We studied the P21 gene expression at the mRNA level in biopsy specimens from colitis ulcerosa, adenomas and adenocarcinomas using TaqMan assay. Our results showed, that quantity of the P21 transcript was evidently lower in the cases with adenocarcinomas than in the cases with colitis ulcerosa or adenomas.

Normal colonocytes in primary culture--an experimental model for molecular pharmacology and biology of large intestine.

A simple method of whole intestinal crypts isolation from rat's colonic tissue has been developed. Culture of viable epithelial cells (colonocytes) was obtained from intact crypts using method providing colonocytes for apoptosis. Satisfactory results have been obtained if the crypts were isolated using collagenase I. Under conditions applied, spontaneous release of the colonocytes took place. Liberated cells underwent almost immediate adhesion to microcarrier surface. The primary culture of normal colonocytes indicating metabolic activity for long time (> 10 days) has been obtained.