Sonia Y. Archer

Histone acetylation and cancer.

In the past year, several papers have been published which implicate a link between alterations in chromatin structure and the development of cancer. Both histone hyperacetylation and hypoacetylation appear to be important in the neoplastic process, depending on the target gene involved. In the case of colon cancer, induction of the p21 gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis.

Butyrate inhibits colon carcinoma cell growth through two distinct pathways

BACKGROUND Dietary fiber and the resultant increase in colonic butyrate levels protect against colon carcinogenesis. Previous studies have shown that p21 and histone hyperacetylation are important in basal growth inhibition by butyrate. This study was designed to elucidate other mechanism underlying the butyrate effects on cell growth. METHODS HT-29 colon carcinoma cells (standard medium or medium lacking serum) were treated with sodium butyrate (NaBu), epidermal growth factor (EGF), or both. Northern blot analyses were performed with cDNA probes specific for c-fos, c-jun, and actin. Cell growth was measured by 3H-thymidine incorporation. Enzyme-linked immunosorbent assay (ELISA) was used to quantify EGF receptor levels. RESULTS Butyrate and serum starvation (SS) both induced a cell cycle withdrawal by 24 hours. In response to EGF treatment, SS cells exhibited a growth spurt and induced c-fos and c-jun proto-oncogene expression, whereas butyrate-treated cells exhibited minimal growth response to EGF. This relative unresponsiveness to EGF in butyrate-treated cells corresponded to a dramatic decline in EGF receptor levels when compared to untreated controls. CONCLUSIONS Butyrate appears to inhibit colon cancer cell growth by two mechanisms, one involving histone hyperacetylation and p21 induction and the other related to impaired EGF-responsiveness.

Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation. Modulation of Cl- secretory capacity by butyrate.

The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.

Enterocyte response to ischemia is dependent on differentiation state.

Enterocytes at the tips of microvilli are more sensitive to an ischemic insult than those cells residing in the crypts, an effect thought to be due to a relative lack of collateral flow. We speculated that this increased cellular sensitivity to ischemia might be an intrinsic feature of the cells related to their differentiated phenotype. To test this hypothesis, enterocyte response to ischemia was determined using both in vivo and in vitro models. For the in vivo studies, male Sprague-Dawley rats underwent laparotomy, and small intestinal ischemia was induced by clamping the superior mesenteric artery for 30 or 60 minutes, after which reperfusion was allowed for various time points up to 4 days. Injury was assessed histologically, as well as with Northern blots, probing for the enterocyte differentiation markers intestinal alkaline phosphatase and lactase, as well as the gut-epithelial marker villin. Mucosal changes consistent with ischemia/reperfusion injury were evident—that is, a rapid inflammatory response followed by progressive villus cell loss beginning at the tips and progressing to the crypts, depending on the degree of insult, with an eventual return to normal microanatomy. Intestinal alkaline phosphatase and lactase were lost immediately after ischemia and returned with reperfusion, confirming that the differentiated cells are particularly sensitive to ischemic injury. The in vitro studies employed two separate models of enterocyte differentiation: sodium butyrate-treated HT-29 cells and Caco-2 cells maintained for 7 days after confluence. In both models, undifferentiated and differentiated cells were subjected to treatment with 2-deoxyglucose and oligomycin-A (in vitro model of ischemia) and apoptosis was assessed by fluorescence-activated cell sorting analysis. Differentiation of both cell lines resulted in a significantly greater apoptotic response to ischemia compared to undifferentiated cells exposed to an identical insult. We conclude that differentiated enterocytes may be inherently more sensitive to ischemia-induced injury than their undifferentiated counterparts. These findings call into question the popularly held belief that villus tip cells are more susceptible to ischemia because of their location relative to the microvascular anatomy.

43 – Intestinal Regeneration and Adaptation Models

The small intestinal epithelium is the most rapidly proliferating tissue in the body, with a turnover time of 2 to 3 days in mice and rats, and 3 to 6 days in humans. This turnover rate is so rapid that cells do not normally enter the prolonged G 0 phase seen in other, more slowly dividing tissues. This renewal is critical for maintenance of the functional integrity of the gut and is very tightly regulated along the crypt–villus axis through the migration of pluripotent stem cells located within the crypts of Lieberkuhn. Crypt and villus architecture is controlled, in part, by stromal and epithelial elements. The cells acquire a differentiated phenotype as they migrate toward the villus tip (absorptive enterocyte, goblet, and enteroendocrine cells), and are eventually extruded into the lumen. Paneth cells do not migrate, however, but differentiate within the base of the crypts, eventually degenerate, and are lost through phagocytosis. Thus, a steady-state equilibrium is maintained between cell production and cell loss. Villus height follows a gradient from the duodenum, where it is greatest, to the ileum, where the shortest villi are encountered. Accordingly, there is a proximal-to-distal gradient in villus height, cell number, and absorptive surface area in the small intestine. These anatomic differences depend not only on the larger amount of ingested nutrients encountered by the proximal gut, but also on an intrinsic genetic program.