Barbara L. Bass

Differentiated intestinal epithelial cells exhibit increased migration through polyamines and myosin II

Early mucosal restitution is a rapid process by which differentiated intestinal epithelial cells migrate to reseal superficial wounds. However, most of the in vitro studies for restitution employ undifferentiated intestinal crypt cells as a model. The transcription factor, Cdx2, plays an important role in the regulation of intestinal epithelial differentiation. Forced expression of the Cdx2 gene in undifferentiated intestinal crypt cells induces the development of a differentiated phenotype. The current study was designed to determine changes in differentiated intestinal epithelial cell migration after wounding in the stable Cdx2-transfected IEC-6 cells and then to examine involvement of polyamines and nonmuscle myosin II in the process of cell motility. Cdx2-transfected IEC-6 cells were associated with a highly differentiated phenotype and exhibited increased cell migration after wounding. Migration of Cdx2-transfected IEC-6 cells were approximately four times that of nontransfected IEC-6 cells. Migration after wounding was associated with significant increases in polyamine synthesis. Depletion of cellular polyamines by 5 mM alpha-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, inhibited cell migration without affecting the differentiated phenotype. DFMO also decreased levels of nonmuscle myosin II mRNA and protein and resulted in reorganization of myosin II, along with a marked reduction in stress fibers. Exogenous spermidine given together with DFMO not only returned nonmuscle myosin II levels and cellular distribution toward normal but also restored cell migration to control levels. These results indicate that 1) Cdx2-transfected IEC-6 cells exhibit increased cell migration after wounding and 2) cellular polyamines are absolutely required for stimulation of cell migration in association with their ability to modulate the structural organization of nonmuscle myosin II.Early mucosal restitution is a rapid process by which differentiated intestinal epithelial cells migrate to reseal superficial wounds. However, most of the in vitro studies for restitution employ undifferentiated intestinal crypt cells as a model. The transcription factor, Cdx2, plays an important role in the regulation of intestinal epithelial differentiation. Forced expression of the Cdx2 gene in undifferentiated intestinal crypt cells induces the development of a differentiated phenotype. The current study was designed to determine changes in differentiated intestinal epithelial cell migration after wounding in the stable Cdx2-transfected IEC-6 cells and then to examine involvement of polyamines and nonmuscle myosin II in the process of cell motility. Cdx2-transfected IEC-6 cells were associated with a highly differentiated phenotype and exhibited increased cell migration after wounding. Migration of Cdx2-transfected IEC-6 cells were approximately four times that of nontransfected IEC-6 cells. Migration after wounding was associated with significant increases in polyamine synthesis. Depletion of cellular polyamines by 5 mM α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, inhibited cell migration without affecting the differentiated phenotype. DFMO also decreased levels of nonmuscle myosin II mRNA and protein and resulted in reorganization of myosin II, along with a marked reduction in stress fibers. Exogenous spermidine given together with DFMO not only returned nonmuscle myosin II levels and cellular distribution toward normal but also restored cell migration to control levels. These results indicate that 1) Cdx2-transfected IEC-6 cells exhibit increased cell migration after wounding and 2) cellular polyamines are absolutely required for stimulation of cell migration in association with their ability to modulate the structural organization of nonmuscle myosin II.

Inhibition of polyamine synthesis induces p53 gene expression but not apoptosis

The nuclear phosphoprotein p53 acts as a transcription factor and is involved in growth inhibition and apoptosis. The present study was designed to examine the effect of decreasing cellular polyamines on p53 gene expression and apoptosis in small intestinal epithelial (IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and 12 days. The cellular polyamines putrescine, spermidine, and spermine in DFMO-treated cells decreased dramatically at 4 days and remained depleted thereafter. Polyamine depletion by DFMO was accompanied by a significant increase in expression of the p53 gene. The p53 mRNA levels increased 4 days after exposure to DFMO, and the maximum increases occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA in DFMO-treated cells were paralleled by increases in p53 protein. Polyamines given together with DFMO completely prevented increased expression of the p53 gene. Increased expression of the p53 gene in DFMO-treated cells was associated with a significant increase in G1 phase growth arrest. In contrast, no features of programmmed cell death were identified after polyamine depletion: no internucleosomal DNA fragmentation was observed, and no morphological features of apoptosis were evident in cells exposed to DFMO for 4, 6, and 12 days. These results indicate that 1) decreasing cellular polyamines increases expression of the p53 gene and 2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt cells. These findings suggest that increased expression of the p53 gene may play an important role in growth inhibition caused by polyamine depletion.

Expression of the transforming growth factor-β gene during growth inhibition following polyamine depletion

Polyamine depletion and cytokine transforming growth factor-β (TGF-β) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-β gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-β. Increased TGF-β mRNA in DFMO-treated cells was paralleled by an increase in TGF-β content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-β gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-β in normal cells was ∼65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-β mRNA in IEC-6 cells. TGF-β added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-β antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-β gene through posttranscriptional regulation and 2) increased expression of the TGF-β gene plays an important role in the process of growth inhibition following polyamine depletion.

Neutropenic enterocolitis in adults: Case series and review of the literature

Necrotizing enterocolitis in adults is a rare disease and, in the past, has been associated with nearly uniform mortality. In recent years, necrotizing enterocolitis, now termed neutropenic enterocolitis, in adults has become more prevalent as a complication of aggressive systemic chemotherapy. In this report, we discuss two cases of neutropenic enterocolitis secondary to the administration of systemic chemotherapy in adult cancer patients: one with lung carcinoma, the other with leukemia. Both patients were successfully treated with early surgical intervention for resection of all necrotizing enteric lesions, and subsequent aggressive critical care support. Our experience suggests that early surgical intervention in adult patients with intestinal necrosis due to chemotherapy is essential to avoid mortality from this condition. Given the widespread, aggressive use of systemic chemotherapy in the neoadjuvant setting, patients at risk for this potentially lethal complication of neutropenic enterocolitis are increasingly common.

Bile salts regulate intestinal epithelial cell migration by nuclear factor–κB–induced expression of transforming growth factor–β☆

Abstract Background Mucosal restitution is an important repair modality in the gastrointestinal tract. We have shown that taurodeoxycholate increases intestinal epithelial cell migration by increasing TGF-β expression, and that the transcription factor NF-κB regulates TDCA induced cell migration after injury. The objectives of this study were to determine if this is a property shared by other bile salts or an effect specific to TDCA, and to determine if NF-κB regulates TGF-β expression. Study design Studies were conducted in IEC-6 cells. Cell migration was examined using an in vitro model. TGF-β protein and mRNA expression was determined by ELISA and Northern blot analysis. Sequence-specific NF-κB binding activity was measured by gel shift assays. Results Taurocholate and deoxycholate at physiologic concentrations significantly increased intestinal epithelial cell migration 6 hours after wounding (p Conclusions We conclude that bile salts at physiologic conditions increase cell migration after injury, an effect regulated by NF-κB. Further, NF-κB elicits TGF-β gene transcription during cell migration. These data support a physiologic role of bile salts in the maintenance of intestinal mucosal integrity.

PEG ileus - A new cause of small bowel obstruction

SummaryA case of small bowel obstruction due to a lodged percutaneous endoscopic gastrostomy tube inner bumper is described. Most probably inner bumper lodgement in the terminal ileum is related to its size. Laparotomy was required to remove the bumper and relieve the obstruction. We suggest that all percutaneous endoscopic gastrostomy bumpers be retrieved endoscopically when the PEG tube is removed or replaced unless a collapsible inner bumper is used.

Matrix alters the proliferative response of enterocytes to growth factors

BACKGROUNDnEnterocyte growth and differentiation along the crypt-villus axis is a highly controlled process. Crypt cells divide to produce enterocytes that migrate along the villus axis prior to terminal differentiation. Growth factors and basement membrane may be involved in the regulation of this process.nnnMETHODSnIEC-6 proliferation was measured in cells grown on laminin or collagen I by measuring the conversion of Owens reagent to formazan. Cells were grown with no growth factor, one growth factor (ie, epidermal growth factor, insulin-like growth factor-I, or transforming growth factor-alpha), or a combination of factors.nnnRESULTSnEnterocyte proliferation was increased in all groups given mitogenic growth factors with additive increases in those given combinations. Enterocytes grown on laminin showed a significantly decreased proliferative response to mitogenic growth factors in all experimental groups. Matrix did not alter seeding efficiency or cell viability.nnnCONCLUSIONnBasement membrane may play a role in the regulation of cell proliferation along the crypt-villus axis.

Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to approximately 2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.

What’s new in general surgery: gastrointestinal conditions

The years 2001 and early 2002 brought continued advances in many areas of gastrointestinal surgery. Progress was evident in the definition of data-based strategies to improve surgical and nonsurgical management of the diverse conditions of organs in the gastrointestinal system. These developments in gastrointestinal surgery were presented at the annual meetings of major surgical organizations, including the Clinical Congress of the American College of Surgeons, the Society for Surgery of the Alimentary Tract, the American Surgical Association, and other leading surgical meetings. This article, organized by organ, will summarize the highlights of these reports published over the last year.

The pancreatic duct epithelium in vitro: bile acid injury and the effect of epidermal growth factor.

BACKGROUNDnPancreatic duct epithelial cells form a barrier against parenchymal injury. The capacity of these cells to respond to injury has not been investigated. We hypothesized that epidermal growth factor (EGF), normally found in pancreatic juice, could protect the duct epithelium from damage.nnnMETHODSnAn explant system of duct cell culture developed in our lab with the bovine main pancreatic duct was used. Explants were exposed to bile acid (taurodeoxycholic acid [TDCA] 0, 0.05, 0.5, and 1 mmol/L) in the presence or absence of EGF (0, 1, 10, and 100 nmol/L) for 48 hours. Epithelial proliferation, damage, and growth out from the explant edge were assessed histologically. Expression of ductal markers and the extent of cell proliferation were determined by immunohistochemistry using specific antibodies.nnnRESULTSnExplant duct cells proliferated and demonstrated continued expression of key duct antigens in culture. TDCA produced dose-dependent mucosal damage and reduced epithelial density and growth from the edge. EGF increased cellular density in the native epithelium, but did not significantly alter growth from the edge. Mucosal damage created by TDCA exposure was significantly decreased with EGF and both growth from the edge and cell density were preserved.nnnCONCLUSIONSnExplants created from the bovine main pancreatic duct serve as an excellent model for the study of duct epithelial cells in vitro. These cells proliferate in response to EGF and are damaged by TDCA at concentrations below those normally associated with detergent-like activity and below levels observed in bile and duodenal secretions. The ability of EGF to protect from this injury suggests a potential physiologic role in the maintenance of the pancreatic duct mucosal barrier.