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M D Basson

Publications and source records attributed to M D Basson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Human Caco-2 intestinal epithelial motility is associated with tyrosine kinase and cytoskeletal focal adhesion kinase signals.

Intestinal epithelial cells assume a specialized phenotype adapted to motility and mucosal healing during mucosal restitution. Since cell-matrix interactions initiate tyrosine kinase (TK) signals, we hypothesized that the regulation of the intestinal epithelial migratory phenotype may also involve TK signals, particularly via focal adhesion kinase (FAK). Caco-2 cells were seeded simultaneously at 26,000 and 6000 cells/cm2. After 4 days, the first cells were confluent, while cells in the second population were not contact-inhibited and expressed migrating lamellipodia. Cells were fractionated into Triton X-100-soluble (membrane/cytoskeletal) and -insoluble (cytosolic) fractions. TK activity in each fraction was assayed by ELISA using a synthetic substrate. FAK protein was assessed by immunoprecipitation with monoclonal anti-FAK and Western blotting. Because active FAK autophosphorylates, we also measured FAK tyrosine phosphorylation, immunoprecipitating with anti-FAK and then Western blotting for phosphotyrosine. TK activity was increased in both cytosolic and membrane/cytoskeletal fractions of migrating cells by 17.6 +/- 3.6 and 28.9 +/- 4.1%, respectively, compared to static cells (n = 11, P < 0.01). FAK protein increased in the cytosolic fraction by 90.4 +/- 20.0% (n = 5, P = 0.01), but did not change in the membrane/cytoskeletal fraction. Tyrosine phosphorylated FAK increased by 62.8 +/- 21.4% in the cytosolic fraction of migrating cells but also by 46.6 +/- 38.4% in the membrane/cytoskeletal fraction (n = 5, P < 0.05). These data suggest that intestinal epithelial cell migration is associated with increases in both cytosolic and cytoskeletal TK activity and upregulation of cytosolic FAK protein. The increase in cytoskeletal FAK phosphorylation without increased FAK protein suggests autophosphorylation and increased cytoskeletal FAK activity. The migrating intestinal epithelial phenotype may therefore be modulated by TK signals including cytoskeletal FAK activity.

Amino Acid Sequence↗
PubMed1998

Tyrosine kinase inhibitors reverse butyrate stimulation of human Caco-2 intestinal epithelial cell alkaline phosphatase but not butyrate promotion of dipeptidyl dipeptidase.

Short chain fatty acids such as sodium butyrate are concentrated in the colonic lumen and may protect against colon carcinogenesis by maintaining colonocytic differentiation, but the mechanisms by which they act are not fully understood. It has recently been suggested that short chain fatty acids modulate cellular tyrosine kinase activity in addition to altering chromatin structure via regulation of histone acetylation and DNA methylation. Therefore, the authors evaluated the influence of tyrosine kinase inhibition on the effects of 10 mM butyrate on human Caco-2 intestinal epithelial differentiation, using alkaline phosphatase and dipeptidyl dipeptidase specific activity as markers of differentiation, and two tyrosine kinase inhibitors, of different mechanisms of action and different effects on Caco-2 brush border enzyme specific activity, to block tyrosine kinase activity. As expected, butyrate stimulated both alkaline phosphatase and dipeptidyl dipeptidase specific activity. The tyrosine kinase inhibitors prevented, and indeed one inhibitor reversed the effects of butyrate on alkaline phosphatase specific activity. However, tyrosine kinase inhibition did not prevent butyrate stimulation of dipeptidyl dipeptidase specific activity. Different pathways are likely to regulate the effects of butyrate on expression of these two brush border enzymes. Butyrate stimulation of alkaline phosphatase, but not dipeptidyl dipeptidase, may involve tyrosine phosphorylation signaling.

Adenocarcinoma↗
PubMed1998

Short chain fatty acids inhibit human (SW1116) colon cancer cell invasion by reducing urokinase plasminogen activator activity and stimulating TIMP-1 and TIMP-2 activities, rather than via MMP modulation.

BACKGROUND: Short chain fatty acids derived from dietary fiber may protect against invasive colon cancer by modulating degradative matrix metalloproteinases (MMPs) and protective tissue inhibitor matrix metalloproteinases (TIMPs). Since invasion depends on the MMP/TIMP ratio, we hypothesized that short chain fatty acids inhibit colon cancer invasion by inhibiting MMPs and stimulating TIMPs. MATERIALS AND METHODS: SW1116 colon cancer cells were seeded onto Matrigel-coated Boyden chambers and treated with unsupplemented media or media containing 10 mM acetate, propionate, or butyrate. SW1116 invasion was quantitated by light microscopy and conditioned media were assayed by ELISA for MMP-1,2,3,9; TIMP-1,2; MMP/TIMP complex; and urokinase plasminogen activator (uPA). All data are expressed as mean percentage of control +/- SE (n > 6). RESULTS: Although all three short chain fatty acids inhibited invasion, butyrate was more potent than either acetate or propionate, inhibiting SW1116 invasion by 35 +/- 1% of control (n = 18, P < .0001) vs. 18 +/- 9% (n = 7, P < .05) for acetate and 10 +/- 6% (n = 7, P < .05) for propionate. MMP-2 was not modulated by any of the short chain fatty acids while MMP-1 was modulated only by butyrate and MMP-3 by propionate. Acetate did not modulate MMPs, TIMP-1, or uPA, but stimulated TIMP-2. In contrast, propionate and butyrate stimulated MMP-9 and TIMP-2 by 119-233% and both inhibited uPA by 8-16%. TIMP-1 was stimulated only by butyrate and actually inhibited by propionate. Only butyrate stimulated both TIMP-1 and TIMP-2. CONCLUSIONS: These data suggest that dietary fiber may protect against invasive colon cancer through stimulation of TIMP and inhibition of uPA activities, rather than through short chain fatty acids effects on the activities of the MMPs studied.

Acetates↗
PubMed1998

Effects of modulation of tyrosine phosphorylation on brush border enzyme activity in human Caco-2 intestinal epithelial cells.

Intestinal epithelial cell differentiation is closely regulated during normal cell renewal, maturation, and malignant transformation. Since tyrosine phosphorylation influences differentiation in other cell types and has been reported to vary between crypt cells to differentiated villus tip cells, we investigated the influence of tyrosine phosphorylation in colonocyte differentiation, by using human colonic Caco-2 cells as a model and expression of the brush border enzymes alkaline phosphatase (AKP) and dipeptidyl peptidase (DPDD) as differentiation markers. We studied three tyrosine kinase inhibitors with different modes of action and specificities, viz., genistein, erbstatin analog (EA), and tyrphostin, and the tyrosine phosphatase inhibitor sodium orthovanadate. AKP- and DPDD-specific activities were assayed in protein-matched cell lysates by synthetic substrate digestion. We also correlated the effects of these agents on brush border enzyme activity with tyrosine phosphorylation of phosphoproteins by Western blotting. Genistein (5-75 mg/ml) dose-dependently stimulated AKP and DPDD with a maximal stimulation at 75 mg/ml by 158.6+/- 17.5% and 228.6+/-37.1% of control values, respectively (n=12, P<0.001). The inactive analog genistin had no effect. Tyrphostin (25 mM) similarly stimulated AKP and DPDD by 138. 6+/-6.6% and 131.8+/-1.5% of control values (n=12, P<0.001). Unexpectedly, EA (0.1-10 mM) had the opposite effect, inhibiting AKP- and DPDD-specific activity significantly at 10 mM with a maximal 14.8+/-6.4% and 26.5+/-2.5% of control values (n=12, each P<0.001). Sodium orthovanadate had a discordant effect on these two differentiation markers. Orthovanadate dose-dependently increased AKP to a maximal 188.5+/-16.1% of basal activity at 1.5 mM but decreased DPDD activity at 1.5 mM to 47.2+/-3.8% (n=9, P<0.001 each). The effects of each agent were preserved when proliferation was blocked with mitomycin C, suggesting that the modulation of phenotype by these agents was independent of any effects of proliferation. The tyrosine phosphorylation of several phosphoprotein bands was affected differently by these agents. In particular, the tyrosine phosphorylation of one 70-kDa to 71-kDa band was increased by genistein and tyrophostin but deceased by EA. The different effects of these modulators of tyrosine kinase activity raise the possibility that at least two independent enzymes or pathways regulating tyrosine phosphorylation modulate intestinal epithelial differentiation. Furthermore, tyrosine phosphorylation of the 70-kDa to 71-kDa phosphoprotein may be important in the intracellular signaling by which intestinal epithelial cell differentiation is controlled.

Alkaline Phosphatase↗
PubMed1998

Effects of short-chain fatty acids on human rectosigmoid mucosal colonocyte brush-border enzymes.

Short-chain fatty acids produced by bacterial fermentation of dietary fiber may provide a tonic stimulus to colonocyte differentiation that contributes to the protective effect of fiber against colorectal malignancy. Since brush-border enzymes are common markers of colonocytic differentiation, we compared the effects of equimolar (10 mmol/L) concentrations of the three most common short-chain fatty acids, acetate, butyrate, and propionate, on the alkaline phosphatase and dipeptidyl dipeptidase specific activity of human colonic mucosal biopsies obtained from normal volunteers. Only butyrate significantly stimulated alkaline phosphatase specific activity (50.4% +/- 18.6%, P < .05). Short-chain fatty acid stimulation of dipeptidyl dipeptidase did not achieve statistical significance. Fibers yielding high colonic butyrate levels could have different effects on human colonic mucosal differentiation.

Aged↗
PubMed1998

Differential effects of mucosal pH on human (Caco-2) intestinal epithelial cell motility, proliferation, and differentiation.

Mucosal pH abnormalities are associated with anastomotic dehiscence, ischemia, and malignancy. We postulated that intraluminal pH influences intestinal epithelial motility, proliferation, and differentiation and studied extracellular pHo (7.0-8.5) effects on human (Caco-2) intestinal epithelial motility, proliferation, and differentiation. Mucosal healing was modeled by sheet migration and differentiation by alkaline phosphatase and dipeptidyl dipeptidase specific activity. In parallel differentiation and motility studies, we inhibited proliferation with mitomycin to dissociate indirect mitogenic effects. Intracellular pHi was quantitated using BCECF/AM at varying extracellular pHo and in migrating cells. Motility was maximal at pHo 7.6 and proliferation at 7.2. Each decreased with acidity and alkalinity. By contrast, brush border enzyme activity was lowest at pHo 7.0 and highest at pHo 8.5. pHi was highest at pHo 8.5. Migrating cell pHi was higher than static cell pHi. Thus, extracellular pHo deviations perturb Caco-2 pHi homeostasis and motility. Alkalinity promotes differentiation while acidity induces proliferation and limits differentiation.

Caco-2 Cells↗
PubMed1998

Strain induces Caco-2 intestinal epithelial proliferation and differentiation via PKC and tyrosine kinase signals.

Although the intestinal epithelium undergoes complex deformations during normal function, nutrient absorption, fasting, lactation, and disease, the effects of deformation on intestinal mucosal biology are poorly understood. We previously demonstrated that 24 h of cyclic deformation at an average 10% deformation every 6 s stimulates proliferation and modulates brush-border enzyme activity in human intestinal Caco-2 cell monolayers. In the present study we sought potential mechanisms for these effects. Protein kinase C (PKC) activity increased within 1 min after initiation of cyclic deformation, and the PKC-alpha and -zeta isoforms translocated from the soluble to the particulate fraction. Cyclic deformation also rapidly increased tyrosine kinase activity. Tyrosine phosphorylation of several proteins was increased in the soluble fraction but decreased in the particulate fraction by cyclic deformation for 30 min. Inhibition of PKC and tyrosine kinase signals by calphostin C, G-06967, and erbstatin attenuated or blocked cyclic deformation-mediated modulation of Caco-2 DNA synthesis and differentiation. These results suggest that cyclic deformation may modulate intestinal epithelial proliferation and brush-border enzyme activity by regulating PKC and tyrosine kinase signals.

Caco-2 Cells↗
PubMed1998

Coculture conditions alter endothelial modulation of TGF-beta 1 activation and smooth muscle growth morphology.

We examined whether endothelial cells (ECs) inhibit smooth muscle cell (SMC) transforming growth factor-beta 1 (TGF-beta 1) activation in bilayer coculture. Western analysis showed that SMCs cocultured with ECs as a bilayer had lower amounts of active TGF-beta 1 protein compared with SMCs cultured alone and SMCs cocultured with ECs as a monolayer. EC inhibition of TGF-beta 1 activation could be blocked with plasminogen activator inhibitor-1 (PAI-1) antibody. Similarly, SMC hill-and-valley growth, a marker for TGF-beta 1 activity, was present in SMCs cultured alone and SMCs cocultured with ECs as a monolayer but was absent in SMCs cocultured as a bilayer. SMCs cocultured with ECs as a bilayer migrated at a greater rate than SMCs cultured either alone or cocultured as a monolayer. The EC effect on SMC migration was inhibited by the addition of 5 ng/ml TGF-beta 1. ECs had no effect on SMC RNA levels of TGF-beta 1. PAI-1 levels were increased in ECs and ECs cocultured with SMCs compared with SMCs cultured alone. ECs inhibit TGF-beta 1 activation in bilayer coculture. This appears to be mediated through an increase in EC PAI-1 release. Alterations in coculture conditions, in particular the degree of EC-SMC cell contact, have profound effects on this process.

Animals↗
PubMed1998

Differential modulation of human (Caco-2) colon cancer cell line phenotype by short chain fatty acids.

Fermentation of dietary fiber within the colonic lumen yields short chain fatty acids (SCFA) such as butyrate, which may modulate colonic mucosal biology and inhibit the development of a malignant phenotype. However, different fibers yield varying proportions of various SCFA. We studied the effects of the three most common SCFA, acetate, butyrate, and propionate, on the proliferation, adhesion, and motility of the human intestinal Caco-2 cell line, as well as the effects of these SCFA on alkaline phosphatase and dipeptidyl dipeptidase specific activity (common laboratory markers of differentiation). In addition, we examined the modulation of c-myc protein and the tyrosine phosphorylation of cellular proteins by these SCFA in order to determine whether the variations in the potency of these three SCFA for phenotypic change extended to variations in effects on intracellular signaling and protooncogene expression. All three SCFA tended to slow proliferation, promote brush border enzyme activity, and inhibit both adhesion to and motility across a type I collagen matrix substrate. However, we observed substantial differences in the potency of these three SCFA with regard to these effects. In particular, butyrate was uniformly more potent than an equimolar concentration of acetate whereas equimolar propionate achieved comparable effects with regard to proliferation and brush border enzyme activity but was intermediate between butyrate and acetate with regard to modulation of cell-matrix interactions. Similarly, the SCFA downregulated c-myc protein levels and modulated the phosphorylation of several intracellular tyrosine phosphoproteins, but the effects of the three SCFA varied substantially for these parameters. These results suggest that the common short chain fatty acids are not equipotent in their effects on human Caco-2 colon cancer cell biology. Such differences in potency could contribute to the observed differences in effects of different dietary fibers in vivo.

Acetates↗
PubMed1997

Basal nutrition promotes human intestinal epithelial (Caco-2) proliferation, brush border enzyme activity, and motility.

OBJECTIVES: Provision of nutrients to the apical membrane of intestinal epithelial cells by the enteral route is critical for normal gut mucosal function and for the sheet migration required for mucosal healing. The present work attempts to determine whether supplemental nutrient delivery to the basal epithelial surface is important for intestinal epithelial biology. Since attempts to regulate intestinal epithelial cell biology by manipulation of parenteral nutrition solutions have met with some success, we hypothesized that basally delivered nutrients might also be important for intestinal epithelial biology. DESIGN: To test this hypothesis, we compared the brush border enzyme activity, proliferation, and motility of human intestinal epithelial (Caco-2) cells cultured on a type I collagen substrate either on cell culture dishes with culture medium above the apical side of the cell monolayer or in culture inserts on 0.45-mu semipermeable membranes with culture medium beneath the monolayers as well as above them. Proliferation was assessed by serial hematocytometric counts over 13-day period. Doubling times were calculated by logarithmic transformation of cell counts 48 hrs apart. The specific activity of the brush border enzymes, dipeptidyl dipeptidase and alkaline phosphatase, was assayed by the digestion of synthetic chromogenic substrates in protein-matched aliquots of cell lysates. Sheet migration was quantitated by the expansion of Caco-2 monolayers across collagen. Motility was dissociated from the proliferative component of monolayer expansion by blocking proliferation with mitomycin C. SETTING: Laboratory for gastrointestinal mucosal biology. SUBJECTS: A well-differentiated subclone of cells derived from the established human Caco-2 colonic epithelial cell line. MEASUREMENTS AND MAIN RESULTS: Basal nutrient delivery promoted Caco-2 proliferation, brush border enzyme activity, monolayer expansion, and cell motility. Proliferation was actually increased by 694 +/- 9.89% (n = 90, p < .0001) in cells nourished apically and basally compared with a 314 +/- 3.31% increase (n = 90, p < .0001) in those cells receiving only apical nutrition. The addition of basal nutrient delivery to the cell culture system augmented both alkaline phosphatase and dipeptidyl dipeptidase specific activity by 116 +/- 5.4% and 256 +/- 14.0%, respectively (p < .0001, n = 6 for each group). The effects of basal nutrient delivery were maintained after mitomycin blockade of proliferation for both alkaline phosphatase (392 +/- 89.8% of control, n = 3, p < .0005) and dipeptidyl dipeptidase (374 +/- 79.1% of control, n = 3, p < .005), suggesting that the increased digestive enzyme-specific activity reflected differentiation rather than indirect effects of slowing of proliferation. Epithelial sheet migration increased by 389 +/- 8.8% and proliferation-blocked cell motility also increased by 76.5 +/- 1.56% (p < .0005, n = 12 for each) compared with apical nutrient delivery only. CONCLUSIONS: These results suggest that although apical nutrition may be critical for intestinal epithelial cell biology, nutrient delivery to the basal surface of intestinal epithelial cell membranes may also promote intestinal epithelial differentiation, proliferation, and mucosal healing.

Basement Membrane↗
PubMed1997

Sucralfate impedes intestinal epithelial sheet migration in a Caco-2 cell culture model.

Sucralfate, which binds to the matrix of the ulcer bed, is theoretically advantageous for duodenal ulcer therapy, but has not fulfilled its promise clinically. We examined the effects of sucralfate and related compounds in a human intestinal epithelial (Caco-2) cell culture model of restitution on sheet migration across and adhesion to collagen I. Migration was quantitated across a collagen I matrix treated with sucralfate or related compounds and correlated with cell adhesion. Caco-2 motility was significantly and dose-responsively inhibited by sucralfate at therapeutic luminal concentrations. Sucrose octaacetate, the sucralfate backbone, and lactose octaacetate exhibited similar effects while the beta-bonded disaccharide maltose octaacetate had little effect. Sucrose itself slightly stimulated motility. Adhesion effects paralleled motility. Thus, sucralfate may inhibit intestinal epithelial motility by sterically interfering with adhesion to collagen I. A sucralfate analog with a lactose octaacetate backbone might retain growth factor binding without inhibiting enterocyte motility, perhaps improving its clinical efficacy.

Anti-Ulcer Agents↗
PubMed1996

Regulation of human (Caco-2) intestinal epithelial cell differentiation by extracellular matrix proteins.

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.

Alkaline Phosphatase↗
PubMed1996

Octreotide differentially modulates human Caco-2 intestinal epithelial cell proliferation and differentiation by decreasing intracellular cAMP.

Somatostatin modulates gastrointestinal mucosal growth and differentiation indirectly via inhibition of bioactive peptides and directly by less well understood mechanisms. We studied the direct effects of the somatostatin analog octreotide on proliferation, brush-border enzyme activity, cell-matrix interactions and intracellular cAMP in Caco-2 human intestinal epithelial cells. Proliferation was assessed by cell counting and [3H]thymidine uptake. The brush-border enzymes alkaline phosphatase (AP) and dipeptidyl dipeptidase (DP) were quantitated by synthetic substrate digestion. Adhesion and migration on purified matrix proteins were also measured. Octreotide (10(-9)-10(-5)M) shortened doubling time (46.5 +/- 6.2% at 10(-5) M, n = 20, P < 0.0001) and stimulated [3H]thymidine uptake. Octreotide decreased intracellular cAMP by 19.4 +/- 5.0% (n = 7, P < 0.0001) while dibutyryl-cAMP (10(-6) M) prolonged doubling time by 10.1 +/- 1.5% (n = 8, P < 0.0001), and blocked the octreotide effect. Octreotide decreased AP and DP with maximal effect at 10(-6) M (36.8 +/- 8.3% and 20.5 +/- 9.1%, n > 7, P < 0.0005 respectively). However, mitomycin proliferative blockade prevented octreotide inhibition of AP and DP, suggesting that the mitogenic effects of octreotide had simply decreased average maturity of the cells. Octreotide did not alter Caco-2 adhesion, EGF-or matrix-modulated motility, or integrin surface expression. Octreotide appears to directly stimulate Caco-2 proliferation by decreasing cAMP. These proliferative effects modulate Caco-2 differentiation but do not affect cell-matrix interactions.

Alkaline Phosphatase↗
PubMed1996

Topoisomerase II inhibition differentially modulates Caco-2 intestinal epithelial cell phenotype.

Caco-2 intestinal epithelial cells differentiate spontaneously after confluence when contact inhibition slows proliferation. We hypothesized that such reversible differentiation might be dependent on DNA synthesis and repair. We studied the effects of the topoisomerase II inhibitor etoposide on Caco-2 proliferation and on the differentiation markers alkaline phosphatase and dipeptidyl dipeptidase specific activity, as well as cell motility. Etoposide (0.3-10 microM) dose-dependently inhibited proliferation and alkaline phosphatase activity. However, etoposide (0.7-3 microM dose-dependently stimulated dipeptidyl dipeptidase activity. Above this concentration, dipeptidyl dipeptidase was also inhibited. Similar effects on enzyme activity were observed when proliferation was blocked with mitomycin C. Etoposide (1-10 microM) also dose-dependently inhibited cell motility. The selective stimulation of dipeptidyl dipeptidase activity by etoposide may offer a clue to the regulation of intestinal brush border enzyme expression at the molecular level.

Alkaline Phosphatase↗
PubMed1996

Regulation of human Caco-2 intestinal epithelial brush border enzyme activity by cyclic nucleotides.

Regulation of intestinal epithelial differentiation is critical to normal function, malignant transformation, and healing. However, the intracellular regulation of intestinal epithelial differentiation is incompletely understood. We studied the effects of intracellular cyclic AMP and cyclic GMP on brush border enzyme activity in the human Caco-2 intestinal epithelial cell using pharmacologic agonists and antagonists of cAMP and cGMP mediated pathways as probes. The stable cyclic nucleotide analogs dibutyryl cAMP and dibutyryl cGMP selectively decreased Caco-2 dipeptidyl dipeptidase specific activity while increasing alkaline phosphatase. The inhibitors of adenylate and guanylate cyclase KT5720 and KT5823 each exerted the opposite effects. Combinations of dibutyryl cAMP and dibutyryl cGMP demonstrated synergistic effects on each brush border enzyme but KT5720 and KT5823 were less than additive. Thus, cAMP and CGMP may regulate human intestinal epithelial differentiation by interacting pathways.

Adenylyl Cyclase Inhibitors↗
PubMed1996

Amplitude-dependent modulation of brush border enzymes and proliferation by cyclic strain in human intestinal Caco-2 monolayers.

Little is known about the effects of repetitive deformation during peristaltic distension and contraction or repetitive villus shortening on the proliferation and differentiation of the intestinal epithelium. We sought to characterize the effects of repetitive deformation of a physiologically relevant magnitude and frequency on the proliferation and differentiation of human intestinal epithelial Caco-2 cells, a common cell culture model for intestinal epithelial biology. Human intestinal epithelial Caco-2 cells were cultured on collagen-coated membranes deformed by -20 kPa vacuum at 10 cycles/minute, producing an average 10% strain on the adherent cells. Proliferation was assessed by cell counting and 3H-thymidine incorporation. Alkaline phosphatase and dipeptidyl dipeptidase specific activity were measured in cell lysates. Since cells at the membrane periphery experience higher strain than cells in the center, the topography of brush border enzyme histochemical and immunohistochemical staining was analyzed for strain-dependence. Cyclic strain stimulated proliferation compared to static cells. Proliferation was highest in the membrane periphery where strain was maximal. Strain also modulated differentiation independently of its mitogenic effects, selectively stimulating dipeptidyl dipeptidase while inhibiting alkaline phosphatase. Strain-associated enzyme changes were also maximal in areas of greatest strain. The PKC inhibitors staurosporine and calphostin C ablated strain mitogenic effects while intracellular PKC activity was increased by strain. The strain-associated brush border enzyme changes were attenuated but not blocked by PKC inhibition. Thus, strain of a physiologically relevant frequency and magnitude promotes proliferation and modulates the differentiation of a well-differentiated human intestinal epithelial cell line in an amplitude-dependent fashion. PKC may be involved in coupling strain to increased proliferation.

Alkaline Phosphatase↗
PubMed1996

Regulation of human colonic cell line proliferation and phenotype by sodium butyrate.

Colonic butyrate may maintain mucosal differentiation and oppose carcinogenesis. We characterized butyrate effects on differentiation, proliferation, and matrix interactions in Caco-2 and SW620 human colonic cells. Differentiation was assessed by brush border enzyme activity and doubling time by serial cell counts. Motility across matrix proteins was quantitated by monolayer expansion and correlated with adhesiveness to matrix. Integrin subunit surface pools were measured by immunoprecipitation. Butyrate-stimulated differentiation inhibited proliferation and was significantly more potent than acetate in this regard. Butyrate also inhibited motility across collagen I, collagen IV, and laminin, as well as decreasing adhesiveness to these matrices and beta 1, alpha 1, and alpha 2 integrin subunit surface expression. Butyrate acts in cultured cells at clinically relevant concentrations to oppose classical malignant behavior, inhibiting proliferation and motility while promoting differentiation. Since butyrate is derived from fermentation of dietary fiber, such mechanisms may contribute to the apparent protective action of fiber against colon carcinogenesis.

Butyrates↗