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The SRC-related intestinal kinase sik is present in the nucleus and phosphorylates the RNA-binding protein Sam68
April 2000
was I.S :t 0.4 Nand 1.3 :t 0.6 N in control and WT.3 treated rats, respectively. Taken together, these findings suggest that specific inhibition of CD 18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine did not change the breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound healing process differently in specific organs. This needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.
2914 HUMAN GASTRIC EPITHELIAL PROLIFERATION IS COORDI· NATED BY INSULIN·LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS. Eric Tremblay, Pierre Chailler, Daniel Menard, Univ de Sherbrooke, Sherbrooke, PQ, Canada. IGF peptides (IGFl,IGF2) are known to be produced in fetal and adult gastric tissues and IGF2 is an autocrine factor for gut-derived cell lines. However their possible contribution to the regulation of cell proliferation during development or regeneration of human gastric mucosa is unknown, as the modulatory effect of IGF binding proteins (IGFBPs). AIMS: To evaluate the ability of human gastric mucosa to produce IGFBPs and to test the effects of native IGFs on epithelial cell proliferation as well as that of truncated IGFs which do not interact with IGFBPs. METHODS: Western! FarWestern blots and immunofluorescence were performed to characterize the expression of IGFl-receptor and IGFBP-l to -6. The effects of growth factors on DNA synthesis were determined in fetal gastric explants maintained in serum-free organ culture. RESULTS: All gastric epithelial cells expressed the IGFI-R. IGFBP-l protein detected in gastric tissue likely derived from an extragastric source: immunoreactivity was found in all mucosal cell types (endocrine pathway of delivery) and its total amount did not increase in culture. IGFBP-2 to -6 proteins were rather associated with epithelial cells and differentially localized along the pit-gland axis. They were produced endogenously and modulated in culture. Addition of IGFI to gastric explants stimulated 3H-thymidine incorporation into DNA after 24-48 h and increased the epithelial labeling index. Illustrating the counter regulatory effect of endogenous IGFBPs on IGF action, truncated R3IGFl was more potent than IGF1. DeI(l-6)IGF2 was the most potent inducer whereas IGF2 remained without significant effect (even at 200ng/mL). The last finding probably reflects the presence of IGFBP-2 and -6, two IGF2 carriers intensely synthesized in the pit/neck region comprising the proliferative compartment and abundantly secreted during culture. CONCLUSION: This study provides new evidence for the involvement of an intragastric IGF/lGFBP system in the fine regulation of epithelial cell division whereby the specific influence of IGF2 appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells. (Supported by the Medical Research Council of Canada; D.M. is a member of the MRC Research Group on Functional Development and Physiopathology of the Digestive Tract.)
2915 THE SRC-RELATED INTESTINAL KINASE SIK IS PRESENT IN THE NUCLEUS AND PHOSPHORYLATES THE RNA·BINDING PROTEIN SAM68. Angela L. Tyner, Jason Derry, Stephane Richard, Taiping Chen, Xin Ye, Univ of Illinois Coil of Medicine, Chicago, IL; McGill Univ, Montreal, Quebec, Canada. Sik is an epithelial-specific intracellular tyrosine kinase that is expressed in differentiating cells of the gastrointestinal tract. Using a variety of techniques including transfection, immunoprecipitation. immunoblotting and confocal microscopy, we have identified the first substrate of Sik, the RNA binding protein Sam68. The cellular function of Sam68 (Src-associated in mitosis, 68 kDa) is not well understood, but its ability to functionally substitute for the HIV-l Rev protein suggests a role in RNA export and posttranscriptional gene regulation. Introduction of Sik and Sam68 expression constructs into the HT-29 human colon adenocarcinoma cell line results in Sam68 tyrosine phosphorylation. As a consequence of phosphorylation by Sik, the ability of Sam68 to bind RNA is inhibited. Sik and Sam68 colocalize in distinct nuclear structures termed SNBs (Sam68 Nuclear Bodies) in HT·29 cells. We found that Sik interacts with Sam68 through its SH3 and SH2 domains, and that the proline rich P3 region of Sam68 is required for Sik SH3 binding. While Sam68 may be phosphorylated by Src-family members during mitosis when the nuclear membrane breaks down, Sik is the first identified tyrosine kinase that is capable of phosphorylating Sam68 within the nucleus where it resides during most of the cell cycle. Sik phosphorylation of Sam68 within the nucleus may regulate gene expression associated with intestinal epithelial cell differentiation at the posttranscriptional level.
AGAA555
2916 MEVASTATIN POTENTIATES BUTYRATE·INDUCED ANTI· PROLIFERATIVE EFFECT AND INDUCES APOPTOSIS IN CO· LON CANCER CELLS. Astrid Waechtershaeuser, Bora Akoglu, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA-) inhibitors (HRI) prevent formation of mevalonate from HMG-CoA and thereby inhibit cholesterol synthesis. Intermediates in the cholesterol synthetic pathway, like farnesyl- and geranylgeranylpyrophosphate, are needed for isoprenylation of cellular proteins, a crucial step for membrane attachment. The aim of this study was to evaluate the effect of HMG-CoA inhibition in combination with butyrate on cell proliferation, differentiation and apoptosis in the human colorectal adenocarcinoma cell line Caco-2. Methods. Caco-2 cells were incubated with butyrate (I mM), mevastatin,(a HRI) or mevalonate. Cell growth was determined by crystal violet staining, alkaline phosphatase (AP) was used as a differentiation marker. Cytotoxicity was excluded by a commercial kit measuring lactate dehydrogenase activity in the supernatant of damaged cells. Apoptosis was quantified by use of a photometric ELISA, measuring cytoplasmic histone-associated DNA fragments. Cell cycle analysis was performed by propidium iodine stanining and flow cytometry. Cyclin-dependent kinase (Cdk) 4 and Cdk 6 were detected by immunoblotting. Results. Inhibition of HMG-CoA-reductase by mevastatin potentiated the antiproliferative effect of butyrate in a doseand time-dependent manner (22861 :t2866 cells/em" vs. 4476S:t5470 in butyrate group, p
2917 EXPRESSION OF 5·LIPOXYGENASE DURING BUTYRATE·IN· DUCED CELL DIFFERENTIATION OF HUMAN CARCINOMA CELLS. Astrid Waechtershaeuser, Stefan M. Loitsch, Dieter Steinhilber, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. Butyrate, a short-chain fatty acid produced by colonic fermentation of dietary fiber, modulates proliferation and differentiation of normal and neoplastic colonocytes. The underlying molecular mechanisms, however, are not clear. 5-Lipoxygenase (S-LO), besides its function as a key enzyme in leukotriene synthesis, has been shown to interact with cellular proteins, suggesting a possible role in the regulation of cellular growth. The aim of our studies was to investigate the differentiation-dependent expression of 5-LO and cyclooxygenase isoforms (COX-I, COX-2) in the human colorectal adenocarcinoma cell line Caco-2, which undergoes cell differentiation in the presence of sodium butyrate (NaBT). Methods. Human colon adenocarcinoma cells (Caco-2) were incubated with NaBT (2 mM). 5-LO, COX-I, COX-2, and FLAP (5-LO activating protein) on mRNA level were quantified by RT competitive multiplex PeR. The corresponding proteins were determined by immunoblotting. Cell differentiation was evaluated by analyzing alkaline phosphatase (AP) activity. TGF-13 secretion was measured by a commercial ELISA. Results. COX-2, COX-l and FLAP mRNA levels were not significantly influenced by NaBT treatment. In contrast, SoLOwas highly expressed on mRNA (78.3:t5.27-fold after 8d; p
was I.S :t 0.4 Nand 1.3 :t 0.6 N in control and WT.3 treated rats, respectively. Taken together, these findings suggest that specific inhibition of CD 18 function and reduced neutrophil recruitment may improve the survival of experimental skin flaps and, thus, may represent a potential target for therapeutic intervention. In contrast, we also found that blocking CD18-dependent neutrophil infiltration in the intestine did not change the breaking strength of colonic anastomosis. Thus, neutrophils may influence the wound healing process differently in specific organs. This needs to be considered when applying an anti-inflammatory treatment regime in order to improve tissue healing.
2914 HUMAN GASTRIC EPITHELIAL PROLIFERATION IS COORDI· NATED BY INSULIN·LIKE GROWTH FACTORS AND THEIR BINDING PROTEINS. Eric Tremblay, Pierre Chailler, Daniel Menard, Univ de Sherbrooke, Sherbrooke, PQ, Canada. IGF peptides (IGFl,IGF2) are known to be produced in fetal and adult gastric tissues and IGF2 is an autocrine factor for gut-derived cell lines. However their possible contribution to the regulation of cell proliferation during development or regeneration of human gastric mucosa is unknown, as the modulatory effect of IGF binding proteins (IGFBPs). AIMS: To evaluate the ability of human gastric mucosa to produce IGFBPs and to test the effects of native IGFs on epithelial cell proliferation as well as that of truncated IGFs which do not interact with IGFBPs. METHODS: Western! FarWestern blots and immunofluorescence were performed to characterize the expression of IGFl-receptor and IGFBP-l to -6. The effects of growth factors on DNA synthesis were determined in fetal gastric explants maintained in serum-free organ culture. RESULTS: All gastric epithelial cells expressed the IGFI-R. IGFBP-l protein detected in gastric tissue likely derived from an extragastric source: immunoreactivity was found in all mucosal cell types (endocrine pathway of delivery) and its total amount did not increase in culture. IGFBP-2 to -6 proteins were rather associated with epithelial cells and differentially localized along the pit-gland axis. They were produced endogenously and modulated in culture. Addition of IGFI to gastric explants stimulated 3H-thymidine incorporation into DNA after 24-48 h and increased the epithelial labeling index. Illustrating the counter regulatory effect of endogenous IGFBPs on IGF action, truncated R3IGFl was more potent than IGF1. DeI(l-6)IGF2 was the most potent inducer whereas IGF2 remained without significant effect (even at 200ng/mL). The last finding probably reflects the presence of IGFBP-2 and -6, two IGF2 carriers intensely synthesized in the pit/neck region comprising the proliferative compartment and abundantly secreted during culture. CONCLUSION: This study provides new evidence for the involvement of an intragastric IGF/lGFBP system in the fine regulation of epithelial cell division whereby the specific influence of IGF2 appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells. (Supported by the Medical Research Council of Canada; D.M. is a member of the MRC Research Group on Functional Development and Physiopathology of the Digestive Tract.)
2915 THE SRC-RELATED INTESTINAL KINASE SIK IS PRESENT IN THE NUCLEUS AND PHOSPHORYLATES THE RNA·BINDING PROTEIN SAM68. Angela L. Tyner, Jason Derry, Stephane Richard, Taiping Chen, Xin Ye, Univ of Illinois Coil of Medicine, Chicago, IL; McGill Univ, Montreal, Quebec, Canada. Sik is an epithelial-specific intracellular tyrosine kinase that is expressed in differentiating cells of the gastrointestinal tract. Using a variety of techniques including transfection, immunoprecipitation. immunoblotting and confocal microscopy, we have identified the first substrate of Sik, the RNA binding protein Sam68. The cellular function of Sam68 (Src-associated in mitosis, 68 kDa) is not well understood, but its ability to functionally substitute for the HIV-l Rev protein suggests a role in RNA export and posttranscriptional gene regulation. Introduction of Sik and Sam68 expression constructs into the HT-29 human colon adenocarcinoma cell line results in Sam68 tyrosine phosphorylation. As a consequence of phosphorylation by Sik, the ability of Sam68 to bind RNA is inhibited. Sik and Sam68 colocalize in distinct nuclear structures termed SNBs (Sam68 Nuclear Bodies) in HT·29 cells. We found that Sik interacts with Sam68 through its SH3 and SH2 domains, and that the proline rich P3 region of Sam68 is required for Sik SH3 binding. While Sam68 may be phosphorylated by Src-family members during mitosis when the nuclear membrane breaks down, Sik is the first identified tyrosine kinase that is capable of phosphorylating Sam68 within the nucleus where it resides during most of the cell cycle. Sik phosphorylation of Sam68 within the nucleus may regulate gene expression associated with intestinal epithelial cell differentiation at the posttranscriptional level.
AGAA555
2916 MEVASTATIN POTENTIATES BUTYRATE·INDUCED ANTI· PROLIFERATIVE EFFECT AND INDUCES APOPTOSIS IN CO· LON CANCER CELLS. Astrid Waechtershaeuser, Bora Akoglu, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA-) inhibitors (HRI) prevent formation of mevalonate from HMG-CoA and thereby inhibit cholesterol synthesis. Intermediates in the cholesterol synthetic pathway, like farnesyl- and geranylgeranylpyrophosphate, are needed for isoprenylation of cellular proteins, a crucial step for membrane attachment. The aim of this study was to evaluate the effect of HMG-CoA inhibition in combination with butyrate on cell proliferation, differentiation and apoptosis in the human colorectal adenocarcinoma cell line Caco-2. Methods. Caco-2 cells were incubated with butyrate (I mM), mevastatin,(a HRI) or mevalonate. Cell growth was determined by crystal violet staining, alkaline phosphatase (AP) was used as a differentiation marker. Cytotoxicity was excluded by a commercial kit measuring lactate dehydrogenase activity in the supernatant of damaged cells. Apoptosis was quantified by use of a photometric ELISA, measuring cytoplasmic histone-associated DNA fragments. Cell cycle analysis was performed by propidium iodine stanining and flow cytometry. Cyclin-dependent kinase (Cdk) 4 and Cdk 6 were detected by immunoblotting. Results. Inhibition of HMG-CoA-reductase by mevastatin potentiated the antiproliferative effect of butyrate in a doseand time-dependent manner (22861 :t2866 cells/em" vs. 4476S:t5470 in butyrate group, p
2917 EXPRESSION OF 5·LIPOXYGENASE DURING BUTYRATE·IN· DUCED CELL DIFFERENTIATION OF HUMAN CARCINOMA CELLS. Astrid Waechtershaeuser, Stefan M. Loitsch, Dieter Steinhilber, Wolfgang F. Caspary, Juergen Stein, J W Goethe Univ, Frankfurt/Main, Germany. Butyrate, a short-chain fatty acid produced by colonic fermentation of dietary fiber, modulates proliferation and differentiation of normal and neoplastic colonocytes. The underlying molecular mechanisms, however, are not clear. 5-Lipoxygenase (S-LO), besides its function as a key enzyme in leukotriene synthesis, has been shown to interact with cellular proteins, suggesting a possible role in the regulation of cellular growth. The aim of our studies was to investigate the differentiation-dependent expression of 5-LO and cyclooxygenase isoforms (COX-I, COX-2) in the human colorectal adenocarcinoma cell line Caco-2, which undergoes cell differentiation in the presence of sodium butyrate (NaBT). Methods. Human colon adenocarcinoma cells (Caco-2) were incubated with NaBT (2 mM). 5-LO, COX-I, COX-2, and FLAP (5-LO activating protein) on mRNA level were quantified by RT competitive multiplex PeR. The corresponding proteins were determined by immunoblotting. Cell differentiation was evaluated by analyzing alkaline phosphatase (AP) activity. TGF-13 secretion was measured by a commercial ELISA. Results. COX-2, COX-l and FLAP mRNA levels were not significantly influenced by NaBT treatment. In contrast, SoLOwas highly expressed on mRNA (78.3:t5.27-fold after 8d; p