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Serum response factor is alternatively spliced in colon cancer
252
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
man colon cancer cells were studied. DOX and INDO were used at clinically achievable concentrations. Beta-catenin levels were determined by western blotting. Beta-catenin-dependent transcription was measured by TOPFlash reporter assay. Cell viability was determined by tiazolyl blue (MTT) reduction and growth was determined by direct cell counting. In vivo, tumor growth was determined by measurement of subcutaneous xenografts in BALB/c nude mice. DOX was administered in drinking water, and INDO by IP injection. Results: In vitro, both DOX and INDO exposure result in a dose dependent down-regulation of beta-catenin protein and beta-catenindependent gene transcription, resulting in inhibition of cell viability and proliferation. A synergistic relationship of combining these two agents is evident. In vivo studies show an enhanced anti-tumor effect of combined DOX and INDO treatment on SW480 xenograft growth (Figure 1). Conclusions: DOX and INDO suppress beta-catenin expression and transcription activity in colon cancer cells, inhibiting cancer cell growth in vitro and in vivo. DOX and INDO may be of clinical use in therapeutic or chemopreventative strategies for colon cancer.
41. Serum Response Factor is Alternatively Spliced in Colon Cancer. L. C. Patten, M.D., N. S. Belaguli, Ph.D., M. Baek, M.D., D. H. Berger, M.D. Baylor College of Medicine, Houston, TX. Introduction: Serum response factor (SRF) is a transcription factor important in cellular differentiation and cell cycle regulation. SRF function is regulated in part by alternative splicing. Little is known about the expression or role of these alternatively spliced forms during tumorigenesis. We hypothesized that there is a change in expression of splice variants during intestinal tumorigenesis, and that this change in splice variant expression promotes the tumor phenotype. Methods: The expression of SRF was determined by western blotting of normal intestinal cells and human colon cancer cell lines. To determine the effect of alternatively spliced forms of SRF on intestinal growth and proliferation, the major alternatively spliced isoform of SRF (SRF‚5) seen in cancer cells was transfected into IEC-6 cells. IEC-6 and IEC-6SRF‚5 cells were plated on matrigel on day 0. Cell numbers were determined at four timepoints. Results: Western blotting demonstrates that full length SRF is the predominant form of SRF in IEC-6 cells and the well-differentiated human colon cancer cell line HT-29. In poorly differentiated human colon cancer cells (WiDr, HCT 116, LoVo, and SW480) SRF‚5 is the dominant form expressed (Figure 1). There was a marked increase in cell growth kinetics in IEC-6 cells transfected with SRF‚5 compared to the parental cells (Figure 2). Conclusion: This data demonstrates that an alternatively spliced form of SRF, SRF‚5 is expressed in human colon cancer cell lines. Additionally, this data demonstrates that expression of SRF‚5 may contribute to the tumor phenotype by affecting cell growth. This is the first study to document a change in alternative splicing of SRF in human malignancy.
RESIDENT AWARD SESSION 42. Treatment of Bile Acid Malabsorption Using Ileal Stem Cell Transplantation. J. R. Avansino, M.D., V. Hoagland, B.S., J. Woolman, B.A., W. G. Haigh, Ph.D., M. Stelzner, M.D. University Of Washington/VAPSHCS. Purpose: To determine if ileal stem cells transplanted into a segment of jejunum can be used to treat bile acid malabsorption. Methods: In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3 mM EDTA solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neo-natal Lewis rats and transplanted into these segments. After 4 weeks, rats underwent an ileectomy and the isolated segment was anastomosed in its place. After an additional 4 weeks, a 48 hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein (by immunohistomorphometry) and IBAT mRNA quantitation (by RTPCR). Data was analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy or sham operation served as controls. Results: Total bile acid loss in the stool was significantly lower in rats with a neo-ileum compared to rats with an ileectomy (p ⬍ 0.004; Fig. 1). Na ⫹-dependent taurocholate uptake was significantly increased in the neo-ileum compared to the jejunum (p ⬍ 0.001). IBAT protein signal intensity was significantly higher in the neo-ileum compared to jejunum (p ⬍ 0.001). IBAT mRNA amounts were significantly higher (p ⬍ 0.004) than in the jejunum however comparable to the ileum. Conclusion: Ileal stem cells were used to establish a new zone of active bile acid uptake and IBAT expression in a jejunal segment. This neo-ileum eliminated loss of bile acids in the stool after ileectomy. This is the first time intestinal stem cell transplantation has been shown to correct a clinical malabsorption syndrome.
43. Cigarette Smoke Increases Intimal Hyperplasia (IH) and Homocysteine (Hcy) in a Rat CEA Model. J. A. Davis, M.D., A. T. Brown, Ph.D., H. Chen, M.D.. Y. Wang, B.S., J. F. Eidt,
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
man colon cancer cells were studied. DOX and INDO were used at clinically achievable concentrations. Beta-catenin levels were determined by western blotting. Beta-catenin-dependent transcription was measured by TOPFlash reporter assay. Cell viability was determined by tiazolyl blue (MTT) reduction and growth was determined by direct cell counting. In vivo, tumor growth was determined by measurement of subcutaneous xenografts in BALB/c nude mice. DOX was administered in drinking water, and INDO by IP injection. Results: In vitro, both DOX and INDO exposure result in a dose dependent down-regulation of beta-catenin protein and beta-catenindependent gene transcription, resulting in inhibition of cell viability and proliferation. A synergistic relationship of combining these two agents is evident. In vivo studies show an enhanced anti-tumor effect of combined DOX and INDO treatment on SW480 xenograft growth (Figure 1). Conclusions: DOX and INDO suppress beta-catenin expression and transcription activity in colon cancer cells, inhibiting cancer cell growth in vitro and in vivo. DOX and INDO may be of clinical use in therapeutic or chemopreventative strategies for colon cancer.
41. Serum Response Factor is Alternatively Spliced in Colon Cancer. L. C. Patten, M.D., N. S. Belaguli, Ph.D., M. Baek, M.D., D. H. Berger, M.D. Baylor College of Medicine, Houston, TX. Introduction: Serum response factor (SRF) is a transcription factor important in cellular differentiation and cell cycle regulation. SRF function is regulated in part by alternative splicing. Little is known about the expression or role of these alternatively spliced forms during tumorigenesis. We hypothesized that there is a change in expression of splice variants during intestinal tumorigenesis, and that this change in splice variant expression promotes the tumor phenotype. Methods: The expression of SRF was determined by western blotting of normal intestinal cells and human colon cancer cell lines. To determine the effect of alternatively spliced forms of SRF on intestinal growth and proliferation, the major alternatively spliced isoform of SRF (SRF‚5) seen in cancer cells was transfected into IEC-6 cells. IEC-6 and IEC-6SRF‚5 cells were plated on matrigel on day 0. Cell numbers were determined at four timepoints. Results: Western blotting demonstrates that full length SRF is the predominant form of SRF in IEC-6 cells and the well-differentiated human colon cancer cell line HT-29. In poorly differentiated human colon cancer cells (WiDr, HCT 116, LoVo, and SW480) SRF‚5 is the dominant form expressed (Figure 1). There was a marked increase in cell growth kinetics in IEC-6 cells transfected with SRF‚5 compared to the parental cells (Figure 2). Conclusion: This data demonstrates that an alternatively spliced form of SRF, SRF‚5 is expressed in human colon cancer cell lines. Additionally, this data demonstrates that expression of SRF‚5 may contribute to the tumor phenotype by affecting cell growth. This is the first study to document a change in alternative splicing of SRF in human malignancy.
RESIDENT AWARD SESSION 42. Treatment of Bile Acid Malabsorption Using Ileal Stem Cell Transplantation. J. R. Avansino, M.D., V. Hoagland, B.S., J. Woolman, B.A., W. G. Haigh, Ph.D., M. Stelzner, M.D. University Of Washington/VAPSHCS. Purpose: To determine if ileal stem cells transplanted into a segment of jejunum can be used to treat bile acid malabsorption. Methods: In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3 mM EDTA solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neo-natal Lewis rats and transplanted into these segments. After 4 weeks, rats underwent an ileectomy and the isolated segment was anastomosed in its place. After an additional 4 weeks, a 48 hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein (by immunohistomorphometry) and IBAT mRNA quantitation (by RTPCR). Data was analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy or sham operation served as controls. Results: Total bile acid loss in the stool was significantly lower in rats with a neo-ileum compared to rats with an ileectomy (p ⬍ 0.004; Fig. 1). Na ⫹-dependent taurocholate uptake was significantly increased in the neo-ileum compared to the jejunum (p ⬍ 0.001). IBAT protein signal intensity was significantly higher in the neo-ileum compared to jejunum (p ⬍ 0.001). IBAT mRNA amounts were significantly higher (p ⬍ 0.004) than in the jejunum however comparable to the ileum. Conclusion: Ileal stem cells were used to establish a new zone of active bile acid uptake and IBAT expression in a jejunal segment. This neo-ileum eliminated loss of bile acids in the stool after ileectomy. This is the first time intestinal stem cell transplantation has been shown to correct a clinical malabsorption syndrome.
43. Cigarette Smoke Increases Intimal Hyperplasia (IH) and Homocysteine (Hcy) in a Rat CEA Model. J. A. Davis, M.D., A. T. Brown, Ph.D., H. Chen, M.D.. Y. Wang, B.S., J. F. Eidt,