BACKGROUND: Smad proteins play a key role in TGF-beta signaling that regulates cell proliferation, differentiation, and apoptosis. Mice deficient in Smad3 develop colonic adenocarcinoma. MATERIALS AND METHODS: We developed a Smad3-deficient colonocyte cell line that was used to study TGF-beta-mediated growth inhibition and induction of apoptosis was compared to young adult mouse colonocyte (YAMC) control cells. Growth inhibition was assessed by cell count and ((3)H)-thymidine incorporation assay. Transcriptional response to TGF-beta was measured by transfecting the reporters p3TP-Lux and p(CAGA)(9)-MLP-luc. TGF-beta-induced apoptosis was assessed using ELISA and Hoechst staining. Mediators of cell-cycle arrest and apoptosis were assayed by Western blot. RESULTS: Smad3-/- cells were resistant to TGF-beta-mediated growth inhibition compared to control cells. Ninety-eight percent of cell count growth inhibition observed in YAMC cells, while 34% inhibition was observed in Smad3-/- cells after TGF-beta treatment. ((3)H)-thymidine incorporation was inhibited by 61% in YAMC cells, while Smad3-/- cells showed 25% inhibition after TGF-beta treatment. Smad3-/- cells were deficient in luciferase reporter induction by TGF-beta. TGF-beta induced apoptosis 8-fold in YAMC cells, but had no effect on apoptosis in Smad3-/- cells. p21(Cip11) and PAI-1 are induced in YAMC cells by TGF-beta, but unchanged in Smad3-/- cells. TGF-beta decreases cyclin D1 levels in YAMC cells but does not affect levels in Smad3-/- cells. CONCLUSIONS: Our findings suggest that the loss of Smad3 contributes to resistance of TGF-beta growth inhibition and apoptosis in colonic epithelium. This may represent a mechanism by which cells are able to escape antiproliferative controls and embark on a pathway toward neoplasia.
BACKGROUND: Smad proteins play a key role in TGF-beta signaling that regulates cell proliferation, differentiation, and apoptosis. Mice deficient in Smad3 develop colonic adenocarcinoma. MATERIALS AND METHODS: We developed a Smad3-deficient colonocyte cell line that was used to study TGF-beta-mediated growth inhibition and induction of apoptosis was compared to young adult mouse colonocyte (YAMC) control cells. Growth inhibition was assessed by cell count and ((3)H)-thymidine incorporation assay. Transcriptional response to TGF-beta was measured by transfecting the reporters p3TP-Lux and p(CAGA)(9)-MLP-luc. TGF-beta-induced apoptosis was assessed using ELISA and Hoechst staining. Mediators of cell-cycle arrest and apoptosis were assayed by Western blot. RESULTS:Smad3-/- cells were resistant to TGF-beta-mediated growth inhibition compared to control cells. Ninety-eight percent of cell count growth inhibition observed in YAMC cells, while 34% inhibition was observed in Smad3-/- cells after TGF-beta treatment. ((3)H)-thymidine incorporation was inhibited by 61% in YAMC cells, while Smad3-/- cells showed 25% inhibition after TGF-beta treatment. Smad3-/- cells were deficient in luciferase reporter induction by TGF-beta. TGF-beta induced apoptosis 8-fold in YAMC cells, but had no effect on apoptosis in Smad3-/- cells. p21(Cip11) and PAI-1 are induced in YAMC cells by TGF-beta, but unchanged in Smad3-/- cells. TGF-beta decreases cyclin D1 levels in YAMC cells but does not affect levels in Smad3-/- cells. CONCLUSIONS: Our findings suggest that the loss of Smad3 contributes to resistance of TGF-beta growth inhibition and apoptosis in colonic epithelium. This may represent a mechanism by which cells are able to escape antiproliferative controls and embark on a pathway toward neoplasia.
Authors: Curtis A Thorne; Alison J Hanson; Judsen Schneider; Emilios Tahinci; Darren Orton; Christopher S Cselenyi; Kristin K Jernigan; Kelly C Meyers; Brian I Hang; Alex G Waterson; Kwangho Kim; Bruce Melancon; Victor P Ghidu; Gary A Sulikowski; Bonnie LaFleur; Adrian Salic; Laura A Lee; David M Miller; Ethan Lee Journal: Nat Chem Biol Date: 2010-10-03 Impact factor: 15.040
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Authors: Sergio Kaiser; Young-Kyu Park; Jeffrey L Franklin; Richard B Halberg; Ming Yu; Walter J Jessen; Johannes Freudenberg; Xiaodi Chen; Kevin Haigis; Anil G Jegga; Sue Kong; Bhuvaneswari Sakthivel; Huan Xu; Timothy Reichling; Mohammad Azhar; Gregory P Boivin; Reade B Roberts; Anika C Bissahoyo; Fausto Gonzales; Greg C Bloom; Steven Eschrich; Scott L Carter; Jeremy E Aronow; John Kleimeyer; Michael Kleimeyer; Vivek Ramaswamy; Stephen H Settle; Braden Boone; Shawn Levy; Jonathan M Graff; Thomas Doetschman; Joanna Groden; William F Dove; David W Threadgill; Timothy J Yeatman; Robert J Coffey; Bruce J Aronow Journal: Genome Biol Date: 2007 Impact factor: 13.583