Robert Beauchamp
Principal Investigator
Last active: 10/31/2018

Smad3 has a critical role in TGF-beta-mediated growth inhibition and apoptosis in colonic epithelial cells.

Mithani SK, Balch GC, Shiou SR, Whitehead RH, Datta PK, Beauchamp RD
J Surg Res. 2004 117 (2): 296-305

PMID: 15047135 · DOI:10.1016/S0022-4804(03)00335-4

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.

MeSH Terms (15)

Animals Apoptosis Cell Division Cell Line Colon DNA-Binding Proteins Growth Inhibitors Intestinal Mucosa Mice Mice, Transgenic Mutation Promoter Regions, Genetic Smad3 Protein Trans-Activators Transforming Growth Factor beta

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