Abstracts
1998 Digestive Disease Week

#1067

A POSSIBLE ROLE FOR p21 IN ALTERATION OF THE ENTEROCYTE DIFFERENTIATION ASSOCIATED WITH PREMATURE CELL CYCLE WITHDRAWAL. S. Archer, S. Meng, R.A. Hodin. Dept. of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.

Normal enterocyte differentiation is characterized by a simultaneous withdrawal from the cell cycle. Premature crypt cell cycle withdrawal occurs under conditions of mucosal atrophy, e.g., prolonged starvation, and results in alteration of the differentiation process, i.e., a specific decrease in intestinal alkaline phosphatase (IAP). We sought to examine the role of the cell cycle inhibitor, p21, in this process. METHODS: HT-29 cells were grown in standard media or induced to early cell cycle withdrawal by one of two methods: (1) 48 hrs. of serum starvation or (2) forced overexpression of the cell cycle inhibitor, p21. p21 overexpression was accomplished by cotransfecting HT-29 cells with plasmids expressing the human p21 cDNA and/or the NEO resistance gene, and selecting stable lines in G418. Cells from each group were treated with 5mM sodium butyrate (NaBu) to induce enterocyte differentiation. 3H-thymidine incorporation was measured and Northern blot analyses were performed using cDNA probes specific for the markers of differentiation, IAP and villin, p21, and the actin control. RESULTS: Under standard growth conditions, NaBu induced enterocyte differentiation, with increases in IAP and villin mRNAs (15-and 2.5-fold respectively, p<0.001), and concomitant cell cycle arrest (90% decrease in 3H-thymidine incorporation, p<0.001). Serum starved control HT-29 cells and the p21 stable transfectants were prematurely withdrawn from the cell cycle, e.g., a 50% decrease in 3H-thymidine in the p21 transfectants compared to the controls, and displayed an approximately 5- and 3-fold induction of p21 mRNA respectively (p<0.001). In both the serum starved cells and p21 transfectants, NaBu caused an increase in villin (2.5-fold, p<0.001), but no increase in IAP. CONCLUSIONS: Premature cell cycle withdrawal alters the normal course of enterocyte differentation, such that induction of IAP is blocked despite normal increases in villin. The cell cycle inhibitor, p21, may play a role in the alterations in enterocyte phenotype seen under conditions of mucosal atrophy.

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