USE OF NORMAL AND TRANSGENIC MICE TO EXAMINE THE RELATIONSHIP BETWEENTERMINAL DIFFERENTIATION OF INTESTINAL EPITHELIAL-CELLS AND ACCUMULATION OF THEIR CELL-CYCLE REGULATORS

A spatially well organized continuum of proliferation, differentiation , and death is displayed along crypt-villus units in the adult mouse s mall intestine, This continuum provides an opportunity to examine in v ivo the mechanisms by which proliferative status changes as a function of cellular differentiation. Immunohistochemical studies of normal FV B/N mice revealed that as epithelial cells complete their terminal dif ferentiation during a 48-72-h migration up villi, there is a marked an d rapid fall in the levels of two important regulators of the G(1)/S t ransition, cyclin D-1 and cyclin-dependent kinase (cdk) 2, However, ce llular levels of their partners, cdk4 and cyclin E, remain unchanged a s does the level of pRB, Adult FVB/N transgenic mice were studied that contained an intestinal fatty acid binding protein gene promoter (Fab pi) linked to wild type Simian virus 40 large T antigen (SV40 TAgWt) o r a mutant TAg with Lys for Glu substitutions at residues 107 and 108 (SV40 TAgK107/8) that fails to bind pRB and related pocket proteins, B oth transgenes are expressed only in villus enterocytes, SV40 TAgWt ca uses these terminally differentiated cells to re-enter the cycle, Re-e ntry is accompanied by a reduction in un/hypophosphorylated pRB, an in duction of cyclin D-1 and cdk2, but no change in cdk4, cyclin E, or E2 F-1. In contrast, SV40 TAgK107/8 fails to induce reentry and does not produce changes in un/hypophosphorylated pRB, cyclin D-1, or cdk2 accu mulation, These results suggest that un/hypophosphorylated pRB is an i mportant mediator of the cell cycle arrest that normally occurs as ent erocytes exit the crypt and complete their differentiation. Fabpi-dire cted expression of E2F-1 does not cause villus enterocytes to return t o the cell cycle, alter their suppression of cyclin D-1 or cdk2, or af fect their state of differentiation emphasizing the insensitivity of t hese cells to the effects of E2F-1, Analyses of p53(-/-) and p58(+/+) mice containing Fabpi-SV40 TAgWt and Fabpi-SV40 TAgK107/8 established that the proliferation induced by SV40 TAgWt does not require p53 and is associated with increased (p53-independent) apoptosis, The presence of cyclin E and cdk4 in differentiating villus enterocytes emphasizes that these cells retain part of their proliferative heritage expresse d 24-72 h earlier in the crypt, The data suggest that down-regulation of cdk2 and/or cyclin D-1 expression may be important for control of p roliferative status and/or execution of terminal differentiation.