CONDITIONALLY IMMORTALIZED INTESTINAL EPITHELIAL-CELLS - NOVEL-APPROACH FOR STUDY OF DIFFERENTIATED ENTEROCYTES

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32-degrees-C but ceased to proliferate between 48 and 72 h of culture at 39-degrees-C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/Al, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39-degrees-C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.