Immortalization of primary epithelial cells requires first- and second-exon functions of adenovirus type 5 12S

Abstract

Immortalization of primary cells is a multistep process. The adenovirus EIA 12S gene product is a member of the class of oncoproteins that have the ability to establish primary cells as cell lines in culture. It is encoded by two exons. Extensive mutational analysis demonstrates that four regions of the ElA 12S gene, encoded by both exons, are necessary for immortalization of primary epithelial cells. Expression of two regions is necessary to activate quiescent cells into the cell cycle but is unable to extend the life span of these cells in culture and thus cannot immortalize them. These regions are encoded by the first exon. A third first-exon region, for which no function has yet been identified, is also required. These three regions are also required for 12S to cooperate with an activated ras gene to bring about tumorigenic transformation. The fourth region is required to maintain the cells in a proliferative mode, extend their life span in culture, and induce an autocrine growth factor. These functions are encoded by the second exon. The cells immortalized by wild-type 12S and immortalizationcompetent mutants retain their epithelial morphology and expression of keratin and vimentin intermediate filament proteins.

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

“…Inactivation of pl05Rb by binding to tumor virus proteins is consistent with reports that the CR2 domain of ElA is required to stimulate quiescent cells to transit the cell cycle during immortalization by ElA (Quinlan and Douglas, 1992) and with the CR2-dependent inhibition of TGF-j3. This hypothesis is also consistent with our results that the plO5Rb binding domain is required for inhibition of NGF activity.…”

“…Transformation, and Inhibition of Growth Factor Action? Mutational analysis of E1A has been used by others to study the mechanisms by which E1A immortalizes primary cells (Quinlan and Douglas, 1992), transforms cells in cooperation with other oncogenes (Ruley, 1983;Montell et al, 1984;Moran et al, 1986;Moran and Zerler, 1988;Stein et al, 1990), or inhibits growth factor activity. Immortalization of primary BRK cells by ElA or transformation in cooperation with the activated ras oncogene requires intact p300 and plO5Rb binding sites (Moran et al, 1986;Moran and Zerler, 1988;Stein et al, 1990), the same sites required to inhibit differentiation of PC12 cells in our experiments.…”

“…ElA is a potent oncogene that can transform established cells on its own and primary cells in cooperation with other oncogenes (Montell et al, 1984;Moran et al, 1986;Moran and Zerler, 1988;Quinlan and Douglas, 1992). ElA can transactivate gene transcription (Lillie and Green, 1989) and bind nuclear proteins (Harlow et al, 1986;Whyte et al, 1988), some of which have been associated with mitotic control (Whyte et al, 1988;Giordano et al, 1989;Goodrich et al, 1991).…”

Expression of the pRb-Binding Regions of E1A Enables Efficient Transformation of Primary Epithelial Cells by v- src

“…Five functional regions of E1A have been described, and mutations in all of these are represented by the E1A mutants shown in Fig. 1 and have been previously described (64,88). As shown in Fig.…”

“…The adenovirus (Ad) E1A gene is one of several DNA tumor virus oncogenes that have been used to study oncogene cooperative transformation of primary cells (for a review, see reference 2). E1A has been shown to immortalize primary cells, such that they maintain many of their differentiated characteristics (35,64,65). In addition, E1A can cooperate with Ha-ras and Ad E1B, as well as polyomavirus mT (pmT), to transform primary cells (14,71,81,88).…”