Biol Cell (1991) 73, 7-14 7 © Elsevier, Paris Original article Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene* Bertrand Schwartz ~, Patrick Vicart ~, Claude Delouis 2 Denise Paulin a** i Laboratoire de Biologie Moldculaire de ia Diffdrenciation de I'Universitd Paris 7 et de I'Institut Pasteur, 25, rue du Docteur-Roux, 75754 Paris; Z INRA, Station de Physioiogie animale, 78352 Jouy-en-Josas Cedex, France (Received 3 January 1991 ; accepted 21 August 1991) Summary - The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused down- stream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides - 878 to + 93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained charac- teristics of differentiated cells: binding of Uiex europaeus lectin and synthesis of Factor VII! for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells. immortuli~tion i endothellum I SV40 1 vimentin geu Introduction Cells removed from a foetus or an adult animal and cul- tured in vitro proliferate initially, but stop after a finite number of divisions. Even cells derived from tumors, in most cases, exhibit such a pattern of limited division and proliferation. In contrast, expression of simian -~'irus 40 (SV40) early genes has been shown to transform cells and allow establishment of permanent cell lines [1, 2]. The maintenance of the immortalized phenotype is dependent on the presence of the large T protein [2], involving gene activation of transcription factors, ie stimulating Ha-ras and trans-acting factor Spl at the mRNA level and affect- ing the transcription factor PEAl, and also involving protein-protein interaction with "anti-oncogene products" such as p53, pll0 Rb and p120, and with enhancer-binding proteins such as AP2 (reviewed in [3-5]). The SV40 early genes have been introduced into a wide range of cells, and the induced phenotypes have been extensively studied ([6-20], human cells reviewed in [21, 22]), showing that these cells may retain some of their initial characteristics of differentiated state, including responses to inducers: angiotensin-converting enzyme was occasionally detectable in transformed endothelial cells [6], Schwann cell lines synthesised specific proteins [16], major keratins in transformed epidermal cells were similar to those found in their normal counterparts [17], bronchial epithelial cells retained the ability to undergo squamous diffel'entiation when exposed to TPA or TGF-~ [15], trans- formed vascular smooth muscle cells had normal dose- * In memoriam to Daniel Sandoz ** Correspondence and reprints response curves to heparin [19], breast epithelial cell lines can differentiate along the myo-epithelial-like cell path- way [20], embryonal cells may still be induced to differen- tiate with retinoic acid [13]. The large T antigen has been shown to be effective to immortalize a very broad range of cells by using recom- binant genes with promoters that are either tissue-specific or ubiquitous: eg, respectively, the insulin promoter, or 5' upstream sequences of the metaliothionein gene. The association of these promoters with large T sometimes in- duced unexpected tissue specificities. For example, GRF and vasopressin regulatory regions linked to SV40 early genes did not exhibit appropriate expression in hypothala- mus, but did in thymic epithelium and pancreas, respec- tively ([23, 24] reviewed in [251). The gene encoding vimentin is expressed in vivo in mesenchymal cells and in muscular and neural precursor cells, before the acquisition of specific networks of other intermediate filaments (for references, see [26]). Further- more, filaments of the vimentin type occur or can be formed in vitro in a vast majority of cultured cells of ver- tebrate origin, irrespective of the tissues from which they were originally derived [27]. The synthesis of vimentin pro- tei~ is enhanced in most transformed cells and vimentin could play a role in cellular transformation and differen- tiation [28-34]. The amount of vimentin is regulated main- ly at the transcriptional level [35, 36], and the vimentin gene is a growth regulated gene whose activity is increased by such molecules as platelet derived growth factor (PDGF), phytohemaggiu~mi~ {PHA), phorbol esters (TPA), butyrate, and the lax protein from HTLV-I [37-43]. In the present work, SV4~ cat I~ ~cnc~ were fused to the vimentin promoter which a~t, ~.tt~cnt I~ in most cultured