Tumorigenic transformation of established murine fibroblasts by transfection with amplification promoting DNA...

Deregulated replication of the cellular genome is assumed to result in tumorigenic transformation of the cell. We tested this hypothesis by using mouse DNA elements that promote amplification of transfected DNA in mammalian cells when linked to a selectable marker gene that is driven by a truncated promoter (Holst et al., Cell 52, 355-365 (1988); Wegner et al., Nucl. Acids Res. 17, 9909-9932 (1989)). Here, the DNA elements muNTS-1, e-1, e-2, e-4, e-5, and e-12 were inserted into the plasmid p-hyg, which contains the gene for resistance to hygromycin B driven by a constitutive promoter. After transfer into established rat fibroblasts (208F), transfected DNA constructs persist in low copy numbers (1-10 copies per cell) integrated into high molecular weight DNA. We observed a neoplastically transformed phenotype in 40% to 70% of hygromycin-resistant colonies of 208F cells which is dependent on the DNA element transfected. 208F cells transfected with vector DNA exhibit a "normal" phenotype and are not tumorigenic. The transformed cells, on the other hand, induced malignant tumors after injection into immunodeficient NMRI nu/nu mice. In contrast to established cells, primary rat embryo fibroblasts (REF) with limited life span are neither neoplastically transformed nor immortalized after transfection of e-4 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)