Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene

Cyclin D1 gene overexpression is a frequent event in a number of human canc ers. These observations have led to the suggestion that cyclin D1 alteratio ns might play a role in the etiology of cancer. This possibility is support ed by the finding that transfection of mammalian cells with cyclin D1 can a ccelerate progression through the G1 phase of the cell cycle. Moreover, cyc lin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 tra nsgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cy clin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene wa s expressed in mammalian cells under the control of tet-repressor. This fus ion gene immortalized primary REFs, and cooperated with activated Haras to transform primary REFs, in terms of anchorage-independent growth in vitro a nd formation of tumors in vivo. Utilizing a tet-regulated gene expression s ystem, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.