Processing of cyclin E differs between normal and tumor breast cells

Cyclin E is a G(1) cyclin essential for G(1) to S-phase transition of the c ell cycle with a profound role in oncogenesis. In tumor cells and tissues, cyclin E is overexpressed and present in its lower molecular weight (LMW) i soforms, and it can be used as a prognosticator for poor patient outcome. I n this study, we have examined differences in the processing of cyclin E be tween normal mammary epithelial and breast cancer cell lines. Five NH2-term inally deleted epitope-tagged (FLAG) cyclin E vectors were constructed span ning the range of LMW forms observed in tumor cells. These constructs were transfected into normal and tumor cells and analyzed for the production of cyclin E-FLAG protein products by Western blot analysis with FLAG and cycli n E antibodies. Our results show that only tumor cells had the machinery to process these cyclin E-FLAG constructs to their LMW forms, whereas normal cells mainly expressed the full-length unprocessed form of each protein. Tu mor and normal cells always process the cyclin E-FLAG protein in the same w ay as endogenously expressed cyclin E. This phenomenon is consistent with a ll of the fell lines used, regardless of transfection efficiency, time of p rocessing posttransfection, or method of transfection. Furthermore, measure ment of FLAG-associated kinase activity in the transfectants revealed that the protein products of the cyclin E-FLAG constructs are 10 times more acti ve in tumor cells than in normal cells. These studies suggest that the LMW forms of cyclin E detected at a much higher level in tumor cells arise from posttranslational action of a protease.