Processing of cyclin E differs between normal and tumor breast cells.
Cyclin E is a G1 cyclin essential for G1 to S-phase transition of the cell cycle with a profound role in oncogenesis. In tumor cells and tissues, cyclin E is overexpressed and present in its lower molecular weight (LMW) isoforms, and it can be used as a prognosticator for poor patient outcome. In this study, we have examined differences in the processing of cyclin E between normal mammary epithelial and breast cancer cell lines. Five NH2-terminally deleted epitope-tagged (FLAG) cyclin E vectors were constructed spanning 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 cyclin 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. Tumor and normal cells always process the cyclin E-FLAG protein in the same way as endogenously expressed cyclin E. This phenomenon is consistent with all of the cell lines used, regardless of transfection efficiency, time of processing posttransfection, or method of transfection. Furthermore, measurement of FLAG-associated kinase activity in the transfectants revealed that the protein products of the cyclin E-FLAG constructs are 10 times more active 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.
Harwell
R M
RM
Division of Molecular Medicine, Wadsworth Center, Albany, New York 12201-0509, USA.
Porter
D C
DC
Danes
C
C
Keyomarsi
K
K
D015153:Blotting, Western; D001940:Breast; D001943:Breast Neoplasms; D002460:Cell Line; D019927:Cyclin E / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D019927:Cyclin E; D011994:Recombinant Proteins