Proliferation marker pKi‐67 affects the cell cycle in a self‐regulated manner

The proliferation marker pKi‐67 is commonly used in research and pathology to detect proliferating cells. In a previous work, we found the protein to be associated with regulators of the cell cycle, controlling S‐phase progression, as well as entry into and exit from mitosis. Here we investigate whether pKi‐67 has a regulative effect on the cell cycle itself. For that purpose we cloned four fragments of pKi‐67, together representing nearly the whole protein, and an N‐terminal pKi‐67 antisense oligonucleotide into a tetracycline inducible gene expression system. The sense fragments were C‐terminally modified by addition of either a nuclear localization sequence (NLS) or a STOP codon to address the impact of their intracellular distribution. FACS based cell cycle analysis revealed that expression of nearly all pKi‐67 domains and the antisense oligonucleotide led to a decreased amount of cells in S‐phase and an increased number of cells in G 2 /M‐ and G 1 ‐phase. Subsequent analysis of the endogenous pKi‐67 mRNA and protein levels revealed that the constructs with the most significant impact on the cell cycle were able to silence pKi‐67 transcription as well. We conclude from the data that pKi‐67 influences progression of S‐phase and mitosis in a self‐regulated manner and, therefore, effects the cell cycle checkpoints within both phases. Furthermore, we found pKi‐67 mediates an anti‐apoptotic effect on the cell and we verified that this marker, although it is a potential ribosomal catalyst, is not expressed in differentiated tissues with a high transcriptional activity. J. Cell. Biochem. 87: 334–341, 2002. © 2002 Wiley‐Liss, Inc.