Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.
The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements.
Duchrow
M
M
Surgical Research Laboratory, University Clinic of Luebeck, Luebeck, Germany. Duchrow@medinf.mu-luebeck.de
Schmidt
M H
MHH
Zingler
M
M
Anemüller
S
S
Bruch
H P
HP
Broll
R
R
D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000032:analysis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016372:RNA, Antisense / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D020080:Tandem Repeat Sequences; D014162:Transfection
D019394:Ki-67 Antigen; D016372:RNA, Antisense; D011994:Recombinant Proteins; D001973:Bromodeoxyuridine