title: The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats.
abstract: The Ki-67 antigen, pKi-67, is one of the most commonly used markers of proliferating cells. The protein can only be detected in dividing cells (G(1)-, S-, G(2)-, and M-phase) but not in quiescent cells (G(0)). The standard antibody to detect pKi-67 is MIB-1, which detects the so-called 'Ki-67 motif' FKELF in 9 of the protein's 16 tandem repeats. To investigate the function of these repeats we expressed three of them in an inducible gene expression system in HeLa cells. Surprisingly, addition of a nuclear localization sequence led to a complete absence of signal in the nuclei of MIB-1-stained cells. At the same time antibodies directed against different epitopes of pKi-67 did not fail to detect the protein. We conclude that the overexpression of the 'Ki-67 motif', which is present in the repeats, can lead to inability of MIB-1 to detect its antigen as demonstrated in adenocarcinoma tissue samples. Thereafter, in order to prevent the underestimation of Ki-67 proliferation indices in MIB-1-labeled preparations, additional antibodies (for example, MIB-21) should be used. Additionally, we could show in a mammalian two-hybrid assay that recombinant pKi-67 repeats are capable of self-associating with endogenous pKi-67. Speculating that the tandem repeats are intimately involved in its protein-protein interactions, this offers new insights in how access to these repeats is regulated by pKi-67 itself.
authors.affiliation: Henry Ford Hospital, Hermelin Brain Tumor Center, Department of Neurosurgery, 2799 W Grand Blvd, Detroit, MI 48202, USA.
authors.forename: Mirko H H
authors.initials: MH
authors.lastname: Schmidt
authors.forename: Rainer
authors.initials: R
authors.lastname: Broll
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Bruch
authors.forename: Michael
authors.initials: M
authors.lastname: Duchrow
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000032:analysis; D002455:Cell Division; D017124:Conserved Sequence; D004318:Doxycycline / Q000494:pharmacology; D000939:Epitopes; D005091:Exons; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D019394:Ki-67 Antigen / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D020080:Tandem Repeat Sequences / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D000939:Epitopes; D019394:Ki-67 Antigen; C440408:MIB-1 antibody; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D004318:Doxycycline