| |
The monoclonal antibody
Ki-67, originally described by Gerdes et al in 1983, selectively reacts
with nuclei of proliferating cells in human tissues. Detailed cell cycle
analysis has shown that this nuclear protein, designated as Ki-67 protein,
is exclusively expressed during the cell division cycle in the G1, S, and
G2 phases as well as in mitosis and that it is absent in the G0 phase (Gerdes
et al, 1984). Because of this property, the monoclonal antibody Ki-67 has
become a useful tool for estimating the growth fraction in malignant disease.
Recently, Bloch et al (1995) reported that the Ki-67 protein is a target
of murine autoantibodies. In sera from MRL/MpJ-+/+ and MRL/MpJ-lpr/lpr mice,
they detected autoantibodies with reactivity against a recombinant peptide
of the human Ki-67 protein. The MRL mouse strain shows features that are
closely related to the clinical syndrome of the autoimmune disease systemic
lupus erythematosus (SLE; Theofilopoulos and Dixon, 1985). Thus, Bloch et
al (1995) suggested that the detection of Ki-67 protein-specific autoantibodies
in the MRL mouse strain and further characterization of this phenomenon
may give clues to the etiology and pathogenesis of autoimmune disease in
these animals. The present study demonstrates that sera from several SLE
patients contained autoantibodies against the Ki-67 protein, indicating
that this cell proliferation-associated protein is present as an autoantigen
under the pathogenic conditions that result in SLE.
An immunoprecipitation method was established that allowed the enrichment
of the Ki-67 protein from proliferating cells and the electrophoretic-based
demonstration of both polypeptides constituting the Ki-67 protein (Schluter
et al, 1993). Figure 1 shows a silver-stained SDS-PAGE from a sequential
immunoprecipitation experiment. In the first step, the Ki-67 polypeptides
were precipitated from a lysate of IM-9 cells with the monoclonal antibody
MIB-21, which is specific for the carboxyterminus of the Ki-67 protein.
The resultant immunoprecipitates were further purified by a second immunoprecipitation
step using the monoclonal antibody MIB-24, which recognizes the aminoterminus.
The apparent molecular weight was calculated by computer-aided analysis
of several silver-stained SDS-PAGEs, yielding an average apparent molecular
weight of 350 kd for the longer polypeptide and 315 kd for the smaller form
of the Ki-67 protein isoform. This finding indicates that our previously
published molecular weights, as determined using IM-9 lysates instead of
immunoprecipitates, were slightly overestimated (Schluter et al, 1993).
Figure 2 shows the results obtained from the modified immunoprecipitation
protocol mentioned above. A single immunoprecipitation step with the monoclonal
antibody MIB-1, which was found to be equivalent to the prototype antibody
Ki-67, was performed, and immunoprecipitates were immobilized onto nitrocellulose
filter by Western blotting (Key et al, 1993). Sera from 20 healthy blood
donors and from 18 patients suffering from SLE were analyzed for reactivity
against the MIB-1 immunoprecipitates. Whereas the sera of 19 blood donors
were negative by immunoblot analysis, the presence of autoantibodies could
not be excluded in one control serum (data not shown). In contrast, immunoblot
analysis showed that 6 of 18 sera samples reacted with the Ki-67 protein
isoforms, indicating that these patients had developed antibodies against
this cell proliferation-associated nuclear protein. In our series, we found
a preferential reactivity with the small type of the Ki-67 protein in two
of six positive sera. Furthermore, some sera showed strong reactions to
proteins of lower molecular weight, which represented either fragments of
the Ki-67 protein or different proteins coprecipitated with the monoclonal
antibody MIB-1. The precise characterization of these proteins is currently
underway.
In summary, not only are there Ki-67 protein-specific antibodies in the
sera from MRL mice, but autoantibodies with specificity against both Ki-67
protein isoforms could also be found in sera from SLE patients. These findings
provide further evidence that an immune response directed against the Ki-67
protein may be a common phenomenon in SLE and related diseases. Further
investigations should be able to clarify whether there is a relationship
between the pathogenesis and/or severity of this disease and the production
of autoantibodies specific for the cell division cycle-associated Ki-67
protein.
Acknowledgements
We thank R. Da[beta]ler and B. Baron for excellent technical assistance.
References
Bloch DB, Rabkina D, and Bloch KD (1995). The cell proliferation-associated
protein Ki-67 is a target of autoantibodies in the serum of MRL mice. Lab
Invest 73:366-371.
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, and Stein H (1984). Cell
cycle analysis of a cell proliferation-associated human nuclear antigen
defined by the monoclonal antibody Ki-67. J Immunol 133:1710-1715.
Gerdes J, Schwab U, Lemke H, and Stein H (1983). Production of a mouse monoclonal
antibody reactive with a human nuclear antigen associated with cell proliferation.
Int J Cancer 31:13-20.
Key G, Becker MHG, Baron B, Duchrow M, Schluter C, Flad HD, and Gerdes J
(1993). New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated
against bacterially expressed parts of the Ki-67 cDNA containing three 66
Bp repetitive elements encoding for the Ki-67 epitope. Lab Invest 68:629-635.
Laemmli UK (1970). Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680-685.
Leary JJ, Brigati DJ, and Ward DC (1983). Rapid and sensitive colorimetric
method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA
immobilized on nitro-cellulose: Bio-blots. Proc Natl Acad Sci USA 80:4045-4049.
Muro Y, Kano T, Sugiura K, and Hagiwara M (1997). Low frequency of autoantibodies
against Ki-67 antigen in Japanese patients with systemic autoimmune diseases.
J Autoimmunity 10:499-503.
Schluter C, Duchrow M, Wohlenberg C, Becker MHG, Key G, Flad HD, and Gerdes
J (1993). The cell proliferation-associated antigen of antibody Ki-67: A
very large, ubiquitous nuclear protein with numerous repeated elements,
representing a new kind of cell cycle-maintaining proteins. J Cell Biol
123:513-522.
Theofilopoulos AN and Dixon F (1985). Murine models of systemic lupus erythematosus.
Adv Immunol 37:269-390.
Towbin H, Staehelin T, and Gordon J (1979). Electrophoretical transfer of
proteins from polyacrylamide gels to nitro-cellulose sheets: Procedure and
some applications. Proc Natl Acad Sci USA 76:4350-4354.
Received September 24, 1997.
Affiliations: Department of Neurosurgery (CG), Medical University of Lubeck,
Lubeck, Germany; National Cancer Institute (MK), Frederick, Maryland; Medizinische-
und Poliklinik der Christian-Albrechts-Universitat im Stadtischen Krankenhaus
Kiel (US), Kiel, Germany; Department of Immunology and Cell Biology (HDF,
JG), Forschungszentrum Borstel, Borstel, Germany; and Institut fur Chemo-
und Biosensorik, Division of Immunotechnology (GK), Munster, Germany.
Note added in proof: After we submitted the present contribution, Muro et
al (1997) reported similar observations in the October issue of the Journal
of Autoimmunity.
The study was supported in part by the Dr. Mildred Scheel Stiftung fur Krebsforschung
Project W23/93/G3.
Address reprint requests to: Dr. J. Gerdes, Department of Immunology and
Cell Biology, Forschungszentrum Borstel, Parkallee 22, 23845 Borstel, Germany.
Fax: 49 4537 188 724 |
