American Journal of Pathology, Vol. 138, No. 4, April 1991 Copyight X American Association of Pathologists

Immunobiochemical and Molecular Biologic Characterization of the Cell Proliferation-associated Nuclear Antigen That Is Defined by Monoclonal Antibody Ki-67 Johannes Gerdes, Li Li, Carsten Schlueter, Michael Duchrow, Claudia Wohlenberg, Christiane Gerlach, Ingrid Stahmer, Sabine Kloth, Ernst Brandt, and Hans-D. Flad

in the right reading frame. (Am J Pathol 1991,

From the Division of Molecular Immunology, Forschungsinstitut Borstel, Borstel, Federal Republic of Germany

Human nuclear antigens that are related to cell proliferation first were defined with the help of human autoantibodies.1'2 The murine monoclonal antibody Ki-67 reacts with a human nuclear antigen that is present in proliferating cells, but absent in quiescent cells.3 A detailed cell cycle analysis showed that the Ki-67 nuclear antigen is expressed in G1, S, G2, and mitosis, but not in Go.4 Thus with the help of this antibody, an exact and rapid determination of the growth fraction of a given human cell population, regardless of whether it is normal or malignant, had become possible. A highly significant correlation between the mean values of the growth fraction as determined with Ki-67 and histopathologic assessment of malignancy has been reported in all tumor entities thus far investigated.5:0 With regard to Ki-67 values of individual patients, however, a remarkable variation within all histologically defined tumor grades has been reported, indicating that the determination of the growth fraction with Ki-67 might be of help in outlining individual prognosis and individual therapy protocols.`20 This suggestion has been substantiated by different retrospective studies, demonstrating that the assessment of the growth fraction with Ki-67 is a new independent prognostic marker.21-3 Despite these well-documented diagnostic and prognostic applications of Ki-67, the antigen that is defined by this antibody is still not known. Thus the aim of this study was to characterize the nuclear antigen that is recog-

The monoclonal antibody Ki-67 detects a human nuclear antigen that ispresent in proliferating cells, but absent in quiescent cells. The aim of this study was to characterize the Ki-67 antigen by means of immunobiochemical and molecular biology techniques. Enzymatic digestion experiments showed that this antigen is highly susceptible to protease treatment, and the antigen cannot be extracted by 0.1 normal HCI indicating that Ki-67 antigen is a nonhistone protein Immunoblot analysis of cell lysates with Ki-67 showed a double band with apparent molecular weights of 395 kd and 345 k*4 regardless of whether the gels were run under reducing or nonreducing conditions. It is noteworthy that these bands were exclusively detectable in lysates prepared from proliferating cells; whereas they were absent in lysates obtained from quiescent cells. These immunobiochemical data are further substantiated by our molecular cloning approaches. By means of immunocloning with Ki-67, the authors isolated and sequenced several cDNA fragments from lambda gt1l libraries. A 1095-bp fragment gave a strong hybridization signal at 7.5 to 9.5 kb in Northern blot analysis with RNA prepared from proliferating cells, whereas it was negative with RNA preparedfrom quiescent cells. This cDNA fragment could be bacterially expresse4 and in subsequent immunoblot analysis Ki-67 reacted exclusively with those fusion proteins that were derivedfrom bacteria containing the insert

138:867-873)

Supported in part by a grant from the Dr. Mildred Scheel-Stiftung fur Krebsforschung, Deutsche Krebshilfe, Project W 32/87/Ge 1. Accepted for publication December 3, 1990. Address reprint requests to Dr. Johannes Gerdes, Division of Molecular Immunology, Forschungsinstitut Borstel, Parkallee 22, D-2061 Borstel, FRG.

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nized by Ki-67 by means of immunobiochemical and molecular biologic methods.

Acetone-fixed cytocentrifuged L428 cells were incubated with 0.1 N HCI for 30 minutes at room temperature to completely extract histone proteins as originally described by Fritzler and Tan.28

Materials and Methods

Cells and Specimens

Preparation of Cell Lysates

Normal human tonsils were provided by Dr. Luhmann, Bad Segeberg, FRG, and Dr. Allin, Bargteheide, FRG. L428 cells (Hodgkin's disease-derived cell line, courtesy of Prof. Diehl, Koln, FRG), MCF-7 (breast carcinoma cell line), and IM-9 (multiple myeloma cell line, courtesy of Prof. Stein, Berlin, FRG) were cultured under standard conditions in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with antibiotics and 10 or 15% of fetal calf serum. Peripheral blood mononuclear cells (PBMNC) were obtained from healthy donors by standard methods, and phytohemagglutinin (PHA) stimulation was carried out as described.4

Pilot studies to solubilize Ki-67 antigen by differential cell solubilization disclosed inconsistent and irreproducible results in subsequent immunoprecipitation or immunoblot experiments. We could overcome these problems by applying the following protocol: 4 to 7 x 107 viable cells were pelleted by centrifugation at 400g for 10 minutes. The pellet was resuspended in 500 ,ul phosphatebuffered saline (PBS) containing 1 mmol/l (millimolar) phenylmethylsulfonylfluoride (PMSF). The cells were snap-frozen in liquid nitrogen and subsequently pulverized in a mortar at - 80°C. The powdered cells were solubilized immediately with 500 ,ul of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with or without 2-mercaptoethanol, as described below.

Antibodies The monoclonal antibody Ki-67, which is of IgG1 subclass, was prepared as described.3 As isotype control we used antibody Ber-H2, which reacts with the CD30 antigen.24 A rabbit antibody to human histone H2B, which is referred to as rabbit anti-H2B in the following, was a gift of Prof. Bustin (Bethesda, MD).25 Rabbit antimouse Ig antiserum was obtained from Dakopatts, Copenhagen, Denmark. Alkaline phosphataseconjugated goat anti-mouse IgG was obtained from Dianova (Hamburg, FRG).

Immunostaining Frozen tissue sections or cytocentrifuged cell preparations were immunostained by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method,26 using New Fuchsin development.27

Enzymatic Digestion of Cell Lysates Aliquots of - 80°C cell powder of 4 x 107 cells were incubated in either 500 p,l PBS containing PMSF or 500 p,l PBS containing 30 pug/ml DNase, 30 ,ug/ml RNase, or 30 ,ug/ml Trypsin for 30 minutes at 37C.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed using a 5% acrylamide Laemmli-9 slab gel system with a 3.5% stacking gel. Sample buffer contained 20% glycine, 0.125 mol/l (molar) TRIS-HCI (pH 6.8), 6% SDS with or without 10% 2-mercaptoethanol. Before electrophoresis, samples were heated to 1 00C for 5 minutes. Molecular weight markers were obtained from Pharmacia, LKB (Freiburg, FRG).

Enzymatic Digestion and Acid Extraction Experiments

Immunoblotting

Acetone-fixed cryostat sections of human tonsils were treated with different enzymes at various concentrations for 30 minutes at 370C. Trypsin, RNase, DNase, alkaline phosphatase, and neuraminidase were purchased from Sigma (Muechen, FRG); papain was a product of Merck (Darmstadt, FRG).

After SDS-PAGE, the proteins were transferred to nitrocellulose membranes by Western blot transfer for 16 hours at 10°C. To block unoccupied binding sites, filters were incubated for 30 minutes in TRIS buffer, pH 7.5 (100 mmol/l TRIS, 100 mmol/l NaCI, 2.5 mmoVl MgCl2, 0.05% Triton X-1 00), containing 3% bovine serum albumin. After

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Table 1. Ki-67 Reactivity of Gerninal Center Cells as Determined by Immunostaining of Enzyme-treated Acetone-fixed Frozen Tonsil Sections*

Enzyme/concentration Trypsin Papain RNAse DNAse Alkalinephosphatase Neuraminidase

0.001%

0.00075%

0.0005%

0.000025%

(w/v) -t -

(w/v) -

0.00001%

(w/v)

(w/v)

(w/v)

-

-+

+

+++ +++ +++ +++

+++ +++ +++ +++

+++ +++ +++ +++

+++ +++

+

+++

+++ +++ +++ +++

+++

+++

TBS control +++ +++ +++ +++ +++ +++

Intensity of staining was evaluated in comparison with TBS control sections. t - = negative. -/+ = very weak positivity. + = weak positivity. + + = strong positivity. + + + = very strong positivity. *

three washes with TRIS buffer, the membranes were incubated with the primary antibodies for 1 hour. Bound antibody was targeted with alkaline phosphataseconjugated goat anti-mouse IgG, and bound alkaline phosphatase was visualized according to the method of Leary et al.30

Immunocloning A lambda gtl 1 library of IM-9 cDNA was obtained from Genofit, Heidelberg, FRG. Immunoscreening was performed according to the guidelines of the manufacturer (Clontech Laboratories Inc., Palo Alto, CA). Preparation of lambda gtl 1 DNA, isolation of cDNA insert, and ligation into M13 vectors were performed as described earlier.31 DNA sequence analysis was carried out by the dideoxychain-termination method.32 Sequences obtained were compared with known sequences using the Microgenie system (Beckman, Muechen, FRG). pEV-vrfl,2,3 vectors' were donated by Dr. Zschunke (Kiel, FRG), and expression of cDNA in all reading frames was performed as described.33

Results

Properties of Ki-67 Nuclear Antigen as Determined In Situ Frozen sections of normal human tonsillar tissue were incubated with different enzymes at various concentrations and subsequently immunostained with Ki-67. Table 1 summarizes the results of a characteristic experiment. It is obvious that the antigen recognized by Ki-67 is highly susceptible to protease, whereas it is resistant to the other enzymes employed. Acetone-fixed cytocentrifuged L428 cells were incu-

bated with 0.1 normal HCI for 30 minutes at room temperature to extract all histone proteins, and subsequently immunostaining was performed with Ki-67 or with rabbit anti-histone H2B antiserum. As demonstrated in Table 2, untreated L428 cells exhibited nuclear staining with rabbit anti-histone H2B antiserum, whereas after acid extraction the nuclei of these cells were negative with this antiserum. In contrast, Ki-67 stained both nuclei of untreated cells and those nuclei after acid extraction. Only a slight decrease in the intensity of Ki-67 staining could be rec-

ognized.

Immunobiochemical Characterization of Ki-67 Nuclear Antigen Cell lysates of unstimulated PBMNC and PHA-stimulated PBMNC and lysates of MCF7 and IM9 were separated on SDS-PAGE under reducing and nonreducing conditions. After immobilization of the separated peptides on nitrocellulose filters by Western blot transfer, the filters were immunostained with Ki-67. Figure 1 demonstrates a characteristic result of such an immunoblot assay. In those blots prepared with cell lysates of proliferating cells, Ki-67 Table 2. Nuclear Staining of L428 Cell Line Cells with Ki-67 and a Rabbit Anti-H2B Antiserum With and Without AcidEJtraction L428 cells L428 cells after untreated acid extraction Rabbit anti-H2B 1:4 + + +1:8 +++ 1:16 ++ Ki-67 supernatant 1:2 +++ ++ 1:4 +++ ++ 1:8 ++ + + + + = very strong staining. + + = strong staining. + = weak but distinct staining. - = negative.

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Figure 1. Immunoblot analysis of reduced lysates obtained from different cells and cell

lines. Blots obtained in identical runs were immunostained with Ki-67 or Ber-H2 or with secondary reagents only. The following cell lysates were applied: lane 1: unstimulatedperipheral blood mononuclear cells (PBMNC); lane 2: unstimulated PBMNC culturedfor 72 hours; lane 3: phytohemagglutininstimulated PBMNC cultured for 72 hours; lane 4: IM-9 cell line; lane 5: MCF-7 breast carcinoma cell line. In cell lysates prepared from proliferating cells Ki-67 immunostains a double band with apparent molecular weights of 395 and 345 kd 7his double band was absent in both controls. 7Te Ber-H2 antibody shows the appropriate molecular weightfor its antigen, which is the CD30 protein.

immunostained a double band with apparent molecular weights of 345 and 395 kd, regardless of whether SDSPAGE was performed under reducing or nonreducing conditions. Immunoblots of unstimulated PBMNC were consistently Ki-67 negative. The control antibody Ber-H2 did not react with the bands detected by Ki-67. This antibody reacted with its appropriate antigen in lysates prepared from IM9- and PHA-stimulated PBMNC, however, demonstrating that this antibody is an adequate isotype control.

with RNA or DNA.

Immunocloning A lambda gtl 1 IM-9 cDNA library was immunoscreened with Ki-67 after IPTG-induction. In 2 x 1 6 plaques, we

TTCTTAGCAC TCAGGAAACT AACACCATCA GCAGGGAAAG GCAGGAGGTG ATGAGAAAGA CATTAAAGCA TTTATGGGAA CTGGCAGGAA CTTTACCTGG CAGCAAAAGA CAGCTACAGA GCTCTAGAAG ACCTGGCTGG CTTTAAAGAG CTCTTCCAGA

CCATGCTTAC CTCCAGTGCA CTCCTAAGGA CTCCTGGTCA

GCCCAAACCA 60 Figure 2. DNA sequence of a 1095 bp DNA GAAACTGGAC 120 fragment of the human Ki-67 gene (a) and DNA sequence of a 429-bp DNA fragment of AAAGGCCCAG 180 the Ki-67 gene (b). 7he 62-bp region presumCACCGAGGAA 240 ably encoding for the Ki-67 epitope is under-

TTAGTGGCTG GACACCCCAA GGAGAACTCT AAACCATCAG CTGGACCTGA GCCCAGGCTC GAAGAAGCAG TCAGCAGACA GTACAGAAGG ACAGATAAAG CAGAAACTGG

CTCCACAGTC TCAGGAAAGC GCAAAGCCAT TGGGAACTCC CACAAACTCC TCCAGACCCC GCGAATCTTC CACCTTTGGA CATCAGGGGA CGTTTAGGGA GGCACCCAAA TCTTCCAGAC CCTGCAGATC GAAGTCTCAG

AGACCCAGTG 300 lined AGATGTAGAG 360 GCACACGCCT 420 AGTGCAGAAA 480 TAAGGAAGAG 540 TGGTCATACT 600 TCCACCAGAA 660 GAAAAGGGAC 720 AACCACACAC 780 AACTGCAAAA 840 AACTAAGGAA 900 ACCAGTATGC 960 ACAACCAGAC 1020 GAAAGTGGAC 1080

AAGGCCCAAC ACTGACAAGC

CCAGTGGACA

A

Experiments involving enzymatic digestion (data not shown) showed that the high molecular weight determined by immunoblotting with Ki-67 was not caused by contamination or complexing of the Ki-67 polypeptide

GTAGAAGAAG

CTGGTAAAAC CAAGCACAAA TAGCGTGCAG TAGGTGAAGA CAGAGAACTT TGGAAGACCT TGGCTGCTGG CCCCAACAAG AGCTCTCAGC TACCAGGAGG ACCCAGCAGC CCCTAGAAGA CCACGACTCA CACCAACAAG AATTC

CTACAAACTC TTCCAGACAC TGCAAATCTT ACATCCCTGG ACGTCAGGGA GCGTTTAAGG AGGTGGCCAA

B GAGCTCTTC

CACTAAAATA CCCTGCGACT GCAACGACCC AAGAGAAGTA GAATCTAATG CCATCAGCAG GAAAGACATC ATCATATTTG AACCGGCAGC AAGAGACGGC GACTGGCTTT AAAGAGCTCT CAAAACTACT AAAATGCCCT CACAAGAAGG CAGCCCAAGA CCTGAAGAAG CTCACACAGA TGAGGATAAA AGCATCAACG AAGTGTAACT GGTAGCAAGA CCTGGCTGGC TGGAAAGAGC CGAGAAAACT ACCAAAATAG CTCCAAGCCA CAGTCCAAGA

CTAAGGAAAA GGCCCAGGCT CTAGAAGACC TGGCTGGCTT GAGGTCACAC TGAGGAATCA ATGACTAACG ATAAAACTGC CACAACCAGA CCCAGACAAA AACCCAGCAA GCTCCAAGCG GGAAAGTGGG CGTGAAAGAA GAGGTCCTAC CAGTCGGCAA AGACCACACA GACACACAGA GAGACAGCAG GAGATGGAAA AATCTGCAAA GCAGATGCTG GACCCAGCAA ACTATGGAAC GAACACCTAA GGAAGAGGCC CAATCACTAG AAGACCTGGC

TAAAGAGCTC CAAAGTAGCC ACGGCTCAAG GCTCACACAG GAGCATCAAA TGGGATGGAG CGGCTTCAAA

60 120 180 240 300 360 420

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at different percentages of homology. Furthermore, the 1 095-bp sequence contains repetitive elements of about 300 bp. The sequence did not show any significant homology with known DNA sequence when compared with the data base within the Microgenie system.

Northern Blot Analysis As shown by Northern blot analysis (Figure 3), the 1095bp fragment reacted with RNA from proliferating cells with an apparent size of 7.5 to 9.5 kb, whereas it was negative with RNA prepared from quiescent cells. The amount of RNA applied in the different lanes was equivalent when hybridized with a cDNA probe for ,3-actin. Figure 3. Northern blot analysis. mRNA prepared from cell line cells (IM-9, lane 1; L428, lane 3) or quiescent peripheral blood mononuclear cells (lane 2) were hybridized with the 1095-bp cDNA fragment isolated witb Ki-67 by immunocloning. A strong hybridization signal was obtained with RNA from the proliferating cell line cells in the range between 7.5 and 9.5 bp, whereas PBMNC were negative.

initially found 18 positive ones, 10 of which could be clonally obtained after tertiary screening. After sequencing, eight of these clones exhibited the identical sequence that is depicted in Figure 2A. Figure 2B demonstrates the sequence of a smaller cDNA fragment isolated by immunocloning with Ki-67. This fragment matches with the other fragment only within 62 bp. Interestingly, these 62 bp were repeated within the larger fragment (underlined) Figure 4. Immunoblot analysis of recombinant Ki-67 material. A 1095-bp fragment was expressed in bacteria in all different reading frames (lanes 1 and 2: correct readingfframe; lane 3: second, lane 4: third reading frame). Ki-67 reacts only with the lysates from bacteria containing the Ki-67-positive cDNA insert in the correct reading frame.

Bacterial Expression of Ki-67-Positive cDNA The 1095-bp fragment was expressed in all different reading frames. To obtain the correct orientation of the isolated cDNA fragment, the pEV-vectors and the fragment were cut with different restriction enzymes (Eco Ri, Bam Hi, Sau 3a) and then ligated. Escherichia cofi HB 101 were transformed with the different constructs, and transformants containing the correct expression plasmids with the appropriate inserts were lysed, applied to SDS-PAGE, and subsequently analyzed by immunoblotting with Ki-67 or control antibody, respectively. Figure 4 demonstrated that Ki-67 reacted only with lysates from

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those bacteria containing the insert in the correct reading frame.

Discussion Because of its well accepted value for diagnostic purposes, numerous efforts have been made to characterize the antigen that is defined by Ki-67. Standard solubilization protocols, however, eg, sequential solubilization, did not result in a consistent immunochemical definition of the antigen (own unpublished observations and recently published by Verheijen et al34). In light of the findings detailed in this contribution, these disappointing experiences may be due to the fact that the Ki-67 antigen is extremely sensitive to proteases. Therefore, we tried to shorten the time of cell lysate preparation, ending up with a protocol to prepare deep-frozen cell powder that was solubilized in SDS-PAGE sample buffer and immediately subjected to SDS-PAGE. Using this procedure, immunoblotting analysis consistently and reproducibly demonstrated two major bands with apparent molecular weights of 345 and 395 kd, regardless of whether the gels were run under reducing or nonreducing conditions. This double band could only be demonstrated in lysates prepared from proliferating cells, but was absent in lysates prepared from quiescent cells (PBMNC). These biochemical results could be substantiated by our molecular biologic approaches, because Northern blot analysis using our Ki-67-positive cDNA disclosed a very large mRNA (7.5 to 9.5 kb). Even though we have not sequenced the entire gene thus far, our results demonstrate that the 1 095-bp fragment described in this study is an integral part of the Ki-67 gene. This fragment contains repetitive sequences of about 300 bp, containing a sequence of 62 bp that is repeated at very high levels of homology. Because the smaller fragment described in this paper matches only with this 62-bp sequence, and as it is present in all other cDNA clones isolated thus far by immunocloning, we assume that this sequence, or parts of it, encode for the epitope that is detected by

Ki-67. Because of the high molecular weight and the sequence thus far known, the Ki-67 antigen is distinct from other proliferation-associated structures such as cyclin,35 p53,36 c-myc, or other oncogene products related with the cell proliferation.37 Recently Verheijen et al34 speculated that the Ki-67 antigen might be a DNA topoisomerase type 11`34 As reviewed by Wang,' DNA topoisomerases II have been characterized from different species as single polypeptide chains with molecular weights in the range of 150 to 180 kd, indicating that a relation of these enzymes to the Ki-67 antigen is quite unlikely. Koike and Ohtsuki39 recently reported on a murine

400-kd nonhistone chromatine protein that is present in large amounts in malignant and normal proliferating cells, but not in resting cells. This structure was found to be a tetramer of 98-kd polypeptide chains and, thus, a relation to the Ki-67 antigen again seems to be improbable. In conclusion, our data show that the Ki-67 antigen is a nonhistone protein assembled by polypeptide chains with an apparent molecular weight of 345 and 395 kd, which is encoded by a thus far unknown gene sequence.

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