nuclear distribution of the ki-67 antigen during the cell ...lation between the percent of...

[CANCER RESEARCH 49, 2999-3006, June 1, 1989]

Nuclear Distribution of the Ki-67 Antigen during the Cell Cycle: Comparison withGrowth Fraction in Human Breast Cancer Cells1

Jan Hein van 1Merendonek,- Rob Keijzer, Cornelis J. H. van de Yelde, and Cees J. Cornelisse

Departments of Surgery [J. H. v. D., R. K., C. J. H. v. d. V.] and Pathology [C. J. C.], University Hospital, P. O. Box 9600, 2300RC Leiden, The Netherlands

ABSTRACT

It has been claimed that the commercially available Ki-67 monoclonalantibody recognizes a nuclear antigen which is solely expressed in cyclingcells. Therefore, at present, Ki-67 is increasingly used as a tool inevaluating growth fractions (GFs) of human tumors. Here we describespecific patterns in the expression and topologica! distribution of thisantigen during the cell cycle in MCF-7 human breast cancer cells. Ourresults support earlier findings that the antigen belongs to a class ofantigens associated with the structural organization of meta- and ana-phase chromosomes, and proteins located near the cortical regions ofprenucleolar bodies and nucleoli. Using 5'-bromodeoxyuridine-labeling

technology, we show that the expression may be undetectably low at theonset of DNA replication.

Comparison of Ki-67 fractions (KFs) and GFs as estimated fromcontinuous S'-bromodeoxyuridine-labeling curves revealed that KF was

invariably higher than GF: in exponentially growing cov362.cl4 humanovary cancer cells, KF was only 3.5% higher than GF; in MCF-7 cells,11.3 Â±4.6%. In MCF-7 cultures either growing under suboptimal conditions or treated with 10 " M tamoxifen, the difference was more

pronounced. Furthermore, we evaluated the decrease of Ki-67-positivecells in nutritionally deprived and cell cycle-specific, drug-treated cultures. Since the results indicate that nonproliferating cells may retain theantigen for a considerable period of time, KF may not always be a reliableindicator of GF.

INTRODUCTION

An increasing number of studies underline the importance oftumor cell kinetics in the prediction of disease-free interval andshort-term survival in breast cancer patients (1-3). Moreover,as cell kinetic parameters determine the effectiveness of chemo-and radiotherapeutic treatment, an estimation of the fractionof tumor cells actively engaged in cell cycle traverse GF3 orDNA synthesis (S-phase fraction) could be helpful in findingan optimal therapeutic approach in individual patients. Furthermore, cell kinetic data on lymph node mÃ©tastasesand thepossible correlation with various characteristics of the primarytumors could be important with respect to chemo- and radiotherapy as an adjuvant to surgery.

Various methods to estimate GFs and S-phase fractions havebeen developed, including pulse- and continuous labeling withtritiated thymidine (4), biochemical analysis of DNA polymer-ase activity (5), and flow cytometric analysis of DNA and RNAcontent (6, 7). However, for routine clinical application thesemethods are either impractical, as they need in vivo administration of a radioactively labeled compound and many serial biopsies from possibly heterogenous tumors, or lack specificity andprecision (8).

Received 11/30/88; accepted 2/27/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported by the Netherlands Cancer Foundation, GrantIKW 87-12.

2To whom requests for reprints should be addressed.3The abbreviations used are: GF, growth fraction; KF, Ki-67-positive fraction;

Moab, monoclonal antibody; TAM, tamoxifen; MTX, methotrexate; FCS, fetalcalf serum; Brill rd, S'-bromodeoxyuridine; PBS, phosphate-buffered saline; RT,room temperature; GA, glutaraldehyde; PLP, periodate-lysine-paraformaldehyde;PE, paraformaldehyde; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzi-

dine; FRG, Federal Republic of Germany; PCN, perichromonucleolin.

A promising recent development is the application of Moabsthat recognize antigens associated with cell proliferation (9-15), therefore allowing an easy and rapid in situ evaluation ofimmunostained tissue samples. One of these Moabs, increasingly used in histopathology, is the commercially available Ki-67 Moab (16, 17). This mouse Moab, raised against crudenuclear fractions of a Hodgkin's lymphoma-derived cell line,

has been used to determine GFs in a number of tumors, i.e.,lymphomas (18, 19), and lung (20), brain (21), and breastcarcinomas (22-26).

Although the biochemical characteristics of the Ki-67 antigenhave not yet been elucidated, it has recently been shown forhuman lung carcinoma and leukemia cells that the antigen isassociated with the nucleolar cortex and the periphery of met-

aphase chromosomes (27, 28). In this paper we describe specificstaining patterns in nuclei of MCF-7 human breast andcov362.cl4 ovarian cancer cells and evaluate in MCF-7 mono-layers the staining patterns of cells present in different phasesof the cell cycle. We have studied the effects of cytostatic agents,such as MTX and TAM, on antigen expression and the correlation between the percent of Ki-67-positive cells and GFs asdetermined from continuous labeling with BrdUrd in MCF-7cultures growing under various conditions.

We show that, although these patterns allow for a subdivisionof the cell cycle, intercellular variability precludes an unambi-gious determination of S-phase fractions. Furthermore, wediscuss the observed discrepancy between Ki-67 positivity and

GF in this cell line.

MATERIALS AND METHODS

Antibodies. Tissue culture supernatant of the mouse monoclonalantibody Ki-67 (IgGl; trade name, DAK-PC; code No. M722) was agift from Dakopatts (Glostrup, Denmark). Anti-BrdUrd monoclonalantibody IU-4 was a gift from Dr. F. Dolbeare (Livermore, CA).

Cell Lines and Growth Conditions. The hormone-responsive humanbreast cancer line MCF-7 (29) was provided by Dr. M. Lippman(National Cancer Institute, Bethesda, MD). The cov362.cl4 humanovary cancer line (30) was a gift from Dr. P. Schrier (UniversityHospital, Leiden, The Netherlands). Cells plated in a density of about0.1 x 10s cells/ml were grown as monolayers on glass slides in Leightontubes at 37 Â°Cin 5 ml of RPMI-1640 culture medium, supplemented

with 5 to 10% FCS (Gibco, Paisly, Scotland), 20 mM 4-(2-hydroxy-ethyl)-l-piperazineethanesulfonic acid buffer (BDH Chemicals, Ltd.,Dorset, England), 15 mM sodium bicarbonate (Baker Chem, Deventer,Holland), 100 lU/ml of penicillin, and 0.1 mg/ml of streptomycinsulfate. The culture medium was (unless stated otherwise) replaced byfresh culture medium every 3 days.

Hormone and Drug Treatments. The antiestrogenic compound TAM,a gift from ICI-Pharma, Macclesfield, England, was added at a finalconcentration of IO"6M in fresh culture medium to MCF-7 cultures 3

days after plating (when about 25% confluency was reached). Recruitment from a TAM-induced G^ block was performed by washing theflasks twice with culture medium before adding fresh medium supplemented with IO"7 M 17/3-estradiol (Sigma, St. Louis, MO). TAM and

170-estradiol were added as stock solutions in ethanol to the mediumat the appropriate concentration, but such that the final concentrationof ethanol in culture was less than 0.02%.

2999

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CELL CYCLE DISTRIBUTION OF Ki-67 ANTIGEN IN MCF-7 CELLS

MTX (Pharmachemie B.V., Haarlem, Holland) was used in finalconcentrations of IO"7 M.

BrdUrd-labeling and Fixation Procedure. Unless stated otherwise, cellcultures were labeled by adding BrdUrd (Sigma, St. Louis, MO) as astock solution in PBS to the culture medium at a final concentrationof 20 mM, 1 day after the cultures had received fresh culture medium.After various time intervals, cells were washed in PBS, fixed 2 timesfor 5 min in acetone at -20Â°Cand at RT subsequently, and air dried.Providing storage under dry conditions, storage of the slides at â€”20Â°C

after fixation did not affect the outcome of the staining procedure.Other fixation protocols tested were: 4% neutral buffered formalin;

Mouin's fixative; 2.5% GA; methanohacetic acid (3:1, v/v) (Carnoy's

fixative); 70% ethanol; 2% PF; and PLP.Immunohistochemistry. In order to evaluate both Ki-67 and BrdUrd

staining patterns in the same cell culture, a fraction of the area of theslides was incubated under a coverslip for 30 to 60 min at RT with Ki-67 Moab appropriately diluted in PBS with 1% BSA (Sigma, St. Louis,MO) and 0.1% Tween-20. Slides were washed in PBS (3 times, 5 min),and subsequently peroxidase-conjugated rabbit anti-mouse immuno-globulin (Dakopatts, Glostrup, Denmark), appropriately diluted in PBSwith 20% normal human serum, was applied for 30 min. After washing3 times for 5 min with PBS, peroxidase activity was detected with asubstrate mixture of DAB (Sigma, MÃ¼nchen,FRG) and 0.05% H2O2in PBS, resulting in the intranuclear deposition of brown pigment.

The protocol for the detection of incorporated BrdUrd has beendescribed previously (31). In short, DNA was denatured by immersingthe slides for 10 min in 0.07 MNaOH containing 70% ethanol, followedby dehydration in a series of ethanol and treatment with proteinase K(0.1 mg/ml; Boehringer, Mannheim, FRG) for 10 min at RT. Thesedenaturation conditions did not affect the DAB stain already presentin the area stained with Ki-67.

After a second dehydration step, a fraction of the slide surface (onlypartially overlapping the area stained with Ki-67) was incubated undera coverslip during l h at RT with IU-4, appropriately diluted in PBSwith 1% BSA. Hereafter, the same protocol was followed as describedabove for the Ki-67 staining.

In additional experiments, BrdUrd incorporation was detected with3-amino-9-ethylcarbazole (EGAchemie, Steinheim. FRG), resulting ina red stain and allowing an easy discrimination between BrdUrd-Iabeledreplication sites and Ki-67 detected with the DAB technique.

Slides were finally counterstained with Mayer's hematoxylin and

mounted with Malinol.Electron Microscopy. For electron microscopy, an MCF7 monolayer

was fixed in PLP (2% paraformaldehyde, 15 min, RT), incubated during15 min in 0.5 M NH4C1 in PBS and 10 min in PBS with 1% BSA, andallowed to react for l h with Ki-67 in the same medium. After washingthe slide with PBS (3 times, 10 min), a peroxidase-conjugated rabbitanti-mouse IgG antibody (Dakopatts, Glostrup, Denmark) was appliedfor 2 h, and cells were washed again in PBS and fixed with 2% GA in0. l M cacodylate buffer (pH 7.4). After another PBS wash step, peroxidase activity was detected with DAB (10 min), and cells werepostfixed during 30 min with reduced OsO4. After dehydration, agelatin capsule was placed upon the monolayer, and cells were embedded in epoxy resin (Epon) and routinely processed for electronmicroscopy.

Morphometrical Analysis. Nuclear projection areas were measuredby tracing the profiles of labeled nuclei on a graphic tablet (MOP-AMOS; Kontron, Echingen, FRG), using a Leitz microscope providedwith a camera lucida drawing tube. About 25 to 50 nuclei of cellsshowing a specific Ki-67 staining pattern were measured.

Frequency distributions of staining patterns were determined bytracing the nuclei that traversed the image field while systematicallyscanning the slide until 250 to 500 nuclei were encountered.

Estimation of Growth Fractions. Growth fractions of both cell lineswere estimated from continuous BrdUrd-labeling curves by extrapolating the more slowly rising component of each curve backwards to theend of the G2 phase (see Fig. 7). The duration of G2 was estimated byscoring the appearance of BrdUrd label in mitotic figures.

Flow Cytometry. Cells were carefully scraped from approximatelyone-fifth part of the area of the slides, frozen, stored in citrate buffer

(pH 7.6) with 10% dimethyl sulfoxide, and prepared for flow cytometryby using the method as described by Vindel0v et al. (32). After stainingwith propidium iodide (Sigma, St. Louis, MO), samples were measuredon an ICP 22 flow cytometer (Ortho, Westwood, MA).

RESULTS

Fixation and Staining Conditions. In order to optimize intranuclear Ki-67 staining in MCF-7 monolayers, we evaluated theeffects of different fixation protocols followed by a standardperoxidase staining. Results are summarized in Table 1. Im-munoreactivity was not detectable after fixation with formalin,glutaraldehyde, or acid-containing fixatives. In contrast, intensestaining was obtained after fixation with paraformaldehyde oracetone. Since most intense staining was obtained after fixationin cold acetone, this protocol was used in further experiments.We also compared a number of immunohistochemical stainingprotocols, namely, the peroxidase-antiperoxidase technique, incubation with biotinylated rabbit anti-mouse IgG followed byincubation with an avidine-biotin-peroxidase complex, and intensification of diaminobcn/Jdini'/HiO: color development

with cobalt chloride. Since these different protocols did notchange the criteria with respect to the assessment of weaklypositive cells (data not shown), the indirect immunoperoxidaseassay as described in "Materials and Methods" was used as

standard.Morphology and Cell Cycle Distribution. The staining pat

terns as observed in nuclei of MCF-7 human breast cancer cellshave tentatively been arranged in Fig. 1.

These highly characteristic patterns were also readily observed in cov362.cl4 ovarian cancer cells (Fig. 2). Postmitoticdaughter nuclei were easily recognized (Figs. 1 and 2, upperlanes); these nuclei were of relatively small size and alwaysdisplayed many, intensely stained spots. Measurements of nuclear areas and frequencies of denned staining patterns inpostmitotic nuclei of MCF-7 cells, summarized in Table 2, aresuggestive of a remarkable rearrangement of the antigen duringthe telophase. As the heavily stained anaphase chromosomesdecondense (and nuclei increase in size), they form a pattern ofapparently homogeneously distributed speckles (Fig. IB). Thelow frequency of this pattern suggests that the small spotsquickly aggregate (Fig. 1C) to form larger, irregularly shapedclusters (Fig. ID), eventually forming nucleoli-like structures(Fig. IE). This staining seems more intense on the nucleolarrim, a phenomenon particularly evident in the relatively largenucleoli of cov362.cl4 cells. The association of the antigen withnucleolar structures is also illustrated by electron micrographs(Fig. 3), showing an MCF-7 nucleus with many DAB deposits,which have a tendency to cluster around distinct regions (A),and two neighboring cells (B), in one of which the nucleoli aresurrounded by a thin layer of electron-dense DAB.

Table 1 Effect of various fixatives on Ki-67 immunoreactivity in MCF7monolayers

FixativeImmune-reactivity*

Buffered formalin (4%, 10 min, RT)Bouin's fluid (10 min, RT)

Glutaraldehyde (2.5%, 10 min, RT)Carnoy's fixative (10 min, RT)Ethanol (70%, 5 min -20'C; 100%, 5 min RT)Methanol (100%, 5 min -20'C, 5 min RT)

PLP (2%, 10 min RT)Paraformaldehyde (2%, 10 min RT)Acetone (100%, 5 min -20'C, 5 min RT)

* Scored as negative (-), weakly positive in a limited number of cells (+/-),

moderately positive (+), strongly positive (++), and very strongly positive (+++).

3000




B

H K

M N OFig. 1. Ki-67 staining patterns in MCF-7 cells. The upper lane shows a putative sequence of cells during mitosis (A) and early (., phase (IS to /â€¢). In the middle

lane nuclei are shown in which staining is absent (C7)or confined to nucleoli (// to /.). Finally, in the lower lane nuclei are shown in which the antigen is present inboth nucleolar structures and the nuclear matrix. The last nucleus in this series represents a cell in prophase, characterized by chromosome condensation (/?). x 500.

/

Fig. 2. Ki-67 staining patterns observed in cov362.cl4 cells. The antigendistribution is essentially the same as in MCF-7 cells (compare with Fig. 1),showing an association with chromosomes during mitosis (A), a clustering duringtelophase and early G, phase (/.( and C), confinement to nucleoli in many cells(/)), and the homogeneous darkening of the nucleus commensurate with adistortion and/or enlargement of the nucleolus (E and F). Mitotic cov362.cl4cells frequently display many small spots around the clustered chromosomes (Aand B, arrows). X 500.

In the largest fraction of exponentially growing MCF-7 cells,the expression of Ki-67 was restricted to a limited number ofrelatively small nucleoli (Fig. 1, middle lane). With increasingnuclear size, however (Table 2), staining became more intenseand was found in the nuclear matrix as well (see also Fig. 1,lower lane). Also the nucleolar size increased dramatically, mostoften showing a highly distorted (worm-shaped) morphology.Occasionally we observed that the large, irregular clusters weregranulated, possibly representing a disintegration of nucleoli(Figs. IP and 2E). A relatively small fraction of nuclei, whichwere most intensely stained (Figs. 1, Q and A, and 2F), alsoappeared to have the largest mean nuclear projection area(Table 2). These nuclei probably represent the transition intoprophase, in which the chromatin condenses into metaphasechromosomes (Fig. IR).

Prevention of chromosomal segregation by Colcemid (an

alkaloid which inhibits polar migration of centrioles) did notappear to affect the redistribution process of the antigen duringtelophase and early d phase (data not shown).

In order to evaluate the frequency and patterns of Ki-67staining during S phase, we labeled MCF-7 cultures during 20min with BrdUrd and subsequently performed an immunocy-tochemical double staining for Ki-67 antigen and DNA-incor-porated BrdUrd. In a previous paper (33), we described that thecharacteristic patterns of incorporated BrdUrd reflect the spa-tional and temporal organization of DNA synthesis. Early S-phase cells are characterized by the presence of 100 to 150distinct replication sites, homogeneously distributed over thenuclear projection area, but with a low density close to thenuclear boundary and nucleoli (Fig. 4A). During mid-S phase,nuclei show a relatively large number of closely spaced spots(Fig. 45), being particularly prominent in perinucleolar chromatin. In late S phase, spots are larger, irregularly shaped, andpreferentially located close to the nuclear boundary and thenucleoli (Fig. 4("). The application of a double staining proce

dure enabled us to allocate Ki-67 staining patterns to thesestages of DNA replication (Fig. 4, D to F; Table 3). Surprisingly,in a majority of cells in early S phase, the Ki-67 staining waseither faint or undetectable, suggesting that, during late GÃ¬phase, the antigen is expressed at a lower level or is even absentin actively proliferating cells. In contrast, in late S phase, amajority of cells showed large, intensely stained nucleoli ofirregular shape and an increased nuclear matrix staining, therefore demonstrating a higher expression of Ki-67 antigen.

A different approach to evaluate the cell cycle distribution ofthe Ki-67 antigen is to analyze synchronized cell populations.For this purpose, MCF-7 cells were treated with TAM, resultingin a significant accumulation of cells in the (MO phase (Fig. 5,upper lane). Release of the d.o block by 17/3-estradiol induceda wave of cells to enter the cell cycle, resulting in an accumulation in early S phase 17 h later. This was accompanied by arelative increase of lightly stained nuclei. Another 12 h later,cells had reached late S phase and G2 phase, evidently showingan increased expression of the antigen (Fig. 5, lower lane).

3001




Table 2 Mean nuclear area and frequency of Ki-67 staining patterns in exponentially growing MCF7 cells

I.II.III.IV.PatterndescriptionMetaphase

chromosomesMultiple,

intensely stainedspotsDenselyspeckledClusteringgranulesIrregularspotsRound

nucleoli- likestructuresLight

nucleus, staining innucleoliAbsentLow

staining level, round-shapednucleoliModeratelypositive, round-shapednucleoliModeratelypositive, irregularshapeIntense

staining, round-shapednucleoliWhole

nucleus stained; large, strongly stainednucleoliRoundshapeIrregularshapeIrregular

and granulatedshapeVerydark nucleus with very dark spotsFig.\A,2A\B,2B1CIDIE,

\F,1C\GIH.2D17\L\J,

\K\M\N,

\O\P,2E\Q,

\R, 2FMNA-

0<m2)74.3

Â±14.0f(28)''93.5

Â±11.6(41)103.2Â±14.5(50)130.4Â±27.2(25)111.2Â±

14.0(50)113.4 Â±23.7(25)134.8Â±

18.2(25)202.1Â±18.2(25)149.0Â±

18.9(25)175.3

Â±24.7(25)191.9Â±38.9(25)186.4Â±27.7(50)203.2Â±40.6 (15)Frequency*1.70.94.98.112.215.319.42.014.514.24.61.80.4

Â°MNA, mean nuclear area.* Percentage of 250 positive nuclei.f Mean Â±SD.'' Numbers in parentheses, number of nuclei measured.

Treatment with TAM resulted in a decrease in the fractionof Ki-67-positive cells (Fig. 6A) and pronounced accumulationof weakly stained cells. As a result of replacement at 72 h with17/3-estradiol-containing culture medium, Ki-67 positivity wasgradually restored to the original level, suggesting that, underthese conditions, the effect of the TAM-induced d-o block onKi-67 expression is completely reversible.

When MCF-7 cells were treated with IO"7 M MTX, the actual

fraction of Ki-67-positive cells remained constant (Fig. 65).However, as judged from values after correction for weaklystained nuclei, the total amount of antigen initially increased(obviously as a result of accumulation of cells in S and Gjphase), but then steadily decreased.

We also evaluated the effect of starvation on Ki-67 antigenexpression and BrdUrd incorporation of nearly confluent MCF-7 cultures. Five days after plating, fresh culture medium wasadded. Thereafter, slides were sampled daily and stained withKi-67 and IU-4. As shown in Fig. 6C, after 2 days both Ki-67positivity and BrdUrd labeling indices steadily decreased.

Comparison of Ki-67 Indices with Growth Fractions as Determined by BrdUrd Labeling. The slowly rising part of continuousBrdUrd labeling curves is determined by the increase of labeledpost-proliferative cells (34). Since for both cov362.cl4 andMCF-7 cultures this increase appeared nearly linear (Fig. 7'A),

GFs were estimated by linear extrapolation backwards to theend of G2 phase. For both cell lines, the mean duration of G2phase was set on 4 h, since 3 h after administration of BrdUrd,mitotic figures were unlabeled, whereas after 5 h, a majority ofmitotic cells was labeled (data not shown). Pulse labeling for20 min of parallel MCF-7 cultures at several time intervalsrevealed a decrease in the S-phase fraction from 40 to 30%,probably as a result of reaching confluency about 2 days afteradding BrdUrd.

We evaluated the effects of reducing the percentage of PCSand treatment with 10~6 M TAM on the shape of continuous

labeling curves; as shown in Fig. IB, particularly the lattertreatment reduced the GF considerably.

In Fig. 1C, Ki-67-positive fractions are depicted as observedduring a continuous BrdUrd-labeling experiment of exponentially growing MCF-7 cells. Since Ki-67 positivity started todecline after 30 h, we used slides fixed within 24 h of BrdUrdlabeling to determine the variability in Ki-67 positivity within

different continuous labeling experiments. The observation,that Ki-67 positivity of cultures grown for as long as 5 days inculture medium with 20 mM BrdUrd was not different fromthat of unlabeled control cultures, suggests that BrdUrd at thisconcentration did not affect cell growth significantly (data notshown). Results of all continuous labeling experiments havebeen summarized in Table 4. In all experimental conditions,Ki-67 positivity appeared higher than the GFs as determinedby the graphical method. This difference was only minor (3.5%)in the ovarian cancer cell line, but varied from 6 to 14.3% inthe exponentially growing MCF7 cells. However, in MCF7cultures growing under less optimal conditions, larger discrepancies were found. In contrast, a very high linear correlationwas observed between GFs and S-phase fractions (Fig. 8).

DISCUSSION

In order to provide a reliable estimate of tumor GFs basedupon immunohistochemical detection of proliferating cell-specific antigens in a single tissue sample, two requirements shouldbe fulfilled, (a) The antigen should be continuously presentduring the cell cycle of all cell types. (Â¿>)A transition to whatevertype of nonproliferating state from any part of the cell cycleshould be followed by a rapid disappearance of the antigen,whereas recruitment of quiescent cells into the cell cycle shouldinduce a comparable fast reexpression.

This may be particularly important if these Moabs are appliedto tumors of which the proliferation patterns may stronglydepend on fluctuating hormone levels.

Since the commercially available Ki-67 Moab is increasinglyused as a GF indicator in various tissue types including breastcarcinoma (22-26), we have addressed in this study both morphological and kinetic aspects of Ki-67 antigen expression inhormone-responsive human breast cancer cells.

Our results confirm recent findings of Verheijen et al. (27)that the Ki-67 antigen not only is a component of the peripheralchromosome scaffold, but also of the peripheral parts of pre-nucleolar bodies and nucleoli. In this respect, Ki-67 resemblesan antiserum recently selected from scleroderma patients byLuji et al. (35), who proposed the term PCN for this type ofantigen. The transition from mitosis to interphase is characterized by a densely speckled staining pattern (Figs. IB and 2B).

3002




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-- !" -w * '";.'.. -! %Â»' 'â€¢:- '

\ â€¢Â»â€¢*L>' *P V ' -

f* Â¿f*t" M * if^k v

' *" Â¿r<- A., ' S ^

' X, ' \r Ãrx/, y r/v/

' 'â€¢' ' iâ€¢-> \i *-

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B

Fig. 3. Transmission electron micrographs of Ki-67-stained MCF-7 cells.Nucleus showing various DAB spots dispersed over the nuclear matrix (A, arrows).Ki-67-negative and -positive cell with DAB staining at nucleolar boundaries (B,arrows). Counterstained with uranyl acetate and lead citrate; A, x. 8000; H. x.1400.

In postmitotic daughter nuclei, enlargement of the spots simultaneously with a decrease in their number is associated with anincrease in nuclear size, therefore suggesting a continuous process of antigen clustering during chromosomal decondensation(preferably around prenucleolar bodies and nucleolar organizing regions). Neither in MCF-7 nor in cov362.cl4 cell populations we could observe weakly stained early d nuclei, even ifGF was significantly reduced as a result of starvation or treatment with TAM, indicating that cells tend to progress throughthis postmitotic phase before entering the Go phase or becomingan end cell.

Ki-67-negative nuclei in exponentially growing MCF-7 cultures were relatively small, suggesting a GÃ¬DNA content.However, double staining with anti-BrdUrd revealed that acertain fraction of these cells could be assigned to early S phase(Table 3), which has a frequency of about 3% in exponentiallygrowing MCF-7 cells (33). It is possible therefore that all cellsare negative at the onset of DNA replication, but then quickly

B

Fig. 4. Patterns observed in MCF-7 cells in S phase, labeled with BrdUrd for20 min. BrdUrd staining only (A to C), and BrdUrd staining in combination withKi-67 staining. The BrdUrd patterns shown represent early S phase (A, D), mid-S (B, E), and late S phase (C, F), respectively. Ki-67-positive nucleoli are indicated(arrows), x. 500.

Table 3 Ki-67 staining intensity of nucleoli in S-subphases of BrdUrd-labeledexponentially growing MCF-7 cells

Ki-67 staining ofnucleoliNot

detectableWeakly stainedMedium, roundMedium, irregularStrongly, roundStrongly, irregularNuclear

BrdUrdpatterns"Early

S38

548000Mid-S0

181416

3517Late

S0

3518

1559

" See Fig. 4; 100 nuclei per BrdUrd pattern.

Jl Jl IIf Bja bâ€” ,

â€”loo, fii-ÃŒL P^ Ã'jJL. E^j H

17 hrs E2 29 hrs E2 45 hrs E2

IFrequency distributions Ki-67 staining patterns

Fig. 5. Partial synchronization of MCF-7 cells. Top, flow cytometric DNAhistograms of untreated control cells, cells treated during 72 h with 10 *M TAM,and cells recruited from the TAM-induced G0 state by treatment with IO"7 M

17/3-estradiol for 17, 29, and 45 h, respectively. Bottom, frequency distributionsof staining patterns in Ki-67-positive nuclei in the following categories (see alsoTable 2): 7, mitotic cells; //, nuclei in early G, phase; ///, antigen restricted tonucleolus, but not detectable in nuclear matrix; IV, large, intensely stained nucleoliin a stained nuclear matrix. At each time interval, 250 randomly chosen labelednuclei were evaluated (control, 500 nuclei). Comparing the subsequent changesin the proportions of specific categories, all differences appeared statisticallysignificant (P < 0.05), with the exception of differences between control and 72-h TAM in Categories I, II, and IV, and between 72-h TAM and 17-h 170-estradiolin Category IV. E2, 170-estradiol.

start to express the antigen. However, an alternative explanation is possible as well. It has been found that mouse fibroblastscomplete growth factor-dependent processes leading to commitment for proliferation during the first 3 to 4 h of GÃ¬phase(denoted G,pm phase) and subsequently may either enter astate of quiescence (G0) or a growth factor-independent pre-

3003




20 40 60 80 100 120 140 20 40 60 80 100 120

Hours after treatment

20 40 60 80 100 120

Fig. 6. Effects on the frequency of Ki-67-positive cells after treatment of exponentially growing MCF-7 cultures with 10"' M TAM (A), with IO'7 M MTX (B),and in unfed plateau-phase cultures (C). Depicted are the total fraction of positive nuclei stored (â€¢)and the total fraction corrected for very weakly stained nuclei (O).After 72 h in a series of TAM-treated cultures, tissue culture medium was replaced by medium containing 10~7M 17j3-estradiol (A, ). inset in B, flow cytometricDNA histogram after 58 h of MTX treatment. C, , fractions of BrdUrd-positive cells after a 20-min pulse labeling period. E2, 17/3-estradiol. Bars, SD.

0 4

O io 20 30 40 50 60 70 Â°_, 10 20 30 40 50

Fig. 7. A, continuous BrdUrd labeling curves of exponentially growing cov362.cl4 cells (V) and MCF-7 cells (â€¢).B, comparison of continuous labeling curves ofMCF-7 cells growing in log phase (â€¢),in 5% PCS (â€¢),and 10"' M TAM (T). C, Ki-67 positivity (â€¢)during continuous labeling (â€¢)of exponentially growing MCF-7cells. D in A and C, 20-min pulse labeling of MCF-7 cells at various time intervals ( ). , linear extrapolation of the slowly rising part of the BrdUrd curvesto the end of G2 phase; the intersect is used as an estimation of GF.

Table 4 Comparison of Ki-67 positivity with the GF as estimated fromcontinuous BrdUrd-labeling curves ofcov362.cl4 cells and MCF- 7 cells, growing

under various conditions

KF

Cell culture TotalMinus weakly

stained GFÂ° SF*

cov362.cl4, 10%PCS'MCF-7,10%PCS'MCF-7,

5%PCS'MCF-7,10%FCSrMCF-7,10"' M TAM*99.5

Â±0.9rf99.3

Â±0.397.7Â±0.885.0

Â±4.298.5Â±0.289.2Â±1.184.3Â±10.894.3

Â±3.1(3)'96.8

Â±1.0(2)91.5Â±2.0(10)7

1.0 Â±0.8(2)92.8Â±1.2(5)72.6Â±9.3(2)55.9Â±13.8(4)968584796260^2650454439323114

" Estimated by linear extrapolation of BrdUrd-labeled fractions at various time

intervals departing from 24 h (see Fig. 7).'' SF, S-phase fraction; obtained by BrdUrd labeling during 20 min and cor

rected for progression of cells into G2 phase.' Exponentially growing cell culture.'' Mean Â±SD of percentages obtained at various time intervals within 24 h of

starting continuous BrdUrd labeling.' Numbers in parentheses, number of cultures evaluated.f Confluent culture (see Fig. 1A). GF in this culture was estimated from the

linear correlation between SF and GF (see Fig. 8).* Tamoxifen was added 48 h before BrdUrd labeling.

DNA-synthetic part of d (Gips), the duration of which showsa large intercellular variability (36). If Ki-67 antigen expressiondiminishes with a similar rate from cells which have entered Goas from cells which have entered a dps phase, then the amountof nucleolar staining observed in early S phase may be determined by variation in the duration of the latter phase. In cancertypes depending on exogenous hormones or growth factors asmitotic stimuli, it may then be impossible to discriminatebetween slowly cycling cells and cells arrested in Go phase.

The level of antigen expression increases from an undetect-

100

80

40

0) 20 r = 0.9 9

V = 0.51X + 0.46

20 40 60 80

Growth fraction

100

Fig. 8. Correlation between growth fractions (obtained by continuous BrdUrdlabeling) and both Ki-67-positive fractions (â€¢)and S-phase fractions (O) in MCF-7 cells growing under various conditions (see also Table 4). Correlation coefficients are 0.65 and 0.99, respectively.

able or low nucleolar staining in early S phase to intensestaining of both nucleoli and nuclear matrix in late S phase andG2 phase (Table 3; Fig. 5). There is ample evidence suggestingthat a number of constituents of the nucleolus may be involvedin the process of condensation and decondensation of chromosomes (37, 41). It has been suggested recently that theantigen for Ki-67 might be type II DNA topoisomerase (27),an enzyme which is also a structural part of the chromosomescaffold (42), but although its presence in interphase nuclei hasbeen confirmed, a nucleolar association has not been described(43). In contrast, the ribosomal Si protein is both associatedwith the interphase nucleolus and distributed over chromo-

3004




somal surfaces during mitosis (44). Moreover, in mitotic cells,distinct Si-containing granules have been described, which werescattered over the cell plasma and therefore resembling thegranules we observed in cov36.2.cl4 cells (Fig. 2, A and B,arrows).

However, within the scope of the potential application ofPCN antigens in cell cycle kinetics, speculation about theirnature seems relevant only if it gives us a better insight into theregulation of their expression in different cell types. Since cellsmay be arrested in all stages of the cell cycle (45), it is seemsparticularly important to study conditions and patterns of antigen reduction at various positions of the cycle.

In MCF-7 cultures, a small fraction of the nuclei, which hada size and nucleolar shape characteristic of G2 phase, appearedonly moderately positive for Ki-67, therefore possibly representing G2-arrested cells.

Treatment of cultures with MTX prevented an increase incell number (data not shown) and resulted in an accumulationof cells with an S-G2 DNA content (see Fig. 6Ã„,inset). Althoughno significant change in the fraction of Ki-67-positive cells wasobserved up to 5 days of MTX treatment, after 2 days the totalamount of antigen steadily decreased, as judged from an increase in the number of weakly stained nuclei (Fig. 6B). Furtherstudies are needed to establish whether a decrease in the levelof antigen expression in late S or G2 phase signifies an inevitableprogression to cellular death or whether these cells are still ableto restore antigen levels in order to enter prophase.

Treatment with the antiestrogenic compound TAM, used toblock a majority of MCF-7 cells specifically in GÃ¬phase (probably by preventing a hormonally induced commitment for proliferation in Gipm), resulted after 3 days in a dramatic reductionin the fraction of cells with an S and G2 DNA content (asdemonstrated by flow cytometric DNA analysis; Fig. 5). However, only a modest reduction in Ki-67 positivity was observedduring this period (Fig. 6/1). When cells were recruited fromthe TAM-induced Go state by replacing the tissue culture medium by fresh medium containing 17/3-estradiol, within 30 to50 h the frequency distribution of Ki-67 positivity was completely restored. These data suggest that, in cells temporallyblocked in G, phase, the Ki-67 antigen expression only slowlydecreases.

This discrepancy between the actual proliferation rate andthe fraction of KF was further substantiated by BrdUrd labelingexperiments. As stressed by Steel (34), in growing cell populations continuous labeling curves do not plateau at a levelindicating GF, since if nonproliferating cells are continuouslyproduced, they will originate also from the labeled compartment. Therefore, GFs were estimated by extrapolating the moreslowly rising component of continuous labeling curves backwards to the end of G2 phase.

The values thus found showed a highly significant linearcorrelation with S-phase fractions measured at the beginningof the labeling experiments (Fig. 8). However, no such correlation was found between GFs and KFs. Furthermore, KF wasinvariably higher than GF, a difference being particularly largein cell populations growing under suboptimal conditions.

These results indicate that, in MCF-7 human breast cancercells, it is not possible to unambiguously determine GFs byevaluation of nuclear Ki-67 staining patterns, since the rate ofdecrease of antigen expression in cells, which have entered aquiescent state, is slow compared to the proliferation rate. Onthe other hand, since antigen expression seems to be down-regulated during d phase, tumor cells with relatively long dpsdurations may have an undetectably low expression of the

antigen and therefore can easily be judged as noncycling. Further experiments are needed to evaluate whether or to whatextent these phenomena are a general problem with respect toother cell types and/or other PCN antigens.

Sasaki et al. (46) have recently derived a (linear) relationshipbetween KFs and BrdUrd labeling indices obtained from varioustumor types pulse labeled in vitro. Although their statementthat, in human solid tumors, GFs can be estimated from S-phase fractions (and vice versa) may be true to a certain degree,their a priori assumption that Ki-67 staining accurately reflectsGFs (and therefore S-phase fractions) does not seem to besubstantiated by our results. As shown particularly in perturbedcell populations, the discrepancy between KF and actual GFmay be quite large.

One should bear in mind though that, especially in neoplasmslike breast cancer, accurate data on GFs may have only limitedmeaning, as these tumors may consist of relatively small prolif-erative pools and display a broad range of intermitotic timesamong tumor cells (47).

Immunohistochemical detection of PCN antigens may thenbe important in characterizing the proliferative activity of primary tumors and lymph node mÃ©tastaseswithin a histolÃ³gica!context. Particularly if combined with immunohistochemicalstaining of other proliferation markers, e.g., enzymes involvedin DNA replication (48), this may provide a useful point-in-time estimate of the fraction of tumor cells assumed to be mostsensitive to proliferation-dependent chemo- and radiotherapy.

ACKNOWLEDGMENTS

The authors thank Dr. P. I. Schrier, Department of Clinical Oncology, and Dr. F. Dolbeare, Lawrence Livermore National Laboratory,Livermore, CA, for providing the ovary cancer cell line and the anti-BrdUrd monoclonal antibodies, respectively. Frans Prins, Joke Beu-kers, and Vincent Smit, Department of Pathology, are gratefully acknowledged for their technical assistance or helpful advice and Dr. A.F. Hermens, Radiobiological Institute REPGO TNO, Rijswijk, forcritically reading the manuscript.

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1989;49:2999-3006. Cancer Res Jan Hein van Dierendonck, Rob Keijzer, Cornelis J. H. van de Velde, et al. CellsComparison with Growth Fraction in Human Breast Cancer Nuclear Distribution of the Ki-67 Antigen during the Cell Cycle:

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