80 Clin Pathol 1992;45:860-865

Papers

Monoclonal antibody JC 1: New reagent forstudying cell proliferation

M C Garrido, J L Cordell, M H G Becker, G Key, J Gerdes, M Jones, K C Gatter,DY Mason

AbstractAim: To characterise a newly developedmouse monoclonal antibody JC1 whichrecognises a nuclear antigen present inproliferating cells in normal tissues andneoplastic lesions, and which is absent inresting cells.Methods: The methodology was estab-lished using a representative range offrozen sections from normal tissues andfrom certain tumours which wereimmunostained with antibodies Ki67 andJC1. The molecular weight of the antigenrecognised by JC1 was obtained by west-ern blot analysis and this was comparedwith that of Ki67. IM-9 cell lysates con-taining Ki67 derived plasmids were alsotested with JC1 antibody.Results: Biochemical investigation indi-cated that the antigen recognised by JClgives two molecular weight bands of 212and 123 kilodaltons, which is distinct fromthe well characterised anti-proliferationmonoclonal antibody Ki67 (395-345 kilo-daltons). In addition recombinant Ki67protein is not recognised by JC1.Immunohistological reactivity was seen inareas known to contain numerous pro-liferating cells such as lymphoid germinalcentres, splenic white matter, corticalthymocytes and undifferentiated sperma-togonia. In tumours many cells from ad-enocarcinomas, oat cell carcinomas,squamous cell carcinomas of lung, andseminomas were labelled by JC1 with adistribution and proportion similar tothat seen with Ki67. In normal tissues theonly apparent difference was in testiswhere JC1 stained a considerably greaternumber of cells than Ki67. In all casesstudied the new antibody showed nuclearreactivity only. JC1 did not show anycytoplasmic crossreactivity with squa-mous cells as is frequently seen withKi67.Conclusion: Antibody JCI, which recog-nises a nuclear antigen present in pro-liferating cells, should provide a usefuladjunct to Ki67 as a marker of prolifera-tion especially in those cases such assquamous cell carcinomas where a Ki67index cannot be determined.

( Clin Pathol 1992;45:860-865)

The rate at which a tumour proliferates has

long been considered to indicate its clinicalcourse.1̀ Histopathologists have thereforesought means of determining this as an adjunctto diagnosis. The development of monoclonalantibodies5- 1 recognising antigens present incells during proliferation has brought thispossibility within the realm of a general diag-nostic laboratory.The monoclonal antibody Ki67 is perhaps

the best known of such reagents and has beenwidely used to detect proliferating cells.'0There are now several studies which indicate acorrelation between tumour proliferation rateas assessed by Ki67 immunoreactivity andclinical outcome. 11-14 However, Ki67 has awell reported crossreaction with some epithe-lial cells, particularly of squamous type. Thiscan lead to strong cytoplasmic reactivity mak-ing the determination of proliferation relatedstaining impossible to define. This has led tothe elimination of cases from experimentalstudy, and, more importantly, will not allowsome individual cases to be assessed in diag-nostic practice. `-16Here we report the production of a new

mouse monoclonal antibody (designated JC 1)that is similar to Ki67 in that it seems torecognise selectively nuclei in proliferatingcells. However, it differs from the latter anti-body in showing little or no cytoplasmiclabelling in squamous or other cell types. Inaddition, it does not react with the sameantigen as Ki67, thus providing an independ-ent assessment of tumour proliferation whichmight be valuable in clinical practice whenindividual cases are being evaluated.

MethodsA representative range of normal tissues wasavailable from the frozen tissue bank stored at- 70°C in the Nuffield Department of Pathol-ogy, John Radcliffe Hospital. Samples of lungcancer (10 squamous cell, seven adenocarcin-omas, and three oat cell carcinomas) andseminoma (10 cases) were obtained from thesame source.Immunostaining with both monoclonal anti-

bodies Ki67 and JC1 was performed using athree stage immunoperoxidase technique asother studies had shown that, unlike Ki67, JC 1did not perform satisfactorily with the APAAPimmunoalkaline phosphatase method. Thepercentage of tumour cells positive for Ki67and JC 1 was established by counting 500tumour cells from separate sections in eachcase.

Nuffield Departmentof Pathology, JohnRadcliffe Hospital,OxfordM C GarridoJ L CordellM JonesK C GatterDY MasonForschungsinstitutBorstel, MolekulareImmunologie, Borstel,GermnyM H G BeckerG KeyJ GerdesCorrespondence to:Dr K C GatterAccepted for publication15 April 1992

860

Use of monoclonal antibody XCI for studying cell proliferation

PREPARATION OF RECOMBINANT PARTS OF THE K167ANTIGEN

Construction ofplasmids: Nucleotides 1-1002 ofthe Ki67 cDNA'7 were cloned between the EcoRl and Pst 1 sites of the pAX4a + vector'8(Medac, Hamburg, Germany). The resultingplasmid was designated pAX X2/1 and wastransformed into E coli JM 109.'9 A 100 basepair fragment, including a 62 base pair repeti-tive element'7 was ligated between the Eco Rland Bam Hl sites of the pEV-vrfvectors20 in allreading frames. The resulting plasmids weredesignated pEV-vrf 11B3, pEV-vrf 21B3, pEV-vrf 31B3 and transformed into E coli RR.1

Preparation of bacterial cell lysates: An ovemightculture of E coli JM109 containing pAX X211was diluted to an OD550 of 0 3, grown to an

OD550 of 1.0, and induced with 1 mM iso-propylthio-fi-galactoside (IPTG), according toMarkmeyer et al.'8 After an additional incuba-tion for two and a half hours at 30°C, bacteriawere harvested by centrifugation, washed oncein phosphate buffered saline (PBS), resus-pended in 1 in 20 volume ofPBS, and stored at- 20°C until further use. E coli RR1 harbour-ing pEV-vrf 1-31B3 plasmids were cultured at37°C in M9 medium22 supplemented with40 pg/ml ampicillin, 0 5% glucose, and casa-mino acids, respectively, until they reached an

OD550 of 0-8. Bacteria were then harvestedand lysates were prepared for sodium dodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and western blots, as describedbelow.IM-9 (multiple myeloma cell line, ATCC

No CCL 159) was cultured under standardconditions in RPMI 1640 medium (Gibco,Berlin, Germany), supplemented with anti-biotics and 10% fetal calf serum.

PREPARATION OF IM-9 CELL LYSATES

IM-9 cell line cells (3 x 107) were harvestedby centrifugation (10 minutes at 400 x g) andsuspended in 250 ,ul PBS containing 1 mMphenylmethylsulfonyl-fluoride (PMSF). Thecell suspension was snap frozen with liquidnitrogen and homogenised in a mortar cooledin liquid nitrogen. The resulting powder wassuspended in 300 pl double concentratedLaemmli-SDS-PAGE sample buffer23 contain-ing 100 mM DTT, boiled for five minutes in awater bath, and briefly sonified to destroycontaminating DNA. Samples were then sub-jected to SDS-PAGE.

SDS-PAGE

SDS-PAGE was performed according to themethod of Laemmli.23 IM-9 cell lysates wererun on 5% separation gels, with 3% stackinggels. Electrophoresis of the two fusion proteinswas performed on 7-5 and 12 5% separationgels, respectively. Molecular weight markerswere obtained from Pharmacia/LKB (Frei-

burg, Germany) and Sigma (Munich, Ger-many).

IMMUNOBLOTTING

Proteins were transferred to nitrocellulosemembranes (BA 85, Schleicher & Schuell,

Dassel, Germany) using the western blottechnique.24 IM-9 cell lysates were blottedovernight at 4°C and 50 mA in a BIO-RADblotting device (BIO-RAD, Munich, Ger-many); the fusion proteins were transferred ina Biometra (Gottingen, Germany) semi-dryblotting apparatus (two hours at 150 mA).Unoccupied protein binding sites of the nitro-cellulose were blocked by incubation for onehour with 1% bovine serum albumin or gela-tine in TRIS-buffered saline (TBS). After onehour of incubation with primary antibody andthorough washing with TBS, the bound anti-body was targeted with goat anti-mouse alka-line phosphatase conjugate, diluted 1 in10 000 in blocking solution. The phosphataseactivity was visualised using nitro blue tetra-zolium (NBT) and 5-bromo-4-chloro-3-indo-lyl phosphate (BCIP) as substrates."

ResultsCHARACTERISATION OF THE JC 1 ANTIBODY ANDANTIGENBiochemical characterisation of antibody JClWestern blot analysis of IM-9 cell lysatesshowed that there were two bands with molec-ular weights of 212 and 123 kilodaltons (fig1A). These are quite clearly different from thebands seen with Ki67 on the same cell lysatepreparation (fig IA). Additional evidence thatJC1 antigen is distinct from Ki67 antigen isshown by western blotting with both anti-bodies of protein preparations prepared fromthe transfection of Ki67 c DNA plasmids intoE coli. In no case were any of the resultantfusion proteins recognised by JC 1 (figs lB andC).

Normal human tissuesIn human tonsils (figs 2A and B) antibody JC 1stained the nuclei of nearly all centroblasts inthe dark zone, a varying number of centrocytesin the light zone of germinal centres, and thenuclei of only a small number of follicularmantle lymphocytes. This pattern of stainingwas virtually identical with that seen in parallelsections labelled by antibody Ki67. In theoverlying squamous epithelium of the tonsil(figs 3A and B) there was a distinct differencein the staining pattern seen with JC 1 and Ki67.With Ki67 there is a strong cytoplasmicreactivity with the epithelial cells especially ofthe basal and intermediate layers. This makes itdifficult to appreciate the nuclear labelling.However, Ki67, in general, seems to labelnuclei preferentially in the basal layer. JC 1 hasno cytoplasmic crossreactivity so that nuclearstaining is clearly seen as being localisedalmost exclusively in the intermediate layer,with only an occasional positive nucleus beingobserved in the basal layer.

In two other lymphoid organs, thymus (figs4A and B) and spleen, both antibodies gave asimilar staining pattern in the areas known tocontain the most proliferating cells-that is,the cortex of the thymus and the white matterin the spleen.

In specimens of five different testes, a sharpdifference was observed between Ki67 and

861

Garrido, Cordell, Becker, Key, Gerdes, Jones, Gatter, Mason

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JC 1. Ki67 generally stains only a small numberof undifferentiated spermatogonia located inthe basal layer of the seminiferous tubule (fig5A). JC 1 labels a much larger number ofundifferentiated spermatogonia extendingtowards the lumen with additional staining ofsome spermatocytes SI and spermatides S3(fig 5B). Neither of the two antibodies stainedmature spermatozoa or Leydig cells.

In other normal tissue, comprising liver,kidney, pancreas, thyroid, prostate, lung andbrain, only an occasional nucleus was labelledby either antibody and no difference was seenin their distribution or number. In proliferating

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normal tissues like gut or bone marrow, nodifference in quantity or quality of stainingbetween the two antibodies was noted. Table 1summarises the results.

Malignant lung and testicular tumoursThe number of labelled nuclei per 500 cellscounted for each tumour type is shown in table2. There was no significant difference betweenthe two antibodies in the adenocarcinomas andoat cell carcinomas of the lung (figs 7A and B).In five of the cases of squamous cell carcino-mas of the lung there was also no difference instaining between Ki67 and JC 1. However, in

862

Use of monoclonal antibody JCI for studying cell proliferation

Figures 2A, B Immunostaining of human tonsil with both Ki67 (A) and JCI (B), respectively. Lymphoid folliculesshow virtually identical pattern of staining by both antibodies in parallel sections, with strong staining of the germinalcentres and a few positive cells from the mantle zone. (Three stage immnunoperoxidase technique).Figures 3A, B Reactivity of the overlying squamous epithelium of tonsil: Note the difference in the staining patternbetwveen Ki67 (A) and J'CJ (B). Ki67 showing a strong cytoplasmic cross reactivity of the epithelial cells from the basaland intermediate layers making the nuclear labelling difficult to appreciate. Occasional nuclei from the basal layer arestained by Ki67. In contrast, JCI shows no cytoplasmic cross-reactivity but clear nuclear staining is localised in theintermediate layer with only an occasional nucleus being observed in the basal layer. (Three stage immunoperoxidasetechnique).Figures 4A, B Immunostaining of the human thymus: both antibodies, Ki67 (A) and JCI (B) show strong positivelabelling of cortical thymocytes. (Three stage immunoperoxidase technique).Figures 5A, B Immunostaining of human testis: Ki67 (A) shows labelling of a small number of undifferentiatedspermatogonia from the basal layer of the seminiferous tubule contrasting with JCl (B) which shows labelling of a largernumber of undifferentiated spermatogonia extending towards the lumen of the seminferous tubule. (Three stageimmunoperoxidase technique).Figures 6A, B Reactivity of squamous cell carcinoma of the lung: Ki67 shows pronounced background staining (A)which obscures the nuclear labelling. A parallel section from the same case stained with JC1 (B) shows reactivityconfined to the nuclei. (Three stage immunoperoxidase technique).Figures 7A, B Immunostaining of a typical case of adenocarcinoma of the lung: both antibodies show almost identicalpatterns of staining with Ki67 (A) and JCI (B).

the remaining five cases it was only possible toachieve a count of nuclei with JC 1 because thelevel of background staining with Ki67 was sohigh. These cases had been selected fromprevious studies of Ki67 labelling in lungcancer where they had been eliminated frominclusion due to this effect (figs 6A and B).

In view of the difference noted betwen Ki67and JC 1 on normal testis 10 cases of seminomawere selected for study. There was a consistentdifference, with a higher number of cells being

labelled by JC1 (table 2). However, this dif-ference was not significant (p > 0-134) andthere was no evidence of the striking differencein distribution of the staining by the twoantibodies as was seen in normal testis.

DiscussionSeveral monoclonal antibodies that identify avariety of cell cycle related antigens haverecently been described as markers of pro-

863

Garrido, Cordell, Becker, Key, Gerdes, j3ones, Gatter, Mason

Table 1 Staining characteristics of Ki67 compared with JCI in sections from normalhuman tissues

Ki67

Squamous epithelium overlying tonsilsBasal layerIntermediateGranular layer

Tonsils:Germinal centres

Testis:Undifferentiated spermatogoniaDifferentiated

Skin:Basal layerIntermediateGranular layer

KidneyLiverBrainLungThyroidPancreasProstateBone marrow and gut

+++ +*

++

+++ +*

JCI

+ I-

++

+ +

+l-

++

All proliferative compartments

Key: + + + > 80% of cells show nuclear staining.+ + + 50-80% of cells show nuclear staining.+ + 10-50% of cells show nuclear staining.+ < 10% of cells show nuclear staining.+ / - Only occasional cells show nuclear staining.- Negative staining.*Cytoplasmic staining also seen.

liferation-Ki67,26 anti-PGNA.927 Of these,Ki67 has been well characterised as recognis-ing polypeptide chains with molecular weightsof 395 and 345 kilodaltons present in thenucleus of cells in all stages of the cell cycleexcept G0..7 28We have produced a new mousemonoclonal antibody JC 1 which shows nuclearreactivity in cells of normal tissues andtumours, with a general distribution very

Table 2 Staining characteristics of malignant tumourswith Ki67 and XCICase No Ki67 (%) JC1 (%)

Adenocarcinoma of the lung:1 12 62 26 273 8 94 31 365 20 246 31 547 No stain 10

(p > 0771)

Oat cell carcinoma of lung:8 35 409 35 3010 46 36

Squamous cell carcinoma of lung:11 32 3412 34 2713 36 2314 52 3815 41 4116 * 2817 * 2318 * 2319 -* 1720 * 32(p > 0-228)

Seminomas:21 34 6222 25 2123 30 7224 13 1825 22 1526 34 4127 16 2328 31 5629 42 5330 48 49(p > 0-134)

*Due to high background cytoplasmic staining, a nuclear countfor Ki67 was not possible in cases 16-20.

similar to that seen with Ki67,26 for example intonsil and thymus, bone marrow and gut.However, we know from the molecular weightof the JC 1 antigen that it differs from Ki67.This is reflected in two important observationsmade in the current study. The first is that JC 1does not cross-react with a cytoplasmic antigenin squamous epithelium. The second is that thedistribution of nuclear staining is not identicalwith Ki67 in all normal tissues such as testisand skin.The lack of cytoplasmic staining with JC 1 on

squamous epithelium may be of considerablevalue in studies of squamous tumours. Severalstudies have reported this effect with Ki67which has led to the elimination of some casesfrom evaluation; examples include cervicalcancers'6 29 and lung carcinomas.'5 Further-more, this effect has also been noted in breastcarcinoma30 and haematological diseases.3'Although these were not specifically addressedin the present preliminary survey with JC 1,there is no reason to suppose that it wouldoccur with this antibody. In this study wespecifically selected a number of squamous cellcarcinomas of the lung that we had previouslybeen forced to eliminate from our investiga-tions with Ki67. In each case JC1 gave a clearnuclear staining without any cross-reaction.Because JC1 correlated well with Ki67 in theother squamous cell carcinomas and in theadenocarcinomas and oat cell carcinomas, it isclear that a combination of these two anti-bodies should be valuable in future studies ofproliferation in these tumour types. Further-more, if it is shown that a measurement oftumour growth fraction is useful in determin-ing some aspect of tumour behaviour, such asprognosis or drug sensitivity, it will be impor-tant to have an antibody such as JC1 for studyof those cases which cannot be evaluated withKi67 alone.The second major difference from Ki67

concerned mainly the testis. JC 1 stained alarger and different number of undifferentiatedspermatogonia than Ki67. The reason for thisis unknown but must obviously reflect adistinction in the localisation of the two anti-gens in testis which is not detectable in mostother tissues. Interestingly, this did not seem toaffect the measurement of proliferating cells inthe series of seminomas, a malignancy directlyarising from seminiferous tubules. However, itemphasises that caution must always beobserved in comparing results from antibodiesrecognising different antigens and that futurestudies would do well to incorporate bothantibodies or at least another independentmeasure of proliferation.

This work was supported by the Leukaemia Research Fund.

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