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NO38expressionandnucleolarcountsarecorrelatedwithcellularDNAcontentbutnotwithproliferationparametersincolorectalcarcinomas

ARTICLEinMOLECULARPATHOLOGY·SEPTEMBER1997

DOI:10.1136/mp.50.4.201·Source:PubMed

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PaulaM.DeAngelis

OsloUniversityHospital

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TrondStokke

OsloUniversityHospital

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olepetterfraasClausen

OsloUniversityHospital

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J Clin Pathol: Mol Pathol 1997;50:201-208

N038 expression and nucleolar counts arecorrelated with cellular DNA content but not withproliferation parameters in colorectal carcinomas

Paula M De Angelis, Trond Stokke, Ole Petter F Clausen

AbstractAims-To investigate the expression ofnucleolar protein N038, to determine thenumbers ofnucleoli per cell, and to exam-ine the relations of these nucleolar para-meters to tumour DNA index, totalcellular DNA content, S phase fraction,and Ki67 labelling index.Methods-36 colorectal tumours and 14normal mucosas were studied. An anti-N038 monoclonal antibody, 31A12, andflow cytometric analysis were used todetect expression of N038 by means of abiotin-streptavidin-FITC (fluoresceinisothiocyanate) staining method. Nucleo-lar counts were determined using fluores-cence microscopy. Flow cytometry wasused to determine tumour DNA indicesand the sizes ofthe S phase fractions. Ki67labelling indices were determined fromtissue sections stained immunohisto-chemically with the MIB-1 antibodyagainst the Ki67 nuclear protein.Results-Generally, tumour cell nucleoliwere larger and more irregular in shapecompared with nucleoli in normal mu-cosal cells. DNA aneuploid and diploidtumours expressed 2.8 and 2.1 times moreN038 than normal mucosa. The mean(SD) values for nucleolar counts werehigher for the DNA aneuploid tumours(3.81 (0.93)) than the diploid tumours(2.62 (0.38)) and normal mucosa (2.34(0.37)). N038 expression and numbers ofnucleoli correlated significantly (r = 0.52,p = 0.01). There were, however, no signifi-cant correlations between these nucleolarparameters and either the sizes oftumourS phase fractions or Ki67 labelling indices.Cell cycle resolved expression of N038 intumours and normal mucosa demon-strated that expression increased approxi-mately in proportion to the DNA contentthroughout the cell cycle. In aneuploidtumours, N038 expression was 43% and98% higher in S and G, phases, respec-tively, compared with the G1 phase. Sort-ing of these populations revealed that thenucleolar count also increased as the DNAcontent increased but by only 29% and47% in S and G2, respectively. Apoptoticcells lacked N038.Conclusions-N038 expression is higherin tumours than in normal mucosa owingto the increased DNA content and largernucleoli in tumours; expression increases

proportionally with DNA content as cellsprogress through the cell cycle from G1through S and G,. However, N038 expres-sion does not correlate with the tumour Sphase fraction or Ki67 labelling index andis lost during apoptosis. Also the resultssuggest that nucleoli grow in size duringthe cell cycle, which would account for thedoubling of N038 expression from GI toG,, as the nucleolar count increased byonly 47%.(7 Clin Pathol:Mol Pathol 1997;50:201-208)

Keywords: N038 expression; nucleolar counts; DNAindex; cellular DNA content; S phase fraction; Ki67labelling index; colorectal tumours

N038, also known as B23, nucleophosmin, ornumatrin is an abundant nucleolar phospho-protein of 38 kDa molecular weight, which islocalised to the granular regions of thenucleolus,l where maturation, processing, andstorage of preribosomal particles have beenshown to take place.2 3 Recent studies haveshown N038 to have DNA binding activity4 5

and to bind to another nucleolar protein,namely p120.6 N038 has been shown also tobe a nuclear substrate of protein kinase C,7mitotic cdc2 kinase,8 and casein kinase II.9 Thefunctional significance of these activities ofN038 is unknown, although it is clear thatN038 plays some role in cellular proliferativeactivity. This is suggested also by another studydemonstrating that N038 expression increasesrapidly during mitogenic stimulation.'° Inaddition, N038 may play a role in theapoptotic pathway, as suggested by a recentstudy" which demonstrated reduced N038expression in apoptotic Jurkat T lymphoblasts.N038 is one of several proteins known to be

associated with nucleolar organiser regions(NORs) and to be stained by the argyrophilicnucleolar organiser region (AgNOR)technique'2 13; only NORs that are transcrip-tionally active are assumed to be identified bythis technique.'4 Quantitative measurement ofthe numbers of NORs in a cell is one methodused currently to assess cellular proliferativeactivity in human tumours. If large numbers ofNORs within malignant cells reflect increasedDNA synthesis (relative to benign tissue) thenit may be reasonable to expect that AgNORcounts could be correlated with other cell pro-liferative markers such as the S phase fraction,BrdU labelling index, Ki67 labelling index, andothers. Tumour ploidy status and S phase frac-tion can be determined from DNA content

Institute for Pathology,The NorwegianNational Hospital,Oslo, NorwayP M De AngelisO P F Clausen

Department ofBiophysics, Institutefor Cancer Research,The NorwegianRadium Hospital,Oslo, NorwayT Stokke

Correspondence to:Dr De Angelis, TheNorwegian NationalHospital, Institute forPathology/University of Oslo,Pilestredet 32, 0027 Oslo,Norway.

Accepted for publication27 May 1997

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cycle. Neither N038 expression nor the num-bers ofnucleoli correlated significantly with thetumour S phase fraction or Ki67 labellingindex.

Materials and methodsCLINICAL MATERIALThirty six surgically removed and previouslyuntreated colorectal carcinomas and 14 normalcolonic mucosal specimens were collected andstudied. All tumours were classified accordingto their Dukes' stage20: one was Dukes' A, 18were Dukes' B, 10 were Dukes' C, and sevenwere Dukes' D. Normal mucosal samples wereselected from macroscopically normal areas ofsurgical specimens. Samples of fresh tumourand normal mucosa were frozen without bufferat -80°C immediately following surgery.

Figure 1 A representative immunoblot of colorectaltumours stainedfor N038 and p83. A major band at38 kDa was seen for all tumours and a minor band directlybelow the major band in two tumours (94-4 and 94-9),representing different phosphorylation states ofN038. p83was used as a concentration and loading standard.

analysis of tumour specimens using flowcytometry. The DNA index is used to reportthe degree of abnormal DNA content in a

tumour. Both the DNA index and S phasefraction have been used as prognostic indica-tors in colorectal cancer; the DNA index hashad only modest impact in this regard.'5Generally, other parameters such as Dukes'stage and histological grade have been shownto be better prognostic factors. However, a highS phase fraction has been shown to correlatewith DNA aneuploidy'6 and, in another study,the S phase fraction proved to be a strongerpredictor of survival than DNA ploidy status.'7Ideally, these factors should be reported alongwith stage and grade. The nuclear proteinwhich is labelled by the Ki67 and MIB-1 anti-bodies is detected in proliferating but not qui-escent cells.'8 '9 Thus, nuclei reacting withthese antibodies are considered to representthe cellular growth fraction both in normal tis-sues and tumours.

In this study we examined the relationsbetween both N038 expression and thenumbers of nucleoli and the tumour DNAindex, total cellular DNA content, S phasefraction, and Ki67 labelling index in colorectaltumours and normal mucosa. N038 expres-

sion and nucleolar counts were higher in thetumours than in normal mucosa and highest inthe DNA aneuploid tumours. In cycling aneu-

ploid tumour cells, N038 expression doubledwhile nucleolar counts increased by only 47%as the cells progressed from G, to G2, implyingthat the nucleoli grow in size during the cell

DETECTION OF N038 PROTEIN BYIMMUNOBLOTTINGSlices of tumour and normal tissue (0.5-1.0 mm) were boiled for three to four minutesin sodium dodecyl sulphate (SDS) samplebuffer.2' The N038 protein and prestainedbroad range molecular weight standards (Bio-Rad, California, USA) were separated by elec-trophoresis in 10% SDS polyacrylamide gels.2'Immunoblotting was performed as describedpreviously," using the mouse antihumanN038 monoclonal antibody 31Al 2 (T Stokke,unpublished) and a biotin-streptavidin alkalinephosphatase staining procedure to detect31A12 (Amersham, Amersham, UK). p83 is aprotein distributed abundantly throughout thenucleoplasm of all human cells tested. Theamount of p83 per cell does not appear to varywith growth rate or tissue origin22 23 and wasdetermined on the same blots as a control forgel loading and cell concentration.

IMMUNOFLUORESCENCE STAINING OF N038 ANDDETERMINATION OF THE MEAN NUMBERS OFNUCLEOLI PER CELL BY FLUORESCENCEMICROSCOPYSingle nuclear suspensions were prepared fromtumour and normal mucosal material by minc-ing tissue slices in 10 mM phosphate buffercontaining 0.1% Triton X100, 5 mM MgCl2,10 mM NaCl, and 0.1 mM phenylmethyl-sulphonyl fluoride (PMSF). Each tumoursample contained a mixture of cells from atleast three separate areas of the tumourspecimen. The suspensions were placed on icefor 10 minutes, filtered, fixed for one hour onice by the addition of 1% paraformaldehyde,washed once with phosphate buffered saline(PBS), centrifuged, resuspended in PBS con-taining 0.1% bovine serum albumin (BSA),and aliquoted as 100 il suspensions (1-2 x 106cells). PBS containing 0.1% Triton X100 wasadded for five minutes on ice followed by addi-tion of dry milk (added to a final concentrationof 5%). The suspension was incubated for 15minutes at room temperature, after which theanti-NO38 monoclonal antibody 3lA12 wasadded at a 1/50 dilution of the stock superna-tant and incubated for one hour on ice. Controlsamples received no primary antibody. Sam-ples were then washed once with PBS/0.1%

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N038 expression and nucleolar counts in colorectal tumours

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Figure 2 Laser confocal micrographs of colorectal tumour nuclei stained with theanti-N038 antibody 31A12 and counterstained with propidium iodide for total DNA.(A) Phase contrast micrograph of the nuclei of interest; (B) propidium iodide fluorescenceof the same nuclei showing the distribution of total DNA in the nucleus; (C) FITCfluorescence of the same nuclei stainedfor N038.

Triton X100, followed by addition of thesecondary antibody (biotinylated horse anti-mouse IgG (Vector Labs, California, USA),made up as a 1/35 dilution in PBS plus 5% drymilk) for 20 minutes. The washing step wasrepeated, followed by addition of the tertiaryreagent, streptavidin-FITC (Amersham) madeup as a 1/35 dilution in PBS plus 5% dry milkfor 20 minutes. The samples were washed withPBS/0.1% Triton X100 and the pellets resus-pended in PBS with propidium iodide (PI)(5 gg/ml). Aliquots of these suspensions wereexamined using a Nikon Labophot fluores-cence microscope (Nikon Corp, Tokyo, Japan)at a 40x magnification, with an FITC emissionfilter setup. The nucleoli were counted in afield of 100 cells per tumour or normalmucosal sample and the mean values calcu-lated.

LOCALISATION OF N038 PROTEIN BY LASERCONFOCAL MICROSCOPYSeveral of the same tumour suspensions usedfor fluorescence microscopy were examinedalso by laser confocal microscopy (Zeiss,Oberkochen, Germany). FITC emission (51 0-550 nm, 488 nm excitation) was measured todetermine the localisation of the N038 proteinin the nucleolus, whereas total DNA was visu-alised with propidium iodide (PI) (emission> 620 nm, 514 nm excitation). Non-confocalinterference contrast images (488 nm) werealso generated for the same cells.

IMMUNOHISTOCHEMICAL DETECTION OF THE KI67PROTEIN AND DETERMINATION OF THE K167LABELLING INDEXFormalin fixed, paraffin wax embedded tissuesections (3 gn) were deparaffinised, dehy-drated in ethanol, and rinsed in distilled water.For antigen unmasking, the slides were micro-waved at 750 W three times for five minuteseach, in 10 mM citrate buffer (pH 6.0). Theslides were cooled for 30 minutes at room tem-perature, followed by washing in Tris buffer(pH 7.4) for five minutes. They were thenincubated with the MIB-1 mouse antihumanKi67 monoclonal antibody (1/50 dilution ofstock antibody which was a gift from Dr J Ger-des, Germany) overnight at 4°C. Positive andnegative controls (without the primary anti-body) were included in the staining protocol.The sections were then washed with Tris bufferand the bound antibody visualised using theconventional alkaline phosphatase antialkalinephosphatase (APAAP) method. Evaluation ofthe sections and scoring for positivity were car-ried out by one of us (OPFC). Tumour cellswith evident nuclear staining were scored aspositive and 300-500 cells were countedrandomly. Preliminary counts showed thatsome cells (< 10%) had relatively weak nuclearstaining, thus all positive cells were pooled andreported as one count. The percentage of posi-tive cells was calculated for each tumour andreported as the Ki67 labelling index.

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Figure 3 (A) Tumour 94-12 stained wzith the anti-NO38 antibody 31A12 (FL1) andIpropidium iodide (PI) for DNA (FL2). The regions R2, R3, R4, and R5 were generatedfor analysis and sorting purposes, and represent the diploid G,, aneuploid G,, aneuploid S,and aneuploid G- components in this aneuploid tumour, respectively; R6 represents cells thathave high PIfluorescence and were shown to be apoptotic. (B) The same tumour, stainedas under (A) but without the primary antibody 31A12 (fluorescence control). (C) Tumour94-22 stained as under (A) with 31A12 primary antibody; R2, R3, R4 represent thediploid G,, S, and G, components, respectively. (D) Fluorescence controlfor the sametumour.

FLOW CYTOMETRIC DETERMINATION OF TUMOUR

DNA INDICES AND PERCENTAGES OF CELLS IN THE

S AND S + G, PHASES OF THE CELL CYCLE

Fresh tumour and normal mucosal cell suspen-sions were stained with PI for DNA accordingto the method of Vindel0v.24 Each tumoursample was a mixture of cells cut from the sameareas of the tumour specimen that were usedfor immunoblotting and immunofluorescencestaining. The samples were analysed using a

Cytofluorograf 50 H laser flow cytometer(Ortho Diagnostic Systems, New Jersey, USA)tuned to an excitation wavelength of 488 nm;PI red fluorescence was collected on a linearscale using a 620 nm long pass filter. DNAcontent histograms (PI fluorescence distribu-tions) were used to calculate both the DNAindices and the percentages of cells in S andS + G, phases. The DNA index was calculatedas the ratio of the mean channel number of thetumour G(/G, peak to the mean channelnumber of the diploid G0,/G, peak (the latterinherent in all aneuploid tumour samples). Asample containing a stem cell line with abnor-mal DNA content was termed DNA aneu-

ploid. Tumours were characterised as DNAdiploid (DNA index 1.0) or DNA aneuploid(DNA index > 1.0) as described previously.25The percentages of S and S + G2 phase cellswere calculated as a fraction of the entire cellpopulation, including both cycling and non-

cycling normal and tumour cells, which was

taken to be 100%. This method is more

representative of the experimental situation(microscopic counting of nucleoli) as we were

unable to microscopically dissect out just thecycling aneuploid population. For the same

reason the S phase fraction of the diploidtumour cells cannot be assessed separatelyunder these experimental staining conditions.

FLOW CYTOMETRIC ANALYSIS AND SORTING OFN038 POSITIVE FRACTIONS BASED ONSIMULTANEOUS DETERMINATION OF N038 ANDDNA CONTENTA Cytofluorograf 50 H laser flow cytometer(Ortho Diagnostic Systems) tuned to anexcitation wavelength of 488 nm was used tomeasure total and cell cycle/ploidy specificN038 expression in nuclei using the sametumour and mucosal suspensions prepared forfluorescence microscopy. Logarithmically am-plified FITC fluorescence of N038 labellednucleoli was collected (and later converted totrue linear values) using a 515-530 nm band-pass filter, and PI fluorescence was collected asdescribed above. Fluorescence values forN038 expression are given as arbitrary fluores-cence units. A FACStar Plus laser flow cyto-meter (Becton-Dickinson, California, USA)tuned to an excitation wavelength of 488 nmwas used to sort various N038 positivefractions (for example, G1, S, and G, phasecells) in 10 tumours (aneuploid and diploid)and three normal mucosa based on bivariatedistributions of green fluorescence (log scaleFITC fluorescence of N038 stained nucleoli)versus red fluorescence (DNA content of PIstained nuclei). The emission filter setups wereessentially the same as for the OrthoCytofluorograf. The mean numbers of nucleoliper cell in each sorted fraction were calculatedbased on 100 cells sorted/examined.

STATISTICAL ANALYSIS

Unpaired non-parametric tests (Mann-Whitney, two tailed) were used to check forsignificant differences between two groups ofdata for a specific parameter. The Bonferronimultiple comparison post test for one wayanalysis of variance (ANOVA) was used if thesame parameter was compared in three ormore groups. Fisher's two-tailed 2 x 2 contin-gency tests were used to check for associationsbetween any two parameters, and correlationanalysis or linear regression analysis were usedto check for the degree of covariation betweentwo variables. All statistical testing was per-formed using Prism software (Graphpad Soft-ware, California, USA). p values < 0.05 wereconsidered to be significant.

ResultsSUBNUCLEOLAR LOCALISATION OF N038 ANDNUCLEOLAR PLEOMORPHISMFigure 1 shows a representative immunoblot ofcolonic tumours stained with the 31A12antibody. The protein recognised by thisantibody banded at 38 kDa (major band); aminor band could be observed directly under-neath the major band in two tumours (94-4and 94-9).Both conventional fluorescence microscopy

and laser confocal microscopy demonstratedthat the 3 lAI2 antibody stained nucleolispecifically (fig 2). Thus, data from immuno-blotting as well as from microscopy was

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N038 expression and nucleolar counts in colorectal tumours

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Figure 4 (A) N038 expression of the total ungated positive cell population (mean FITC fluorescence ofN038 labelled cells measured in arbitraryfluorescence units) is highest in DNA aneuploid colorectal tumours compared with diploid tumours and normal mucosa. (B) Numbers of nucleoli (meannumber of nucleoli per cell) are also highest in DNA aneuploid tumours relative to diploid tumours and normal mucosa. The horizontal lines represent themean value for each dataset.

consistent with the observation that 31A12recognises the nucleolar protein N038. Theminor bands seen in fig 1 probably representeddifferent phosphorylation states of the protein.N038 immunofluorescence appeared to belocalised to the perimeter of the nucleoli, suchthat the nucleolar centre appeared black,surrounded by a ring of positive staining. Thetumour nuclear suspensions were a mixture oftumour and normal mucosal nuclei, and thenumber of nucleoli seen/counted per nucleusranged from one to 16 for the entire tumourset. All specimens contained a certain numberof nuclei that did not demonstrate apparentnucleolar staining (non-fluorescent). In thetumours, there was a distinct heterogeneity innucleolar sizes, ranging from small to very

large, and in shape, with irregular patternsincluding round and elongated nucleoli. Thenucleoli seen/counted were distinct bodies andnot fluorescent "specks". In the normalmucosal specimens the numbers of nucleoliper nucleus ranged from one to six, and theyappeared generally to be smaller and more

rounded than those seen in the tumourspecimens-that is, more homogeneous withrespect to size and shape.

N038 EXPRESSION AND NUCLEOLAR COUNTSFlow cytometric analysis demonstrated that alltumours and normal mucosal specimens ex-

pressed N038, but at variable levels ofintensity (examples are shown in fig 3). N038expression was not restricted to one cell cyclecompartment but was expressed throughoutthe cycle. The diploid, presumably normal,cells in aneuploid tumours also expressed theprotein (fig 3A, region R2). A few aneuploidtumours contained a small population ofN038 negative cells with high PI staining (fig3A, region R6). These were sorted using flowcytometry and examined microscopically; mostof these cells had the morphological character-istics of apoptotic cells.

Total N038 expression for each sample was

obtained from the flow cytometric data as themean FITC intensity of the whole, ungatedpositive population; the mean (SD) values fortotal N038 expression were 142 (15) for theaneuploid tumours, 106 (37) for the diploidtumours, and 50 (6) for normal mucosa (fig

4A). Nucleolar counts were obtained fromfluorescence microscopy data and mean valuesfor this parameter were 3.81 (0.93) for theaneuploid tumours, 2.62 (0.38) for the diploidtumours, and 2.34 (0.37) for normal mucosa

(fig 4B). N038 expression correlated signifi-cantly with the numbers of nucleoli (r = 0.52,p = 0.01). N038 expression and nucleolarcounts were significantly higher in the tumoursthan in normal mucosa (p = 0.009 andp = 0.001, respectively), and highest in theDNA aneuploid tumour group compared withthe diploid tumour group (p < 0.05 andp < 0.001, respectively).No significant differences were observed for

either N038 expression or nucleolar countsbetween Dukes' stage C + D and Dukes'A + B tumours (p = 0.13 and p = 0.43, re-

spectively).

K167 LABELLING INDEX AND S PHASE FRACTIONThe mean (SD) Ki67 labelling indices were 70(18) for the aneuploid tumours, 72 (19) for thediploid tumours, and 38 (4) for the normalmucosal specimens. The mean percentages ofS phase cells were 12.3 (7.0) for the aneuploidtumours, 15.9 (4.2) for the diploid tumours,and 7.0 (2.8) for the normal mucosal speci-mens. There was no significant correlationbetween the tumour S phase fraction and thetumour Ki67 labelling index (r = 0.17,p = 0.40). In the tumour group, there were no

significant correlations between N038 expres-sion and either the size of the S phase fractionor the Ki67 labelling index (r = 0.17 andp = 0.46; r = 0.15 and p = 0.65, respectively);the same was true for nucleolar counts versus

these two parameters (r = 0.29 and p = 0.09;r = 0.10 and p = 0.62, respectively).

N038 EXPRESSION, NUCLEOLAR COUNTS, AND DNA

CONTENTIt is possible that the higher N038 expressionand nucleolar counts seen in the tumour groupcompared with normal mucosa could be a

result of the higher DNA indices in the tumourgroup. Therefore, N038 expression and thenumbers of nucleoli were compared in the dip-loid tumours and normal mucosa. N038expression was significantly higher in theformer than in the latter (p < 0.05), whereas

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the numbers of nucleoli were not. If N038expression and nucleolar counts in the aneu-ploid tumours were divided by the DNA indexand compared with normal mucosa, the sametrend was seen. This implies that tumour cellsexpress higher amounts ofN038 in relation tothe amount of DNA compared with normalmucosal cells, but that they contain the samenumbers of nucleoli relative to the amount ofDNA as do normal mucosal cells.

It was directly evident from the flowcytometric cytograms of N038 fluorescenceversus DNTA content that N038 expressionincreased as the DNA content increasedthroughout the cell cycle of the aneuploidtumour populations (fig 3A). In diploidtumours, diploid cells (fig 3A, R2 and fig 3C,R2) showed a more heterogeneous expressionof N038 than aneuploid cells and the cells inthe S and G2 phases of the cell cycle. Thismight be because in diploid tumours, diploidcells consist of leucocytes and normal mucosalcells in addition to tumour cells. Thus,aneuploid tumour cells represent the mosthomogeneous population, a fact that can bedistinguished clearly by DNA content. How-ever, S and G2 cells from diploid tumours arealso relatively pure because the admixture ofnon-tumour cells comprises mainly non-proliferating leucocytes and mucosal cells.Therefore, the cell cycle resolved expression ofN038 in a presumably homogeneous popula-tion oftumour cells was analysed in more detailin the aneuploid tumours. N038 (SD) expres-sion was 43% (29%) and 98% (48%) higher inthe S and G2 phases, respectively, relative to itsexpression in GI (fig 5A). In addition, theseaneuploid tumours were sorted using flowcytometry to determine the number of nucleoliin the S and G, phases relative to GI. Thenumbers (SD) of nucleoli were 29% (18%)higher in S than in G,, and the numbers ofnucleoli were 47% (23%) higher in G2 than inG, (fig 5B). Hence, on average, the nucleolimust grow in size to contain 35% more N038as the cells proceed from GI to G2,

DiscussionWe examined the relation between both N038expression and nucleolar counts and the DNA

index, total cellular DNA content, S phasefraction, and Ki67 labelling index in colorectalcarcinomas, using a monoclonal antibody thatspecifically labels N038. This is a nucleolarphosphoprotein associated with nucleolar or-

ganiser regions in cells. The immunofluores-cent staining ofN038 was localised specificallyto the perimeter of the nucleoli, which isconsistent with a previous report demonstrat-ing N038 localisation to the outer granularregions (granular and dense fibrillar compo-nents) of the nucleoli.' The pattern of stainingin tumours was clearly different from that seenin normal mucosa. In general, tumour nucleoliwere larger and more irregular in shape (round,oval, or elongated) compared with nucleoliseen in normal mucosa. Nucleolar pleomor-phism has been known for some years to becharacteristic of cancer cells.26 27 The tumourgroup also had significantly higher nucleolarcounts than the normal mucosa group. It hasbeen shown previously with AgNOR stainingthat, generally, highly malignant neoplasmshave smaller and more numerous AgNORsthan benign or less malignant tumours."8 29

Yang et al10 demonstrated a higher number ofAgNOR counts in adenocarcinomas than inadenomas with severe dysplasia. The reasonsfor these differences are still unclear. Althoughwe did not use the more conventional AgNORtechnique to label nucleolar proteins, our studyis consistent with the AgNOR studies as we

demonstrated increased numbers of nucleoli intumour cells compared to normal cells, al-though they were not smaller in the tumourgroup. The relation between N038 stainingandAgNOR counts is not quite resolved, and isa subject of great interest that deserves furtherattention.N038 expression was seen in all phases of

the cell cycle of tumour and normal mucosalcells, and morphologically apoptotic tumourcells lacked N038, as shown by cell sorting.Nucleolar counts correlated significantly withN038 expression, and both parameters weresignificantly higher in the tumours than normalmucosa, being highest in the DNA aneuploidtumours.No significant association was observed

between nucleolar counts and Dukes' stage, in

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N038 expression and nucleolar counts in colorectal tumours

agreement with the results of Kram et al.3' Inaddition, we did not observe significant corre-lations between either N038 expression ornucleolar counts and S or S + G, phasefractions in the tumour cell populations. Theseresults are consistent with previous reportsfailing to demonstrate significant correlationsbetween AgNOR counts and the percentage ofcells in the S + G, phases of the cell cycle in

31 32colonic adenocarcinoma, gastric carcinoma,and breast cancer.33 However, Hasegawa et al34reported a significant linear correlation be-tween the AgNOR count and percentage ofcells in the S + G, phase in testicular germ cellcarcinomas.

It was speculated that the observed differ-ences in N038 expression and nucleolarcounts between the tumours and normalmucosa could be a result of the higher averageDNA index in the tumour group. Both N038expression and the number of nucleoli increasewith DNA content, but only N038 expressionappears to be higher in tumours compared withnormal mucosa after normalising for DNAcontent. That is, per amount of DNA, bothtumour cells and normal mucosal cells containthe same numbers of nucleoli. Thus, the higherN038 expression seen in the tumours is mostlikely related to the larger sizes of the tumournucleoli.

It was clear from the N038 flow cytometricsorting analysis of aneuploid tumour cells thatwhile N038 expression increases by - 100%from GI to G(, the nucleolar count increases byonly 47%. Thus, the nucleoli must increase insize to account for the observed increase inN038 expression from G, to G,. N038expression increases by only 85% and 70%from G, to G2 in diploid tumours and normalmucosa, respectively. This is probably becauseof the admixture of mainly non-cycling leuco-cytes in the diploid component of these groups,which results in a heterogeneity of N038expression. Unlike the aneuploid tumours, itwas not possible to examine homogeneouspopulations of cycling cells in these twogroups. However, future studies with three-parameter N038, keratin, and DNA flowcytometry might be possible in order to distin-guish leucocytes from epithelial cells forfurther analysis of N038 expression andnucleolar frequency.

Neither N038 expression nor nucleolarcounts correlated with the sizes of the tumourS or S + G2 phase fractions, or with the Ki67labelling index in colorectal carcinomas. Fur-thermore, no significant correlation was foundbetween the tumour S phase fraction and theKi67 labelling index. Thus, even though N038expression and nucleolar counts are increasedin S phase and G, phase cells relative to GIphase cells, the sizes of the S phase (and S + G2phase) fractions are relatively small comparedto the sizes of the GI fractions. This mayexplain the observed lack of correlation be-tween these nucleolar parameters and the Sand S + G, phase fractions. Thus our resultsindicate that N038 expression does not corre-late with cell proliferation as measured by thesizes of the S phase fraction or by the Ki67

labelling index. There was also no correlationbetween the S phase fraction and the Ki67labelling index, indicating that these areindependent parameters, both of which mayreflect cell proliferation.N038 expression and nucleolar counts in-

crease with DNA content, whether this is causedby an aneuploid tumour stemline or passagethrough the cell cycle. The number of nucleoli isnot a marker of neoplasia per se, since theincreased nucleolar counts seen in tumours arethe result of their increased DNA index relativeto normal mucosa. N038 expression, however,is higher in colorectal tumours than in normalmucosa, and this remains true even when theDNA content is taken into consideration. This isconsistent with the observed larger sizes ofnucleoli in tumour cells compared with normalmucosal cells.

This work was supported by the Norwegian Cancer Society.The authors thank Erik Trondsen, of the surgical department,Akershus Hospital, Oslo, Norway, for providing colorectaltumour and mucosal specimens.

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doi: 10.1136/mp.50.4.201 1997 50: 201-208Mol Path

 P M De Angelis, T Stokke and O P Clausen parameters in colorectal carcinomas.content but not with proliferationare correlated with cellular DNA NO38 expression and nucleolar counts

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