Clonality of preneoplastic liver lesions: histological analysis in chimeric

rats

W. C. WEINBERG* and P. M. IANNACCONEt

Northwestern University Medical School, Department of Pathology and Northwestern University Cancer Center, 303 East Chicago Avenue,Chicago, IL 60611, USA

•Present address: Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Building 37, Room 3B 25,Bethesda, MD 20892, USAf Author for correspondence

Summary

The clonality of chemically induced alteredhepatocellular foci was examined in rat liver.Chimeric rats composed of two histologicallydistinguishable cell lineages were placed on aninitiation-promotion protocol for liver cancer in-duction. This resulted in multiple lesions ofaltered enzyme expression. These altered hepa-tocellular foci are generally considered to beinitiated sites susceptible to cancer formation.The cellular origins of these lesions were deter-mined by aligning sections demonstrating celllineage with serial sections stained for alteredenzyme expression. Analysis included 110 areasof deficient ATPase (EC 3.6.1.3) activity and 59glucose-6-phosphatase (EC 3.1.3.9; G-6-Pase) de-ficient lesions, 744 foci of re-expression of y-

glutamyl transpeptidase (EC 2.3.2.2; y-GT), anddecreased glycogen mobilization (187 lesions). Ofthe 1100 focal enzyme alterations, 1054 wereshown to be composed entirely of cells from asingle lineage of the two lineages present in themosaic tissue. Multiple alterations occurredwithin given lesions. Lesions with up to fourphenotypic alterations were found to consist ofcells of a single lineage. These results suggest thatindividual enzyme-altered foci are clonal in ori-gin and that phenotypic heterogeneity withinaltered hepatocellular foci is due to lesion pro-gression within a clonal population and not to amulticellular derivation.

Key words: chemical carcinogenesis, clonal foci, rat liver,enzyme lineage markers, histochemistry, chimeras.

Introduction

A number of current theories of carcinogenesis requirethat cancers arise from the clonal expansion of singleaffected cells. Many experimental and human spon-taneous cancers have been determined to be clonal inorigin (Deamant & Iannaccone, 1985; Iannaccone etal. 1978, 1987a; Iannaccone, 1980; Fialkow, 1976;Tanooka & Tanaka, 1982). The genesis of cancer iscommonly regarded as a multistep process; if any oneof these steps were to impart a rare selective advantageupon a single cell, the resulting tumours would beclonal growths. Thus, to determine if events critical tothe formation of the cancer occur in rare affected cellsfrom the time of initiation, it is important to determineif preneoplastic lesions are clonal in origin.

Journal of Cull Science 89, 423-431 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

Following exposure of a rat to hepatocarcinogens butprior to the onset of hepatocellular carcinoma severalbiochemical and morphological alterations become evi-dent. These include foci of altered enzyme expression,decreased glycogen mobilization, acidophilic and baso-philic foci, and hyperplastic nodules (Peraino et al.1981; Pitote/ al. 1978; Solt & Farber, 1976; Soil** al.1977; Hanigan & Pitot, 1985). A significant role forthese early lesions in cancer development is supportedby the following observations: (1) the alterationsalways precede cancer development (Rabese/ al. 1972;Williams, 1980), (2) they consist of actively dividingcells (Soltef al. 1977; Rabes & Szymkowiak, 1979) and(3) cancerous cells have been noted within hyperplasticnodules (Solt et al. 1977). Altered hepatocellular fociinduced by carcinogen treatment include decreased

423

expression of ATPase (EC 3.6.1.3) and glucose-6-phosphatase (EC 3.1.3.9; G-6-Pase), and increasedexpression of y-glutamyl transpeptidase (EC 2.3.2.2;y-GT; Emmelot & Scherer, 1980). Areas of decreasedglycogen mobilization were evident as lesions thatstained with periodic-acid-Schiff (PAS-positive) infasted liver.

The present study was undertaken to determinewhether the hepatocellular foci showing altered en-zyme activity that are seen in rat liver followingcarcinogen treatment are clonal in origin. An initiation-promotion model was selected resulting in enhancedproduction of foci with multiple phenotypic changes,thus providing an opportunity to examine the lineage ofphenotypic heterogeneity within foci. The recognitionof clonality within a given system requires a lineagemarker capable of distinguishing between cells in thepopulation under study. Chimeras are mosaic animalsproduced experimentally by amalgamating preimplan-tation embryos of distinguishable strains (Iannacconeet al. 1978; McLaren, 1976). For this study, ratchimeras were produced between congenic strainsdiffering in the major histocompatibility complex of therat, designated RT1 (Butcher & Howard, 1988; How-ard et al. 1980). Cells of these strains are distinguishedin tissue section by application of an iodinated mono-clonal antibody directed to class I antigen determi-nants, followed by autoradiography. Sections treated inthis way for lineage distribution can be compared toadjacent serial sections stained for morphological andenzymatic alterations. The lineage of cells in preneop-lastic populations is determined by direct histologicalanalysis, thereby helping to define the issue of clonalityin early steps of the neoplastic process.

Materials and methods

Pmduction of chimeras

Chimeras were produced between congenic strains of rat:PVG and PVG-RTI", differing in expression of class Iantigens of the major histocompatibility complex (Olac Ltd,Shaw's Farm, Bicester, England). Donor females matednaturally; three and a half days after mating eight-cellmorulae were recovered from oviducts under a low powerZeiss stereomicroscope (10— 40 X) with phosphate bufferedmedium (Papaioannou & West, 1981). Zonae pellucidae wereremoved from morulae stage embryos by brief incubation inTyrode's solution acidified to pH 2-5 (Nicolson et al. 1975).Pairs of embryos were placed in drops of rat embryo medium(Yamamura & Markert, 1981) under mineral oil. The embryopairs were placed in contact using a micropipette andmaintained at 37°C in a 5 % CO2 atmosphere overnight.Tetraparental embryos were then transferred into the uteri ofpseudopregnant recipient Holtzman females who had matedwith vasectomized males 24 h after the donor matings.

Antibodies

Monoclonal antibodies R3/13 and R2/15S against class Iantigen determinants of the PVG-.R7711 strain were providedby Dr Jonathan C. Howard (AFRC Institute of AnimalPhysiology; Galfre et al. 1977; Howard et al. 1980).

Tissue preparation

Organs were removed immediately after death, immersed inOCT compound (Miles, Naperville, IL) and quickly frozenin liquid nitrogen. Frozen tissues were stored at —80°C.Registration marks were produced with a warm wire intreated liver-tissue blocks immediately before cryostat sec-tioning.

Lineage marker for histological sectionsCell lineage was detected in histological sections as previouslydescribed (Weinberget al. 1985a,b; Iannaccone et al. 19876;Iannaccone, 1987). Briefly, monoclonal antibodies (50/il,0'5mgml~') were iodinated in a 30-s incubation with 2mCi1ZSI (350-600mCiml"1, 13-17mCi/<g~'; Amersham, Arl-ington Heights, IL) and 10/il chloramine T ( lmgrnl" ' in0-5 % Na2HPO4, pH7-4). The reaction was stopped with theaddition of 100/il tyrosine (saturated, in PBS). The antibodywas then diluted with 1800/d PBS containing 2% foetalbovine serum and 0 1 % sodium azide, and dialysed over-night. Sections 6/lm thick were mounted on gelatin-coatedslides and incubated with the iodinated antibody at 4CC for30 min. Sections were washed extensively, fixed for 10 min inbuffered formalin (Surgipath, Grayslake, IL), allowed to airdry overnight, then dipped in NTB2 emulsion (Kodak,Rochester, NY) 1:1 in water at 42°C. After 2-6 daysexposure the autoradiograms were developed in D-19(Kodak, Rochester, NY) and fixed in Kodak Fixer.

Analysis of tissue sections

Tissue sections were photographed on a Leitz-Epivert micro-scope using EPY 50 film emulsion (Kodak, Rochester, NY).Aggregates of cells of similar lineage (patches), liver lesions,and section perimeters were analysed with a SummagraphicsMM 1200 digitizing tablet connected to an NEC-APC 8088based microcomputer as previously described (Berkwits &Iannaccone, 1985; Weinberg et al. 1987). For analysis ofpatch pattern, the minor component was traced. The compu-tation of patch area was determined from the stored x,ycoordinates (Iannaccone et al. 19876,c; Berkwits & Iannac-cone, 1985). The lineages of circumscribed lesions weredetermined by overlaying the hand tracings of lesions withthose of the lineage-marker sections.

Induction of liver lesions

Liver lesions examined for lineage origin were induced inchimeric rats using the protocol of Pitot et al. (1978) whenthe rats were 10 weeks old. The left and median liver lobeswere excised. Diethylnitrosamine was administered 24 h laterby gastric intubation at a dose of 20rngkg"'.Within 2 weeksthe animals were placed on a high protein diet containing0-05% phenobarbital (Teklad Diets, Madison, WI). Animalswere starved overnight before sacrifice. Chimeric rats weretreated by this protocol along with 14 control rats of the PVGand PVG-RTJ' strains. Chimeras were sacrificed at 13-14-5

424 IV. C. Weinberg and P. M. Iannaccone

Table 1. Cellular lineage of y-GT areas in chimeric liver

Animal

090609090912

Total

a

8040

5

125

<0-05

c

9028

195

313

mixf

1031

14

0

a

1274

7.3

05-0-10

c

131243

68

mix

410

5

0-

a

18133

34

Are;i of lesion10-0-50

c

221170

103

mix

622

10

(mm2)*0-.

a

500

s

50-1

c

558

18

•00

mix

000

0

a

200

7.

1-00-2

c

231

6

•00

mix

000

0

a

300

3

> 2 0

c

248

14

mix

100

1

•Areas were determined by image processing as described in Materials and methods.f a ' refers to lesions composed entirely of cells of the PVG-RTI* lineage; 'c' refers to lesions composed entirely of the PVG lineage and

'mix' refers to those lesions which are composed of both types of cells.

Table 2. Cellular lineage of PAS-positive areas in chimeric liver

a

1210

<0-05

c

1057

mix-f

01

0

a

11

•05-0-10

c

046

mix

03

Area of0

a

60

lesion•10-0-

c

131

(mm2)*50

mix

00

0-

a

00

50-1

c

03

•00

mix

02

1

a

00

•00-2

c

17,

•00

mix

00

Animal

09060912

Total 22 67 1 2 4 6 3 6 32 0 0 3 2 0 3 0

•Areas were determined by image processing as descnbed in Materials and methods.f a ' refers to lesions composed entirely of cells of the PVG-RT1" lineage, V refers to lesions composed entirely of the PVG lineage and

'mix' refers to those lesions which are composed of both types of cells.

Table 3. Cellular lineage of G-6-Pase-deficient areas in chimeric liver

Animal

09060912

a

00

<0-05

c n

013

00

0

a

10

•05-0-

c

013

10

mix

01

0-

a

00

Area of lesion10-0-50

c mix

0 025 0

(mm2)*0-50-1-00

a c

1 10 1

mix

00

1

a

10

•00-2

c

10

•00

mix

00

a

10

>2-00

c

00

mix

00

Total 0 13 0 1 1 3 1 0 2 5 0 1 2 0 1 1 0 1 0 0

•Areas were determined by image processing as described in Materials and methods.f a ' refers to lesions composed entirely of cells of the PVG-RT1* lineage; 'c' refers to lesions composed entirely of the PVG lineage and

'mix' refers to those lesions which are composed of both types of cells.

Table 4. Cellular lineage of ATPase-deficient areas in chimeric liver

Animal

09060912

a

30

<0 05

c mixf

5 09 0

0

a

60

•05-0-

c

811

10

mix

01

0-

a

60

Area of lesion10-0-50

c

1474

mix

25

(mm2)*0-50-1 •

a

20

c

06

00

mix

00

1

a

1

n

•00-2

c

10

•00

mix

10

a

10

>2-

c

13

00

mix

00

Total 3 14 0 6 19 1 6 38 7 2 6 0 1 1 1 140

•Areas were determined by image processing as described in Materials and methods.f a ' refers to lesions composed entirely of cells of the PVG-RT1' lineage; V refers to lesions composed entirely of the PVG lineage and

'mix' refers to those lesions which are composed of both types of cells.

Clonal origin of preneoplastic lesions 425

Fig. 1. Photomicrographs of carcinogen-treated chimeric rat liver. A. Frozen section incubated with an iodinatedmonoclonal antibody that recognizes the PVG-RT1* haplotype. Areas of black are silver grain accumulation in theautoradiogram overlying cells of the PVG-RTI" lineage. The white areas are cells of the PVG lineage. There are two areasof focal proliferation evident (a, b), which are composed exclusively of cells of the PVG-RTI' and PVG lineagesrespectively. The alteration in patch pattern surrounding these lesions demonstrates their expanding nature. B. Serialsection stained for the presence of y-GT as described in Materials and methods. Both lesions a and b are seen to be y-GTpositive. C. Serial section stained with PAS after fasting as described in Materials and methods, demonstrating that lesion bbut not lesion a is an area of aberrant glycogen retention. Lesion b demonstrates heterogeneous expression of thisphcnotype even though the lesion is clearly clonal. D. Serial section stained for the presence of G-6-Pase as described inMaterials and methods. Both lesion a and lesion b appear to be homogeneously deficient in G-6-Pase activity. Bar, 10 mm.

Fig. 2. Computer-generated picture of digitized data fromFig. 1 demonstrating lesions of heterogeneous enzymeexpression and patches of PVG-derived cells. Continuousblack lines ( ), patches of cells of PVG lineage; blackdotted lines ( ), PAS-positive areas; red dashed lines( ), y-GT-positive areas; blue lines ( ), ATPase-deficicnt areas; green lines ( ), G-6-Pase-deficientareas. Bar, 1-0mm.

Fig. 3. Computer-generated picture of digitized dataobtained as described in Materials and methodsdemonstrating the relationship of morpholo.gically alteredareas to patch pattern. Solid lines ( ), patches of cellsof the PVG lineage; dotted lines ( ), PAS-positiveareas; red dashed lines ( ), y-GT-positive areas; greenlines ( ), ATPase-deficient areas. Bar, 1-0 mm.

426 \Y. C. \\'ei>iberg and P. M. lannaccone

8

months of age. Frozen sections of treated livers were cut at athickness of 6fJ.m and mounted serially.

acid-Schiff (PAS) reaction forglycogen was performed usingestablished procedures (Sheehan & Hrapchak, 1980).

Histochemical staining reactions

Glucose-6-phosphatase (G-6-Pase; EC 3.1.3.9) and aden-osine triphosphatase (ATPase; EC 3.6.1.3) were demon-strated using modifications of the methods of Wachstein &Meisel (1957, 1956). For demonstration of G-6-Pase activitysections were incubated for 15min at 37°C in the followingreaction mix: 8-0ml sodium glucose-6-phosphate (l-0mgml"1), l-2ml 2% Pb(NO3)2 and 8-8ml of a working buffercontaining 15-0ml 0-2M-maleic acid, 15-0ml 0-2M-Tr i sHCl , and25-5mlO-2M-NaOH. Slides were then rinsedin tap water and distilled water before a 20 s incubation in 1 %ammonium sulphide. For demonstration of ATPase activity12-5 mg of substrate (Na-ATP) was dissolved in 11 ml waterand mixed with 10-0 ml of 02M-Tris-maleate buffer, 2-5 ml0-lM-MgSO4, and l-5ml 2% Pb(NO3)2. Sections wereincubated in the above mixture for 15min at 37°C, rinsedquickly through two changes of distilled water and reacted for30 s with 1% ammonium sulphide. The slides were thenfixed in buffered formalin, dehydrated and mounted.

y-Glutamyl-transpeptidase (y-GT; EC 2.3.2.2) activitywas elucidated by the method of Rutenberg et al. (1969)using ./V-(y-l-glutamyl)-4-methoxy-2-napthylamide(GMNA; Polysciences, Inc.) as a substrate. The periodic

Results

Patch analysis of chimeric liver

A complete description of the analysis and implicationsof patch patterns of normal liver has been publishedpreviously by us (Iannacconeef al. 19876; Weinberge/al. 19856). The normal patch pattern is highly complexgeometrically. There are linear relationships betweenthe proportion of the two cell types in the mosaic tissueand the size and number of patches. As the proportionof major cell type increases, the area of patches of thattype increases while the area of patches of the minorcell type decreases. Conversely, as the proportion ofthe major cell type increases the number of patches ofthat type decreases, while the number of patches of theminor cell type increases (Iannaccone et al. 19876).Chimeras treated with diethylnitrosamine and partialhepatectomy with phenobarbital promotion displayedlesions of each parental lineage in proportion to thepercentage of cells derived from either lineage in thechimeric liver.

B

Fig. 4. Photomicrographs of serial sections of carcinogen-treated chimeric rat liver demonstrating the lineage of small areasof altered y-GT expression. A. Unstained autoradiogram incubated with an antibody as described in Materials and methodsthat recognizes cells of the FVG-RTJ" lineage. B. Serial section stained for the presence of y-GT activity as described inMaterials and methods. These sections illustrate the clonality of small lesions. Bar, 2 0 mm.

Fig. 7. Computer-generated picture of digitized data fromserial sections obtained from chimeric liver treated asdescribed in Materials and methods. Two lesions ofheterOgeneously altered enzyme expression are evident (aand b). Solid black lines ( ), patches of PVG lineage;red dashed lines ( ), y-GT-positive areas; green lines( ), ATPase-deficient areas; dotted black lines ( ),PAS-positive areas. Bar, 1-0 mm.

Fig. 8. Computer-generated picture of the patch pattern ofa lesion of apparently mixed lineage. Solid black lines( ), patches of PVG lineage; red dashed lines ( ), y-GT-positive areas; green lines ( ), ATPase-deficientareas, a, b, lesions of heterogeneous enzyme expressionclearly of one lineage; c, lesion falls across two patches, buthourglass shape suggests it represents a coalescence of twoadjacent patches. Bar, 1-0 mm.

Clonal origin of preneoplastic lesions \T1

Fig. 5. Photomicrographs of serial sections of carcinogen-treated chimeric rat liver. A. Unstained autoradiogramincubated with an iodinated monoclonal antibody thatrecognizes cells of the P\'G-RTIa lineage. B. Serial sectionstained for the presence of y-GT activity. C. Stained forthe presence of ATPase activity. The arrow points out afocus of altered y-GT and ATPase expression, which isseen to be composed entirely of cells of the PVG-/?77°lineage. A similar area is present in the centre of thesection but does not show altered expression of ATPaseactivity. Bar, 2 0 m m .

Clonal analysis of lesions

A total of 714/744 y-GT-positive foci (Table 1),181/187 PAS-positive foci (Table 2), 58/59 G-6-Pase-deficient foci (Table 3) and 101/110 ATPase-deficient(Table 4) foci were determined to be composed of cells

derived from a single lineage. Hepatocellular focishowing phenotypic alterations were evident in all sizecategories, and ranged in size from smaller than0-05 mm2 to greater than 2-0 mm2. Of 1100 phenotypicalterations resulting from the carcinogenic regimenonly 4-2% (46) showed evidence of cells derived frommore than one lineage. Some single lesions containedmultiple phenotypic alterations (Table 5). The ma-jority of these lesions of mixed lineage were as small asor smaller than the average patch size.

Expression of RTl.Aa antigen in enzyme-altered fociinduced in untreated control rats was examined. A totalof 615 y-GT lesions were examined: 366 were in PVG-RTla rats and 249 were in PVG rats. The lesions weredistributed across several size categories, some reach-ing greater than 2-0 mm2 in area, and no inappropriatereactivity with the antibodies was detected. Thus thecarcinogenic protocol does not alter the expression ofthe marker antigen to a demonstrable degree (Wein-berg etal. 1987).

Several aspects of focus formation become evident inthe examination of the tissue sections. The expandingnature of the lesions is reflected in the distortion of thesurrounding normal patch pattern seen particularlyclearly in Fig. 1 (corresponding computer analysis inFig. 2), but also surrounding smaller lesions such as (a)in Fig. 3. Fig. 4 demonstrates clearly the derivation ofsmall lesions from a single genotype. Foci of y-GTexpression are seen to align directly with patches ofcells of single genotype.

Multiple enzyme alterations corresponding to indi-vidual patches are shown in Figs 1—3. Given thecomplex patch shape and distribution in normalchimeric liver it is exceedingly unlikely that these areaswould be aligned by chance alone, and thus it may beconcluded that the foci are derived from single cells.Serial sections demonstrating cell lineage and enzymealterations were digitized and areas were determined asdescribed in Materials and methods. The digitized datawere used to generate an image of the lesion in each ofthe serial sections. These images were then recon-structed using independent landmarks (Figs 2, 3).This analysis revealed numerous examples of hetero-geneous enzyme expression within regions (Figs 1-3,5-8). When the outline of all overlapping areas weretaken as one large lesion, the entire lesion was seenagain to conform to the patch pattern and directlyoverlie a single patch. This was true regardless of thedegree of phenotypic heterogeneity of the lesion (Table

5).The direct visualization of foci in these studies allows

the unambiguous interpretation of cell lineage in thelesions as shown in Fig. 1 and corresponding digitizeddata in Fig. 2. In these sections two nodules were seento be abutting. These might be interpreted as one largearea of y-GT expression, however examination of the

428 U'. C. Weinberg and P. M. lannaccone

F&Fig. 6. Photomicrographs of serial sections of carcinogen-treated chimeric liver. A. Unstained autoradiogram prepared byincubating the section with iodinated R2/1SS antibody which recognizes cells of the P\'G-RT!" lineage. B. Serial sectionstained to demonstrate the presence of ATPase activity. C. Serial section stained for the presence of G-6-Pase activity.D. Serial section stained for the presence of y-GT activity. Lesion a can be seen to have altered expression of all threeenzymes with alterations heterogeneously distributed within an area composed exclusively of cells of the PVG-RTI" lineage.Bar, 2-0 mm.

60420

11

12613t

2300

2120

Table S. Cellular lineage of phenotypically complexlesions in chimeric liver

Phenotypic complexity*1 2 3 4

af

* Phenotypic complexity refers to the number (1, 2, 3 or 4) ofdifferent alterations in enzyme expression within a given lesion.Histochemical determinations were made as described in Materialsand methods.

•fa' refers to lesions composed entirely of cells of the PVG-RTI'lineage; 'c' refers to lesions composed entirely of the PVG lineageand 'mix' refers to those lesions which are composed of both typesof cells.

X Histological examination of one of these revealed an hourglass-shaped lesion overlying two adjacent patches of PVG and PVG-RT1" origin which strongly suggests coalescence of two individuallesions (sec Fig. 8).

neighbouring section stained for the lineage markerdemonstrates that they are actually two nodules, one ofPVG derivation, the other of ?\'G-RTla origin. Com-puter reconstructions shown in Fig. 8 represent liversections containing lesions of both cell lineages. Thelesion labelled c could be interpreted as mixed lineage,and therefore derived from multiple cells. Upon directexamination, however, it is clear that the hourglass-shaped lesion clearly lies over two patches, and that thelesion is actually two distinct adjacent foci. In a studyin which lineage was determined by homogenizingdissected foci this would have suggested a multiclonalorigin for one lesion. By direct histological examinationthe distinct origins of the two lesions become clear.

Discussion

These results clearly establish that initiation by acarcinogen with subsequent promotion results inaltered hepatocellular foci that are derived from a singlecell. This was demonstrated for nearly all of the fociexamined. This result is obtained irrespective of the

Clonal origin ofpreneoplastic lesions 429

degree of phenotypic heterogeneity of lesions analysed.This observation suggests that the clonal step occursbefore the development of heterogeneity of enzymephenotype within lesions and strongly suggests thatthis heterogeneity is a reflection of progression of thelesion.

The composition of tumours in an individual with atleast two genetically distinct cell populations can beevaluated with respect to the proportion of the differentcell populations present in a tumour. A tumour derivedfrom many cells would be expected to contain both celltypes represented in the normal organ. Conversely, aclonal tumour should only have one cell type present.Much evidence has accumulated regarding tumourclonahty from examining the composition of tumoursin individuals displaying cellular mosaicism.

Lesions smaller than patches of contiguous cells ofcommon lineage might yield inconclusive results, sincethese small lesions might be composed of cells from asingle lineage by chance alone. However the irregularshape of normal patches, and the distinct alignment oflesions onto the patch pattern as well as the sheernumber of lesions composed of a single lineage,strongly supports the thesis that these areas of singleand heterogeneous enzyme altered expression are clo-nal populations.

Tumours in animal models and clinically overthuman tumours have been shown to be clonal(Deamant & Iannaccone, 1985; Iannaccone e/ al. 1978,1987*7; Fialkow, 1976). Hepatocellular carcinoma andhyperplastic nodules deficient in ATPase activity inmouse liver have previously been shown to be clonal inorigin (Howelle/a/. 1985; Kabtsetal. 1982). Carcino-gen-induced foci of altered y-glutamyl transpeptidaseexpression were shown by us to be clonal (Weinberg etal. 1987). In view of the multistep nature of thecarcinogenic process, we have attempted to identifyclonal steps in the sequence leading to neoplasia.Altered enzyme expression observed in chemicallytreated rat liver provides an opportunity to examinepreneoplastic lesions to determine the earliest recogniz-able step at which clonal selection occurs in theformation of cancer.

This work was supported in part by grants CA29078 andES03498 from DHHS, USPHS and by grant 15-49 from theMarch of Dimes Birth Defects Foundation.

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(Received 6 October 1987 -Accepted 16 November 1987)

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