the cellular origin of chemically …jcs.biologists.org/content/joces/29/1/249.full.pdfthe cellular...

y. Cell Sci. V), 249-269 (1978) 249Printed in Great Britain © Company of Biologists Limited 1Q78

THE CELLULAR ORIGIN OF CHEMICALLY

INDUCED TUMOURS

P.M. IANNACCONE, R. L. GARDNER* AND H. HARRISThe Sir William Dunn School of Pathology, University of Oxford,South Parks Road, Oxford OXi 3RE, England

SUMMARYTumours were induced by chemical carcinogens in. chimaeric mice made by the amalga-

mation of two embryos each producing a different electrophoretic variant of the enzymeglucose phosphate isomerase. Although, in these chimaeras, the smallest samples of normaltissue that could be analysed almost invariably contained both isoenzymes, almost all the tumourscontained only one. An analysis of the size of the clones that formed the chimaeric epidermispermitted the conclusion that the epidermal tumours produced could not have risen from morethan 8 cells; and the most probable interpretation of the data is that these tumours wereclonal growths.

INTRODUCTION

Genetic mosaics have been extensively used to study the cellular origin of humantumours (Linder & Gartler, 1965; Gartler, 1974; Fialkow, 1976). Most of thesestudies have exploited polymorphisms of the enzyme glucose-6-phosphate dehydro-genase (G-6-PD) which is coded by a single locus on the X chromosome (Ohno,Poole & Gustavsson, 1965). Several polymorphic variants of G-6-PD have beendescribed (Gartler, 1974). In placental mammals, one X chromosome is inactivatedin the cells of the female early in embryonic development (Lyon, 1961; Cattanach,1975), so that the adult female is a genetic mosaic. The most common G-6-PDvariants are revealed by differences in electrophoretic mobility and are referred to asthe A (rapidly migrating) and B (slowly migrating) variants. The electrophoreticdifference is determined by the substitution of a single amino acid in the polypeptidechain (Yoshida, 1967). Mosaic individuals consist of a mixture of 2 populations ofcells, one having the paternal X chromosome active and the other the maternal Xchromosome active. Where the individual is heterozygous for variants of G-6-PD,one population of cells produces one variant of the enzyme and the other populationproduces the other. It has been argued that tumours arising in such genetic mosaicswould contain both enzyme variants if they arose from a large number of cells, butonly one variant if they arose from a single cell (Linder & Gartler, 1965). During de-velopment, extensive cell mixing must occur after the X chromosome has been in-activated, for very small pieces of normal tissue contain both enzyme variants. Thissuggests that the 'patch' size (the number of contiguous cells having the samefunctional genotype) in normal tissues is very small (Nesbitt, 1974). The patch size of a

• Department of Zoology, University of Oxford.

250 P. M. Iannaccone, R. L. Gardner and H. Harris

number of human tissues has been determined. In human scalp hair follicles it appearsto be between 950-3500 cells (Gartler et al. 1971), in human myometrium approxi-mately io5 cells (Linder & Gartler, 1965) and in human vulvar epithelium approxi-mately 1500 cells (Friedman & Fialkow, 1976). Tumours arising in females dimorphicfor G-6-PD usually contain only a single enzyme variant, although very small samplesof the normal tissue from which the tumour originated contain both variants. Bothbenign and malignant tumours involving many different tissues have been studied(Gartler, 1974). The generality of the finding that the tumours contain only a singleenzyme variant has prompted the conclusion that they are, for the most part, uni-cellular in origin.

No X-linked marker suitable for studies of this kind is currently available for thecommon laboratory animals, so that the question of the cellular origin of chemicallyinduced experimental tumours has not so far been examined by this method. Experi-mental chimaeras provide an alternative approach to the problem (Tarkowski, 1961;Mintz, 1962; Gardner, 1968). These animals are usually tetraparental and are pro-duced by amalgamating embryos carrying suitable genetic markers. If 2 embryoshave different electrophoretic variants of a polymorphic enzyme, then the chimaera pro-duced by their amalgamation is analogous, for experimental purposes, to the geneticmosaic resulting from X-linked polymorphism. In the present paper we describe ananalysis of tumours induced by chemical carcinogens in experimental chimaerasdimorphic for the enzyme glucose phosphate isomerase (Gpi-i).

MATERIALS AND METHODS

Glucose phosphate isomerase (Gpi-i) as a marker

Electrophoretic variants of glucose phosphate isomerase (E.C. 5.3.1.9) have been used asmarkers of chimaerism (De Lorenzo & Ruddle, 1969). In mice there is a slowly migrating anodalvariant of the enzyme (Gpi-ia) and a rapidly migrating cathodal variant (Gpi-ib). The enzymeis dimeric and forms a heteropolymer of intermediate mobility when both alleles are activein the same cell. In heterozygous animals the homopolymers and the heteropolymer arepresent in a ratio of 1:2:1.

The 2 variants of Gpi-i can be separated by horizontal starch gel electrophoresis. Twelveper cent starch gels (w/v in a 1:20 dilution of tank buffer) were used essentially as described byChapman, Whitten & Ruddle (1971). The gels were prepared by the standard procedure andmeasured 7 x 8 x 0 1 cm. The tank buffer was 0 2 M Tris-citrate, pH 6-4. Samples were appliedto the gel by inserting saturated strips of cellulose acetate paper into 1 x 4-mm slots cut intothe gel. Approximately 02 fil of sample were loaded into each channel. A current of 8-12 mAwas applied across a 6-cm gap for 3 h at 4 °C. The gel was blotted dry, and the side that hadfaced downwards during the electrophoresis was stained with a reaction mixture containing1 mg NADP, 20 mg fructose-6-phosphate (glucose free), 15 units of glucose-6-phosphatedehydrogenaae from Torula yeast, 05 mg phenazine methosulphate and 1 mg MTT tetrazolium,in approximately 5 ml of 0-3 M Tris-HCl buffer at pH 80. Six millilitres of 2 % (w/v) Difcobacteriological agar in distilled water were boiled, cooled to 60 °C and added to the reactionmixture, which was then applied to the gel. The gels were developed at 37 CC in the dark fora period of 10-40 min depending on the specific activity of the sample. The gels were photo-graphed on Pan F film (Ilford) through a Wratten No. 58 green filter at intervals after theapplication of the stain. The samples for electrophoresis were prepared by homogenizing smallvolumes of tissue in 10 mM Tris lysate buffer at pH 74 in a o-4-ml Beckman 'microfuge' tube.The homogenate was frozen and thawed 3 times in a dry ice-alcohol bath and then spun for30 s in a Beckman microfuge, model No. 152. The supernatant was applied to the cellulose

Cellular origin of tumours 251

acetate strip on an ice-cold surface. Fig. 1 (p. 254) demonstrates the various isoenzyme patternsof Gpi-i.

The proportion of each isoenzyme in the experimental sample was assessed by comparingthe electrophoretic pattern with that produced by standard mixtures of the 2 isoenzyme variants.These standards were made by mixing in known proportions blood from animals homozygousfor each of the 2 variants. The major electrophoretic band in the tissue sample was matchedagainst the major band in the blood mixture; this permitted the minor bands in the tissue tobe compared with known amounts of the minor component in the blood. The lower limit ofresolution of the minor component was about 1 %.

Production of chimaeric animals

Parental genotypes

Pathology Oxford (PO) mice are Swiss albino derivatives introduced into the Sir WilliamDunn School of Pathology from the Chester Beatty Research Institute in 1937. They havebeen maintained by random mating as a closed colony. Electrophoretic examination of bloodfrom the tail revealed that the animals now segregate at the Gpi-i locus. Either Gpi-iaa orGpi-ibb animals were selected for experiment.

CBA/HT6T6 mice are C3H derivatives which were introduced into the Dunn School in1973 and have since been maintained by sib mating. The characteristics of this inbred line havebeen previously described (Ford, Hamerton, Barnes & Loutit, 1956; Carter, Lyon & Phillips,1955). The animals are Gpi-ibb.

Manipulation of embryos

The animals were mated under natural light, and embryos were collected by flushing theoviducts or the uteri of the pregnant females with phosphate-buffered medium. The embryoswere harvested 2-5, 3-5 or 4-5 days post coitum.

Inner cell masses were removed from 4'5-day-old blastocysts of CBA/HT6T6 and POGpi-ibb genotype by microsurgery. Inner cell masses were removed from 3'5-day-old blasto-cysts of PO Gpi-iaa or Gpi-ibb genotype by treating the blastocysts with rabbit anti-mouseantiserum and guinea pig complement (Solter & Knowles, 1975). This ' immunosurgery' wassometimes done after removal of the zona pellucida by acid Tyrode's solution, pH 2-5, con-taining 0-4 % (w/v) polyvinyl pyrrolidone (Nicolson, Yanagimachi & Yanagimachi, 1975).

The inner cell masses from the CBA/HT6T6 and PO Gpi-ibb embryos were injected into3'5-day-old PO Gpi-iaa blastocysts by the techniques described by Gardner (1978). Innercell masses from PO Gpi-iaa embryos were similarly injected into PO Gpi-ibb blastocysts.

Morulae of PO Gpi-iaa and PO Gpi-ibb genotype were aggregated as described by Mintz(1962). These aggregation chimaeras were produced by Dr V. G. H. Riddle.

The amalgamated embryos were injected into the uterine horns of either PO or CFLPalbino mice (Anglia Laboratories) which had been mated 2-5 days previously with vasectomizedmales. Table 1 describes the genetic constitution and method of production of the chimaerasused.

Chemical carcinogenesis

Two-tenths of a millilitre of a 0-2% solution of 7,12 dimethylbenz[a]anthracene (DMBA)were applied to the shaved backs of the chimaeric animals with an automatic pipette. One weeklater 02 ml of a 025 % solution of croton oil in acetone was applied to the same site, and thisprocedure was repeated twice weekly until tumours appeared. Some of the animals were treated,after application of the carcinogen, with a 0-005 % solution in acetone of i2-O-tetradecanoyl-phorbol-13-acetate (TPA), the active component of croton oil. 20-methylcholanthrene (MCA)was applied in the same way, as a 0-7 % solution in acetone, followed by TPA twice weekly.2-5 mg of MCA in 01 ml of olive oil were injected subcutaneously into both flanks. The MCAwas not completely dissolved at this concentration.

Tab

le I

. In

duct

ion

of t

urno

urs

in c

him

aeri

c mice

Mo

de

of p

rodu

ctio

n T

ota

l do

se,

No.

of

To

tal dose,

No.

of

Ani

mal

G

enet

ic c

onst

itut

ion

of c

him

aera

C

arci

noge

n m

g

appl

icat

ions

P

rom

oter

m

a

appl

icat

ions

S

ite

I In

ject

ion

of i

nner

cel

l m

ass

(DM

BA

D

MB

A

DM

BA

D

MB

A

DM

BA

M

CA

' D

MB

A

DM

BA

D

MB

A

DM

BA

D

MB

A

\MC

A

DM

BA

M

CA

D

MB

A

A~

reg

ati

on

of m

orul

ae

MC

A

DM

BA

M

CA

D

MB

A

Inje

ctio

n of

inn

er c

ell

mas

s D

MB

A

Agg

rega

tion

of

mor

ulae

D

MR

A

? M

CA

Inje

ctio

n of

inn

er c

ell

maw

D

MB

A

DM

BA

CO

I

I

CO

I I

CO

I

I

CO

10

.5

TP

A

0.27

I

CO

36

.5

TP

A

0.41

2

?'P

A

0'42

2 T

PA

0.

422

TP

A

0.43

I

skin

sk

in

skin

P

sk

in

% sk

in

3

subc

ut.

skin

n

sk

in

skin

-

skin

1

skin

> b

su

bcut

. .

skin

0

subc

ut.

Q sk

in

3

subc

ut.

9

skin

su

bcu

t.

skin

?

skin

sk

in

3 3 sk

in

c.

subc

ut.

skin

sk

in

skin

DM

BA

= 7

,12

dim

ethy

lben

zant

hrac

ene;

C

O =

cro

ton

oil;

TP

A =

12-

0-te

trad

ecan

oyl

phor

bol-

13-a

ceta

te;

MC

A =

20-

met

hylc

hola

nthr

ene.


Adult animals of between 20 and 30 g were used. All carcinogens were applied under lightether anaesthesia.

Table 1 describes the regimen for each chimaera.

Sampling of normal tissues

Epidermis and dermis were isolated by treating whole skin with 1 % (w/v) trypsin in phos-phate-buffered saline (PBS) at 4 °C overnight. The skin was rinsed in 5 ml of cold PBS con-taining 50 mg of ovomucoid trypsin inhibitor and in 3 changes of cold PBS; it was thenblotted dry. The epidermis was removed with fine forceps as a sheet and placed on to anice-cold glass surface. Fragments of epidermis 0-5 to 10 mm* in area were teased away fromthe main sheet with fine forceps under a hand lens. The samples were placed on celluloseacetate strips, frozen for 1-5 h at — 20 °C and then subjected to electrophoresis. Fragments ofthe isolated dermis were treated in the same way. Usually, 20 samples of dermis and 20 samplesof epidermis were removed from each animal for analysis. The remaining epidermal anddermal sheets were extracted and were also subjected to electrophoresis. In some animals,small pieces of subcutaneous tissue adjacent to tumours were frozen and subjected to electro-phoresis.

Sampling of tumours

The tumours were removed after the animals had been killed or had died. In some animals,the tumours were removed surgically in order to allow other tumours to develop. For surgicalremoval, the animals were anaesthetized with ether or with tribromoethyl alcohol and amylenehydrate (Avertin) given intraperitoneally. The base of the tumour was tied off with sutureligatures, so that the tumour could be cut out without at the same time removing any normalskin.

Each tumour was cut into two, and one half was fixed in 10% formaldehyde-saline forhistological examination. The other half was teased apart, rinsed in cold PBS and blotted dryin order to remove normal blood elements as far as possible before homogenization.

RESULTS

Histology of the tumours

The tumours produced could be divided into 4 histological groups: papillomas,squamous cell carcinomas, basal cell carcinomas and fibrosarcomas. In 2 animalsextension of fibrosarcomas to the diaphragm caused the death of the animals.Figs. 5-8 show typical histological appearances. The papillomas presented as tumoursraised above the surface of the normal skin. They contained morphologically abnormalepidermal cells grouped around a loose connective tissue core of variable size. Usually,differentiating epidermal cells or cells derived from the basal layer predominated, butoccasionally other cell types were seen. In some cases there was histological evidenceof localized invasion of surrounding tissue. In 1 case (T130) the tumour was com-posed of sweat gland elements; and in 4 cases (T186, T179, T180 and T329) thetumours contained the keratinized material characteristic of cutaneous horns.

Some of the squamous cell carcinomas arose from papillomas; others arose denovo, usually from ulcerating lesions. These tumours were invasive growths. Theyshowed a variable pattern of differentiation, but some keratinization was always pre-sent. One typical basal cell carcinoma was produced. The fibrosarcomas were com-posed of spindle-shaped cells irregularly disposed and showing numerous mitoticfigures. These tumours were clearly invasive and some were ulcerated.

17 CEU 29

2f4 - M- Iannaccone, R. L. Gardner and H. Harris

2 1

# # 0

/ 2 .3

7 2 3 4 5


Glucose phosphate isomerase banding pattern of normal tissue

The Gpi-i activities of small fragments of normal epidermis, dermis and sub-cutaneous tissue were as follows: 456/495 samples of epidermis taken from 28chimaeric animals had both ' a ' a n d ' b ' type activity; 398/417 samples of dermis from 22chimaeric animals had both ' a ' and ' b ' type activity; and 25/25 samples of subcutan-eous tissue from 4 chimaeric animals had both ' a ' and ' b ' type activity. The resultsof the assays are summarized in Table 2, p. 258. The proportions of the two typesof enzyme activity in the normal tissues varied greatly (Fig. 2). The distributionsobserved are set out in Table 3.

Glucose phosphate isomerase banding pattern of the tumours

Unlike the samples of normal tissue, the tumours, with 7 exceptions, showed onlya single electrophoretic variant of the enzyme (Table 2). Tumours were assessed ashaving a single isoenzyme variant if 95 % or more of the enzyme activity was of onetype. A minor band consisting of no more than 5 % of the total activity could beaccounted for by the observed degree of contamination of the tumour with host cells.For the most part, these contaminants were erythrocytes, inflammatory cells andfibroblasts. The tumour samples were all greater than 1 mm in their longest dimensionand were thus always larger, often very much larger, than the samples of normaltissue taken.

PO «-> CBA/HT6T6 chimaeras. An analysis was made of 25 papillomas, 3 carcino-mas and 1 fibrosarcoma which arose in 5 animals produced by amalgamation of POGpi-iaa embryos and CBA/HT6T6 Gpi-ibb embryos. The tumours varied in sizefrom 1 to 29 mm in their largest dimension.

Table 4 shows the Gpi-1 isoenzyme activities of the tumours and of the normal tissuesamples derived from the same animals. In all of these tumours, with the exception ofT255, T256 and T258, 95 % or more of the Gpi-i present was of a single electrophore-tic type. T255 and T258 had also been analysed at an earlier stage and were found at

Fig. 1. Horizontal starch gel electrophoresis of glucose phosphate isomerase (Gpi-i).1, blood from a homozygous Gpi-iaa animal. 2, blood from a homozygous Gpi-ibbanimal. 3, blood from a heterozygous Gpi-iab animal showing a heteropolymericband of intermediate mobility. This is more intense than the homopolymeric bands.4, extract of chimaeric tissue showing both the ' a ' and ' b ' type homopolymers.

Fig. 2. Horizontal starch gel electrophoresis of small samples of normal tissue. Allsamples show both ' a ' and ' b ' activity, but in varying proportions.

Fig. 3. Horizontal starch gel electrophoresis of tumour homogenates and standardblood mixtures: J, blood from a homozygous Gpi-iaa animal; 2, blood from ahomozygous Gpi-ibb animal; 3, homogenate of rumour T181 from mouse C56; 4,homogenate of tumour T180 from mouse C56.

Fig. 4. Horizontal starch gel electrophoresis of rumour homogenates and standardblood mixtures: 1, blood from a Gpi-ibb animal with 5 % (v/v) Gpi-iaa blood added;2, homogenate of a small piece of normal tissue adjacent to tumour T263; 3, 4, 5,homogenates of 3 individual samples from different areas of tumour T263 from mouseC83.

17-2


Fig. 5. Papilloma T120 from mouse C52 showing a warty appearance with epidermalcell overgrowth. Differentiated epidermal cells are present. There is a small core ofloose connective tissue. Haematoxylin and eosin, x 30.Fig. 6. Squamous cell carcinoma T236 from mouse C51. The tumour is relativelyhomogeneous in cell type. Haematoxylin and eosin, x 190.

7,

y

Fig. 7. Basal cell carcinoma T237 from mouse Csi, showing localized invasion of sur-rounding tissues. Haematoxylin and eosin, x 190.Fig. 8. Fibrosarcoma T339 from mouse C87. The tumour is invading muscle tissue.Haematoxylin and eosin, x 170.


that stage to contain only a single isoenzyme. It thus seemed probable that T255,T256 and T258 were formed by the coalescence of smaller primary tumours. MouseC53 developed 11 papillomas and 2 carcinomas over a 52-week period. All of thetumours in this animal contained only the ' a ' type isoenzyme. This may to someextent be a reflexion of the fact that in the normal epidermis in this case approximately

Table 2. Glucose phosphate isomerase isoenzyme banding patterns inchimaeric tissues and tumours derived from them

Gpi-ia Gpi-ib Gpi-ia and b

Normal epidermis 14(28 animals)

Normal dermis 2(22 animals)

Normal subcutaneous tissue o(4 animals)

Tumours 46 (9)*(21 animals)

• The numbers in parentheses refer to additional analyses done on tumours that recurredafter surgical removal of the primary tumour.

Table 3. Distribution of glucose phosphate isomerase isoenzymes insmall samples of normal skin

25

17

0

43(2)

450

398

25

7(2)

EpidermisClass I*Class IIClass III

DermisClass IClass IIClass III

• The division

0-20

353

—

277

into classes is

% of activity present as

20-40

3419—

281 0

—

arbitrary and

A

4O-60

9119

5

364

5

serves merely

Gpi-ia

60-80

—2 2

2 4

—

13

33

to facilitate

80-100

—

936

—7

37

statistical

fvTn n f

animals

51 0

4

464

treatment.

80% of the cells contained ' a ' type isoenzyme, so that there would be a much greaterchance that an ' a ' type cell would give rise to a tumour than a ' b ' type cell. However,since the smallest fragments of normal epidermis that could be analysed still showed20% of the ' b ' type isoenzyme, the tumours could not have been produced by theunselected growth of a large number of epidermal cells.

The papilloma T122 on mouse C51 recurred after surgical removal for analysis andprogressed over a period of 30 weeks to form a basal cell carcinoma. Both the initialpapilloma and the carcinoma produced the same single Gpi-i isoenyzme.

Mouse C61 was given 20-methylcholanthrene by subcutaneous injection. Thisanimal had produced only one tumour after the application of DMBA to the skin


followed by 35 weeks of treatment with croton oil. Twelve weeks after the subcutaneousinjection of carcinogen the animal died. The diaphragm and liver were found to beinvaded by a fibrosarcoma. Several samples of this tumour all showed 95 % or moreof a single Gpi-i isoenzyme.

Mouse C56 and mouse C61 each developed multiple tumours, all of which con-tained a single Gpi-i isoenzyme, but some of the tumours contained one isoenzymevariant and some the other. It is therefore clear that the presence of a single Gpi-iisoenzyme in the tumour is not simply a reflexion of the fact that cells with thisisoenzyme have a selective advantage; single isoenzyme tumours of different typesmay arise in the one animal.

PO Gpi-ia <-> PO Gpi-ib chitnaeras. Fifty-seven papillomas, 4 carcinomas and 6fibrosarcomas arose in 16 animals produced by amalgamating PO Gpi-iaa and POGpi-ibb embryos. These chimaeras were constructed to eliminate as far as possiblethe effects of any differences in the susceptibility of genetically different cell types(PO and CBA/HT6T6) to the carcinogens. Twenty-two of the tumours were Gpi-iaand 39 were Gpi-ib. The enzyme analyses on all the tumours arising in the POGpi-ia «-> PO Gpi-ib chimaeras are set out in Table 5.

Mice C69, C88, C94, C103 and C105 developed single isoenzyme tumours of bothtypes. In mouse C103 the epidermis was at least 80% Gpi-ib, which might accountfor the predominance of ' b ' type tumours. Tumour T356, however, was Gpi-ia.The amount of ' b ' type isoenzyme in this tumour was more than is usually found insingle isoenzyme tumours, but since the isoenzyme pattern of the normal tissues ofthis animal was heavily biased in favour of the ' b ' type isoenzyme, contaminationwith normal host tissue would contribute more ' b ' type activity than usual.

Tumour T322 in mouse C84 and tumours T339 and T340 in mouse C87 wereexplanted in an attempt to remove host cell contamination as far as possible. Cellularelements derived from the blood would not be expected to show much growth duringa short period of cultivation in vitro, and fibroblasts from adult mice do not initiallygrow rapidly either. The explants were grown for 6-9 days in the same bottle, andthe confluent cells were then harvested. Cell lysates were subjected to electrophoresis.The Gpi-i isoenzyme patterns were found to be the same as those produced by thehomogenates of the tumours, but the cell lysates contained 1 % or less of the minorisoenzyme variant. Tumour T375 on mouse C96 was a squamous cell carcinomawhich ulcerated and became secondarily infected. This tumour had infiltrated manynormal subcutaneous structures. The presence of both isoenzyme variants in this casemight therefore reflect infiltration by inflammatory cells or the presence of other hosttissues. Explantation of this tumour failed because of bacterial contamination of theculture. Figs. 3 and 4 show the Gpi-i isoenzyme banding patterns of typical tumours.

In all, 96 independent tumours were analysed. These included both benign andmalignant tumours of epidermal and mesodermal origin. In Table 6 the analyses of allthe tumours arising in both kinds of chimaera are summarized.

260 P. M. Ianmccone, R. L. Gardner and H. Harris

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Tab

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Per

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fro

m i

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est

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nen

t w

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w

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y

C56

M

T

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9b

a

=b

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5 1

2

28

b.p.

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T1

79

3

b I

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T

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o

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64

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5

I2

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ben

ign

pap

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ma;

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= b

asal

cel

l ca

rcin

om

a; S

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. =

sq

uam

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s ce

ll c

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nom

a; c

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= c

uta

neo

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fib

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ma;

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. co

al =

co

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2 o

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llom

as.

t T

hes

e tu

mo

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are

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of

prim

ary

tum

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rs w

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he

des

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tion

of

the

pri

mar

y t

um

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r is

sho

wn

in p

aren

thes

es.

Tab

le 5

. Ana

lysi

s of

turn

ours

in

chiin

aeri

c m

ice

PO

Gpi-la *

PO

Gp

i-~

b

Per

iod

from

ini

tiat

ion

01

,0

en

zym

e T

O

TO

Lon

gest

T

yp

e o

f G

pi-

I is

oenz

yme

pre

sen

t ac

tivi

ty p

re-

appe

aran

ce

tim

e of

di

men

sion

, A

,

sen

t as

min

or

of t

um

ou

r,

anal

ysis

, A

nim

al

Sex

T

um

ou

r m

m

Blo

od

Ep

ider

mis

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erm

is

Tu

mo

ur

com

po

nen

t w

eeks

w

eeks

H

isto

logy

c6

4

M

T2

61

9

a=

b

a=

b

b <

5

I 6

24

b

.p.*

T

26

2

6 b

I I 6

24

b.p.

* T

32

0

3O

b 5

I9

3 I

S.C

.C.

C66

M

T

18

9

3 a

<b

a

=b

b

I 5

I I

b.p

. T

26

8

4 b

I 2 5

27

b

.p.

T3

16

(T

18

9)t

7

b I

5 3 3

b

.p.

C69

F

T

23

8

10

a=

b

a>

b

a I

12

19

b

.p.

T2

59

6

a I

20

2 2

b

.p.

T2

60

4

a 1

20

2 2

b

.p.

Tz9

o (

T2

38

)t

3 a

<5

1

2

26

b.p

. T

z91

(T

26

0)t

5

a <

I

20

26

b

.p.

Tz9

2 (

T2

~g

)t

2

a I

20

2 6

b

.p.

T3

18

I

b I

-

28

b

.p.

c7

" F

T

31

9

7 a

=b

a

=b

a

I 21

3 3

b.p

.

c7

2

F

T3

27

5

a>

b

a>

b

a>

b

a <

I

2 2

2 5

b

.p.

T3

28

17

a

I -

10

fs.

C83

F

T

26

3

10

a>

b

a=

b

a>

b

a I

5 7

S.C

.C.

T2

88

(T

26

3)t

30

a

I 5

12

S

.C.C

.

T2

89

10

a 5

9 12

S

.C.C

.

C84

M

T

32

2

30

a=

b

a<

b

a<

b

b 5

12

1

0

fs.

T3

23

15

b

5 8

10

fs.

a7

M

T

33

9

2 5

a

=b

a

<b

a

=b

a

5 1

2

I 6

fs.

T3

40

I5

a

5 I4

I 6

fs.

(39

M

T2

65

1

0

a<

b

a<

b

b I

5 10

b.p

. T

26

6

3 b

I

9 1

0

b. p

.

Cellular origin gf turnours

Tab

le 5

(co

nt.)

N

OI

P

Per

iod

fro

m i

nit

iati

on

I

A

\

% e

nzy

me

To

L

on

ges

t T

yp

e o

f G

pi-

I is

oen

zym

e p

rese

nt

acti

vit

y p

re-

app

eara

nce

T

o ti

me

of

dim

ensi

on

, ,------

7

sen

t as

min

or

of t

un

lou

r,

anal

ysis

, A

nim

al

Sex

T

um

ou

r m

m

Blo

od

Ep

ider

mis

D

erm

is

Tu

mo

ur

com

po

nen

t w

eek

s w

eeks

H

isto

log

y

c9

4

F

T4

23

5

a

>b

a

=b

a

=b

b

<

I

24

38

b.p

. .b

T4

24

3

a

I I 6

3

8

b.p

. T

42

5

7

b

< I

I 6

3

8

b.p

. T

42

6

4

a+

b

10

O/,b

34

38

b

.p.

r,

R

T4

27

2

b

I 3

6 3 8

b

.p.

;S

T4

28

2

b

I I 6

3 8

b.p

. T

42

9

5

b

5

3 6

3 8

b.p

. b

I

I 6

b.p

. 1

T4

30

7

3

8

"

T4

3 I

7

b

5

24

3 8

b.p

. T

43

2

10

b

5

I 6

38

b

.p.

+' T

43

3

z b

I

24

38

b.p

. P

T

43

4

2

b

5

24

3 8

b.p

. 0

T4

35

2

b

I 24

3

8

b.p

.

c9

6

F

T3

74

2 I

a=

b

a<

b

a<

b

b

5 2

0

24

fs.

% Cb Y

T3

75

15

a

+b

2 2

24

S

.C.C

. D

I 0-2

0

%a

3

c9

9

M

T4

49

10

a=

b

a=

b

a=

b

b 5

15

2

8

b.p

. A

,

T4

50

7

b

5

14

28

b

.p.

T4

5 I

5

b

< I

27

28

b

.p.

3

T4

52

8

b

<

5

I 8

28

b

. p.

3 C

IO

~

M

T4

53

5

a

=b

a

=b

a

=b

b

5

8

28

b

.p.

Ce

T

45

4

4

b

< 5

2 3

28

b

.p.

T4

5 5

5

a

< I

2 5

28

b

.p.

C1

06

M

T

45

6

8

a=

b

a=

b

a=

b

b

< I

I

I 29

b

.p.

T4

57

2

b

< I

29

b

.p.

-

b.p.

= b

enig

n p

apil

lom

a; S

.C.C

. = s

quam

ous

cell

car

cin

om

a; f

s. =

fib

rosa

rcom

a.

t T

hes

e tu

mo

urs

are

rec

urr

ence

s of

pri

mar

y tu

mo

urs

wh

ich

wer

e ex

cise

d. T

he

des

igna

tion

of

the

pri

mar

y t

um

ou

r is

sh

ow

n i

n p

aren

thes

es.

f T

he

rea

sons

fo

r cl

assi

fyin

g th

is t

um

ou

r as t

ype

a ar

e gi

ven

in t

he

tex

t.

Tab

le 6

. Sum

mar

y of

glu

cose

pho

spha

te i

som

eras

e is

oenz

yme

patte

rns

in t

unw

ws

prod

uced

in

chh

eric

mic

e

Pap

illo

mas

C

arci

nom

as &

Sar

com

as

L I

> w

--

7

DM

BA

+ TP

A

MC

A +

TP

A

DM

BA

+ C

O*

MC

A*

D

MB

A + C

O+

r

A

\----

Y

Ty

pe

of c

him

aera

a

+b

a

b a

+b

a

b a

+b

a

b a-

tb

a b

a+

b

a b

Q G -

PO

tt C

BA

/HT

6T

6

1 (

2)t

21 (4

) 3

(1)

o o

o

o o

o

o 3 (

1)

0

o

0

1

5.

(5 a

nim

als)

I

I 0

%

PO

++

PO

4

14 (

3)

8 (1

) o

2

16

I I1

2 (

1)

I 3

3 (1

6 an

imal

s)

DM

BA

= 7

, 1

2 d

imet

hylb

enza

nthr

acen

e; C

O =

cro

ton

oil;

TP

A =

12-

0-te

trad

ecan

oyl

phor

bol-

13-a

ceta

te;

MC

A =

20-

met

hylc

hola

nthr

ene.

t

Th

e n

umbe

rs i

n pa

rent

hese

s re

fer

to a

ddit

iona

l an

alys

es m

ade

on t

umou

rs t

hat

recu

rred

aft

er s

urgi

cal

rem

oval

.


DISCUSSION

In the present series of 96 primary tumours and 13 recurrences following removal ofprimary tumours, all except 9 contained only one of the two electrophoretic variantsof the enzyme glucose phosphate isomerase. The smallest fragments of normalchimaeric tissue that could be analysed almost invariably contained both electro-phoretic variants. Of the 9 tumours showing both isoenzymes, 2 were recurrences oftumours that had originally shown only one. In the case of the remaining 7 tumourscontaining both isoenzymes, there is circumstantial evidence in support of the viewthat these tumours were either heavily contaminated with non-tumourous host cellsor that they resulted from the coalescence of 2 or more primary tumours. If thisevidence is accepted, then we may conclude that none of the primary tumours arisingin these chimaeric animals produced both electrophoretic variants of glucose phosphateisomerase.

In humans dimorphic for the X-linked enzyme glucose-6-phosphate dehydrogenase,the presence of only a single electrophoretic variant in spontaneous tumours has beentaken as evidence that the tumours are of clonal origin. The arguments in support ofthis conclusion have been extensively rehearsed (Gartler, 1974; Fialkow, 1976) andwill not be repeated here. However, the use of experimental chimaeras for the ex-amination of this question raises problems that do not occur in the interpretation ofthe data from heterozygous humans. In the latter, there is no reason to suppose thatthere would be differences in susceptibility to carcinogenesis between the cellsshowing one form of glucose-6-phosphate dehydrogenase and those showing the other;but in the experimental chimaeras this is not necessarily the case. The CBA/HT6T6animals are in general less susceptible to the induction of skin tumours than the POanimals. If this susceptibility resides entirely in the susceptibility of the individualcells, then one would expect that in the CBA/HT6T6 *-* PO chimaeras, tumourswith the PO phenotype, expressing only the PO form of the isoenzyme (Gpi-ia),would predominate. This was indeed found; but, even so, tumours of CBA/HT6T6type, expressing only the CBA/HT6T6 form of the isoenzyme (Gpi-ib), did arise inthese chimaeras. In the case of the PO (Gpi-ia) «-> PO (Gpi-ib) chimaeras, differentialsusceptibility of the 2 cell populations to the carcinogen is not a problem; the incidenceof tumours of Gpi-ia and Gpi-ib type reflected the proportion of the 2 cell types inthe tissues at risk. It could be argued that the presence of only a single isoenzyme inthe tumours merely reflected a selective growth advantage in the cells producing thatparticular isoenzyme; but the fact that in several cases single isoenzyme tumours ofdifferent types arose in the one animal make this argument very difficult to sustain.

Another important difference between humans heterozygous for an X-linked markerand the chimaeras used in the present experiments is in the distribution of the 2parental cell lineages in the normal tissues. It will be obvious that the strength of theargument for a single isoenzyme tumour being of clonal origin rests on the size of theclones that are normally to be found in the tissue from which the tumour arose. InX-linked human mosaics, since inactivation of the X chromosome is essentiallyrandom, about one half of the cells in any tissue would be of one type and one half of

Cellular origin of tumors 267

the other; and the clone size would be expected to be the same for the 2 cell types. Ifthen, one samples tissues in which both enzyme activities are present, the distributionof the ratios of the 2 activities will be binomial; and the power of the expansion thatbest fits the observed data will give an approximate value for the number of clones inthe sample. If there are significant differences in the sizes of the clones, the value givenby the binomial expansion will be an underestimate. Sampling errors in this situationbecome important only when the clone size approaches the sample size and the prob-ability of a sample containing cells of only one type becomes high. In the case of theexperimental chimaeras, the 2 parental cell types need not be equally represented inthe tissues; and there is, in fact, no way of predicting what the proportion of the 2cell types might be at any one site. This consideration becomes crucial when the dis-proportion between the 2 cell types in the tissue being examined is large.

'Patches' of cells of the one type may, of course, be formed either by single clonesor by the coalescence of like clones. West (1975) has shown that the probability of suchcoalescence, and hence of the formation of multiclonal patches in a tissue, is a functionof the proportions of the 2 cell types present. Algebraic solutions for the relationshipbetween the number of clones in a patch and the ratio of the 2 cell types present havebeen developed for large 2-dimensional arrays (the analysis of 3-dimensional arraysis much more complex and cannot satisfactorily be approached by the examination ofsmall samples). Since, to a first approximation, the epidermis can be considered as a2-dimensional array, West's analysis is relevant. For an array of 100 x 100 contiguoushexagons, it can be shown that a homogenous patch would contain on averageabout 25 clones, and this number would go up as the size of the array increased.

In the chimaeric tissues examined in the present study, the ratio of the 2 parentalcell types varied widely, so that the results could not be pooled; but a satisfactorytreatment of the data could be achieved by distributing the ratios into the classesshown in Table 3. As described above, the number of clones (n) in each sample canbe derived from the binomial expansion that best fits each distribution. This distri-bution is (p + q)n where p and q are the relative amounts of 'a ' type and ' b ' typeenzyme activity in the sample; and (n) can be estimated from the mean values and theirvariances:

xx p and s2 x —, so thatn

Pq u 9 S (x — x)2 .... .n =-$ where s* = —-. — (Wegmann, 1070).

s2 n — 1 \ o ? 1 1

The coarse grouping of the ratios tends to overestimate the variance, but this can beadjusted by Sheppard's correction (Kendall & Stuart, 1961). Thus,

2 _ £(*-3c)2 o-22

n'—i 12 '

where 0-2 represents the interval between groups. For the epidermis the valuesobtained for (n) are 11-9, 14-6 and 12-5 for groups 1, 2 and 3 respectively; for thedermis, these values are 15-8, 8-8 and 14-0. The average sample of epidermis in which


'a ' and ' b ' type activity are present in equal amounts therefore contains about 15clones; and, on average, there are about 1-7 samples per homogeneous patch. Bycounting the number of basal cells in a known length of normal epidermis, it couldbe estimated that the average sample contained 8800 cells, so that the average patchsize was about 15000 cells. The average clone size was thus about 600 cells, a figurewhich agrees reasonably well with other estimates in the literature.

The 25 small samples of subcutaneous tissue contained equal amounts of the 2isoenzymes, so that clone and patch size in this tissue are likely to be much the same as,or smaller than, those calculated for the epidermis. However, subcutaneous tissuecontains a variety of structures so that the samples taken might not have representedhomogeneous tracts of tissue. In any event, small pieces of tissue immediatelyadjacent to the tumours were examined at the same time and these invariably showedthe presence of both isoenzymes.

If the tumours showing both forms of the glucose phosphate isomerase isoenzymedo not represent artifacts, then their number may be used to calculate the maximumnumber of cells in the chimaeric tissue from which the tumours can have originated.Hutchison (1973) has developed a model for a circular sample of radius (r) randomlylocated in a field composed of contiguous hexagonal clones. If r is less than onehalf of the length of the hexagon, and the 2 cell types are present in equal proportionsthen the proportion of samples containing cells of both types,

Pm l x = —o-O3iir2+i-i547r (Friedman & Fialkow, 1976).

The analysis can also be applied to contiguous squares or contiguous triangles.If one treats the epidermis as a 2-dimensional system and limits the analysis to those

animals in which the epidermis contains both isoenzymes in approximately equalamounts (Class 2, Table 3), Hutchison's model can be applied. Of 50 epidermaltumours arising in such animals, 6 were considered to be of mixed isoenzyme com-position, so that Pm l x is 0-12, which yields a value for (r) of 0-1042. Thus, to give 6mixed tumours out of a total of 50, the required ratio of target size to clone size (thearea of the circular sample/the area of the hexagonal clone) is o-oi 31. We have calculatedthat there are about 600 cells in an average clone, so that the size of the target (thenumber of cells giving rise to the tumours) is about 8. This figure represents an upperlimit, for some of the tumours we have scored as mixed may have shown both iso-enzymes for artifactual reasons such as host cell contamination or coalescence of 2or more primary tumours. Moreover, Hutchison's analysis is based on clones ofregular and simple shape, whereas in 3-dimensional reality the shapes of the clonesmight be more complex (for example, clones might interdigitate); and this wouldfurther reduce the target size. We can therefore conclude that, at the very least, thechemically induced tumours studied in the present experiments arose from very fewof the tissue cells exposed to the carcinogen. The most probable interpretation of thedata is that these tumours, like the spontaneous tumours studied by similar methods inhumans, are clonal in origin.


We thank Dr F. H. C. Marriot for help with the statistical analysis and Mr Gareth Plantfor technical assistance. P.M.I, was a recipient of Grant GM02050 from the National Instituteof General Medical Sciences as a trainee of the Department of Pathology, College of Physiciansand Surgeons of Columbia University. The work was supported by the Cancer ResearchCampaign.

REFERENCES

CARTER, T. C , LYON, M. F. & PHILLIPS, R. J. S. (1955). Gene-tagged chromosome trans-locations in eleven stocks of mice. J. Genet. 53, 154-166.

CATTANACH, B. M. (1975). Control of chromosome inactivation. A. Rev. Genet. 9, 1-18.CHAPMAN, V. M., WHITTEN, W. K. & RUDDLE, F. H. (1971). Expression of paternal glucose

phosphate isomerase-i (Gpi-i) in preimplantation stages of mouse embryos. Devi Biol. 26,153-158.

D E LORENZO, R. S. & RUDDLE, F. H. (1969). Genetic control of two electrophoretic variants ofglucose phosphate isomerase in the mouse (Mus musculus). Biochem. Genet. 3, 151—162.

FIALKOW, P. J. (1976). Clonal origin of human tumours. Biochim. biophys. Acta 458, 283-321.FORD, C. E., HAMERTON, J. L., BARNES, D. W. H. & LOUTIT, J. F. (1956). Cytological identi-

fication of radiation-chimaeras. Nature, Lond. 177, 452-454.FRIEDMAN, J. M. & FIALKOW, P. J. (1976). Viral ' tumorigenesis' in man: cell markers in

Condylomata acuminata. Int. J. Cancer 17, 57-61.GARDNER, R. L. (1968). Mouse chimaeras obtained by the injection of cells into the blastocyst.

Nature, Lond. 220, 596-597.GARDNER, R. L. (1978). Production of chimaeras by injecting cells or tissue into the blastocyst.

In Methods in Mammalian Reproduction (ed. J. C. Daniel). New York: Academic Press.(In press.)

GARTLER, S. M. (1974). Utilization of mosaic systems in the study of the origin and progressionof tumors. In Chromosomes and Cancer (ed. J. German), pp. 313-334. New York: Wiley.

GARTLER, S. M., GANDINI, E., HUTCHISON, H. T., CAMPBELL, B. & ZECHHI, G. (1971).

Glucose-6-phosphate dehydrogenase mosaicism: utilization in the study of hair folliclevariegation. Ann. hum. Genet. 35, 1-7.

HUTCHISON, H . T . (1973). A model for estimating the extent of variegation in mosaic tissues.J. tlieor. Biol. 38, 61-79.

KENDALL, M. G. & STUART, A. (1961). The Advanced Theory of Statistics, vol. 1. London:Griffen.

LINDER, D. & GARTLER, S. M. (1965). Glucose-6-phosphate dehydrogenase mosaicism:Utilization as a cell marker in the study of leiomyomas. Science, N. Y. 150, 67-69.

LYON, M. (1961). Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature,Lond. 190, 372-373.

MINTZ, B. (1962). Formation of genotypically mosaic mouse embryos. Am. Zool. 2, 432.NESBITT, M. N. (1974). Chimaeras vs X inactivation mosaics: significance of differences in

pigment distribution. Devi Biol. 38, 202-207.NICOLSON, G. L., YANAGIMACHI, R. & YANAGIMACHI, H. (1975). Ultrastructural localization

of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs.J. Cell Biol. 66, 263-274.

OHNO, S., POOLE, J. & GUSTAVSSON, I. (1965). Sex-linkage of erythrocyte glucose-6-phosphatedehydrogenase in two species of wild hares. Science, N.Y. 150, 1737-1738.

SOLTER, D. & KNOWLES, B. B. (1975). Immunosurgery of mouse blastocyst. Proc. natn. Acad.Sci. U.S.A. 72, 5099-5102.

TARKOWSKI, A. K. (1961). Mouse chimaeras developed from fused eggs. Nature, Lond. 190,857-860.

WEGMANN, T. G. (1970). Enzyme patterns in tetraparental mouse liver. Nature, Lond. 225,462-463.

WEST, J. D. (1975). A theoretical approach to the relation between patch size and clone sizein chimaeric tissue. J. theor. Biol. 50, 153-160.

YOSHIDA, A. (1967). A single amino acid substitution (asparagine to aspartic acid) betweennormal (B + ) and the common negro variant (A + ) of human glucose-6-phosphate dehydro-genase. Proc. natn. Acad. Sci. U.S.A. 57, 835-840.

{Received 12 August 1977)

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