Pathogenesis of heterogeneity in human multinodular goiter A study on growth and function of thyroid tissue transplanted onto nude mice

Pathogenesis of heterogeneity in human

multinodular goiter. A study on growth and

function of thyroid tissue transplanted onto

nude mice.

H J Peter, … , H Studer, S Smeds

J Clin Invest.

1985;

76(5)

:1992-2002.

https://doi.org/10.1172/JCI112199

.

Functional and morphologic heterogeneity of human multinodular goiters was investigated

in 300 samples from "cold" and "hot" regions of 20 goiters transplanted onto nude mice.

Transplants were labeled with [3H]thymidine and radioiodine, while the host's

thyroid-stimulating hormone (TSH) secretion was either stimulated or suppressed. Proliferation and

function of follicular cells were assessed in whole follicles reconstructed from

autoradiographs of serial sections. Hot transplants had a higher autonomous iodine uptake

than those of cold tissue in TSH-suppressed hosts. Functional autonomy widely varied

among the follicles, but even more so among individual cells. Hot grafts differed from cold

ones only by a comparatively larger fraction of autonomous cells. Intercellular differences of

iodinating activity were not abolished by TSH. Grafts faithfully reproduced the individual

growth pattern of the original tissue. Between 0.5% and 7% of all follicular cells replicated

despite suppression of TSH. Up to 70% of these cells were clustered, forming scattered foci

of autonomously growing tissue. Other cells only started replicating after long-term TSH

stimulation. Thus, goiters contained subsets of cells with high and others with low growth

response. Progenies of replicating cells remained clustered, sometimes budding outwards

to form new follicles. Autonomy of growth and autonomy of function are independent traits of

epithelial cells. Epithelial cells have their individual growth pattern, replication rate, and

functional capacity. These traits are passed […]

Research Article

Find the latest version:

http://jci.me/112199/pdf

Pathogenesis of Heterogeneity

in Human Multinodular Goiter

AStudyonGrowth and Function of Thyroid Tissue Transplantedonto Nude Mice

HansJ. Peter, Hans Gerber, Hugo Studer, and Staffan Smeds

Department ofInternal Medicine, UniversityofBern, Inselspital, CH-3010 Bern, Switzerland;DepartmentofSurgery,

University Hospital, S-581 85Link6oping, Sweden

Abstract

Functional andmorphologic heterogeneityofhumanmultinodular goiterswas investigated in 300 samples from "cold" and"hot" regions of 20 goiterstransplantedonto nude mice.Transplants werelabeled with[Hjthymidineand radioiodine, while the host's thyroid-stimulating hormone (TSH) secretion was either stim-ulated orsuppressed.Proliferation and function of follicular cells wereassessed in whole folliclesreconstructed from autoradio-graphs of serial sections.

Hottransplants had a higherautonomousiodine uptake than thoseofcold tissue in

TSH-suppressed

hosts.Functional auton-omywidely varied amongthefollicles,but even more so among individual cells. Hot grafts differed from cold ones only by a comparativelylargerfraction ofautonomous cells. Intercellular differences ofiodinating activitywerenotabolished byTSH.

Grafts faithfully reproduced theindividual growth pattern oftheoriginal tissue.Between0.5%and 7%ofallfollicularcells replicated despite suppression of TSH. Up to70% ofthese cells wereclustered, forming scattered foci of autonomouslygrowing tissue.Other cells only started

replicating

after long-term TSH stimulation. Thus, goiters contained subsets of cellswith high andothers with lowgrowth response. Progeniesofreplicating cellsremainedclustered, sometimes buddingoutwards to

form

newfollicles.

Autonomyofgrowth andautonomyof functionare indepen-denttraitsof

epithelial

cells.

Epithelial

cellshave

their individual

growth pattern, replicationrate, andfunctionalcapacity. These traitsare passed on froma mother cell to its progeny

during

follicle

neogenesis.

Tothis main mechanism

accounting

forthe morphologicand

functional heterogeneity

ofhuman

goiters,

in-heritable modifications ofgene

expression

must

probably

be added.

Introduction

Thesinglemost

intriguing

aspectofhuman multinodular

goiter

is the

striking

functionaland

morphologic

heterogeneityamong newly

generated

follicles

(1-3).

In

previous

studieson

experi-mental

goiters

in

animals,

wehaveconcluded thattwo

entirely

Partof this studywaspresentedatthe 14th AnnualMeetingof the Eu-ropeanThyroidAssociation,Rotterdam,TheNetherlands,inSeptember 1984andwaspublishedasanabstract(1984.Ann. Endocrinol. Paris. 45:81),and partwaspresentedatthe 9th InternationalThyroid Congress, SaoPaulo, Brazil,inSeptember 1985.

Dr. Gerber isaSpecialFellow of the Kamillo EisnerStiftung, Her-giswil, Switzerland.

Receivedfor

publication 19 June1985.

different setsof mechanisms may account for part of this het-erogeneity. On theonehand,changes of follicular function may result fromtheimpact of environmentalfactors such as iodine supply (4, 5),failingblood supply in some areas ofgrowing goiters (6), or aging ofcell components (5). On theother hand, the generation ofnewfolliclesfrom genetically different cells of the mother follicle was shown to be a second, basically different mechanism that generates heterogeneity among the newly formed follicles (3).However, these two processes cannot possibly account for all characteristics ofmorphologic and functional variegationobserved in humannodulargoiters. Among the still unexplained features are regionally variable metabolic abnor-malities(7), abnormal growth patterns,and theappearanceof autonomyofgrowth andofiodinemetabolismin many goiters. Serial studieson humangoiters have so far been severely limitedby the lack of animal models reproducingthe growth pattern ofhuman nodular goiters. Thisdifficulty has recently been overcome in that humanthyroid tissuecan now be grown through transplantationontonude mice (8-11). Moreover, be-cause human tissue is sensitive to mouse thyroid-stimulating hormone

(TSH),'

growthandfunction of goiter transplantscan be studied in the presence and absence of stimulators such asTSH.

Theinvestigationsreported here areaimedatproviding in-sight into the mechanisms that amplify-duringtheprocessof transformation of the normal human thyroid into a nodular toxicornontoxic

goiter-the

natural

heterogeneity

ofthenormal thyroid

epithelium

and create new

morphologic

and functional

diversity

amongthe follicular cells. In

particular,

the study

of

humangoiter growth in hostmice shouldallow toclearly dis-tinguish between inbornandenvironmental mechanisms.

Methods

Thyroidtissue from 20patientswithmultinodulargoitersandfromone

patientwithneoplasticdiseaseof the larynx,undergoing thyroidsurgery,

wastransplantedonto150 nude(nu/nu) ICR mice, kept under pathogen-freeconditions ina 12-hlight, 12-hdarkcycle. The animalswerefed with cereal-basedmousebreedingdiet(NafagAG, Gossau, Switzerland) sterilizedby irradiationwith 2.5megarads.

Transplantationprocedure. Thyroidtissuesurgicallyremoved from

thepatientwas immediately immersed into ice-cool Eagle'sminimal essential mediumcontaining 100 IU ofpenicillinand 100 ,g of strep-tomycinperml(Serva,

Feinbiochemica,

Heidelberg,FederalRepublic ofGermany).Specimensselected fortransplantationwerecutinto

frag-mentsmeasuringabout 3 X 1 X 1 mm. Threefragmentsweregrafted subcutaneouslyunder the skinoneach side of the vertebralspine.Goiter specimensfromscintigraphicallyhotgoiterareaswereimplantedonthe left,specimensfrom coldgoiterareas ontherightside.

Labelingwith[3H]thymidine,

125j,

and13)ILTostudytheimpactof host TSH suppression and TSHhypersecretion oniodine metabolism

1. Abbreviations usedin this paper:FLC, fractionof

[3H]thymidine-labeledcells; MMI, methimazole;PAS,

periodic

acid-Schiff;T4,

L-thy-roxine;

TSjI,

thyroid-stimulatinghormone.

J. Clin. Invest.

© TheAmerican Society forClinicalInvestigation,Inc. 0021-9738/85/11/1992/11 $1.00

andgrowth, animalsgrafted3-8 wkpreviouslywith tissue fromasingle goiter,weredividedintotwogroups, each including three to five animals. Subsequently, group I received L-thyroxine (T4) (Fluka AG, Buchs, Switzerland) 0.5

/Ag/ml

in its drinking water, resulting in a daily T4 intakeof~-2.5

1Ag

peranimal, whereas, for group II, methimazole (MMI) (Fluka) 0.075% and sucrose 1% were added to the drinking water. The goiter transplants of additional animals remaining untreated, were pro-cessedforhistologic examination only.

Proliferating cells were labeled byinjecting[3H]thymidine (New En-gland Nuclear, Boston, MA,specificactivity 15 Ci/mmol) i.p. three times daily for 2wk,beginning 1 wk after initiation of the different feeding regimens. Inoneexperiment comprising eight animals, [3Hjthymidine wasadministered continuously by means of miniosmotic pumps (Alzet model 2001, Alzo Corp., Palo Alto, CA) implanted into the peritoneal cavity.Inexperiments involving labelingwith1251Ior

3'I,

MMItreatment

(groupII)wasstopped2d before iodine administration, whereas the T4 feeding(group I)wascontinued.Forinvestigatingiodine metabolism in transplants concomitantly labeled with [3H]thymidine, 50,ACiof ' 'I wereinjected intraperitoneally 1 hbeforesacrifice by chloroform

anes-thesia. Animalsnotlabeled with[3H]thymidinebutotherwiseidentically treated were given 125I because of its more suitable autoradiographic properties.

The transplantswerecarefullyfreed fromadjacent connectivetissue andweighed before fixation. 1251 and 1311 uptakewere measured ina

gamma counting system (MR 480, Kontron,Zurich, Switzerland)and calculated permilligramof transplantweight.

Histologic and autoradiographic procedures. Tissue fixed in 4%

phosphate-bufferedformaline, pH 7.4,wasdehydrated inaseries of al-cohols and embedded in methacrylate (JB-4embedding mixture, Poly-sciences Inc.,Warrington, PA). Forautoradiography,series of upto40 3-,um sections pertransplant weremountedonglassslides,dipped in Kodak NTB-2nuclear emulsion (Eastman Kodak Co., Rochester, NY) and,afterappropriateexposuretimes, developed with Kodak D-19 de-veloper. A mean of 30 serial sections were then counterstained with nuclear fastred, and additional sectionswerestained withperiodic acid-Schiff(PAS).Inordertocorrelateiodine organificationof the follicles with cell proliferation, tissue specimens double-labeled with 13'I and [3H]thymidinewereprocessed as follows: all odd-numbered sections of

aseries wereprocessed immediately for '3'I-autoradiographs, whereas the even-numbered sections were dipped4mo later for[3H]thymidine autoradiography. At that time'"'Iradioactivity had virtually disappeared. Tissuespecimens labeled with[3H]thymidineor125Ialone were processed without delay.

Becausethe grain density in identically exposed autoradiographs di-rectly reflects the relative amount of radioactive iodineorganified, semi-quantitative autoradiography could be done on sections from the left-and theright-sidedtransplants grown in the same animal. In the same way, the relative radioactivity of transplantsfromgoiter samples derived from the same patient but growing in differently treated animals was compared.

Assessment of the fraction and the distribution pattern of [3H]thy-midine-labeledfollicular cells within a transplant. In transplants labeled with[3H]thymidine,20 cell groups from different regions were evaluated, eachgroup comprising 100 follicular cells. Cells containing five or more silvergrains per nucleus were considered as labeled. The fraction oflabeled

cells(FLC)of a transplant is given as the mean value of the 20 samples.

Todetermine whether or not the distribution of the labeled cells among the sample groups corresponded to a random distribution, x2 testing was applied according to Riedwyl(12).

The pattern of distribution of the labeled cells within the shell of individualfollicleswasinvestigated by reconstructing entire follicles from contiguous serial sections by means ofaprojection microscope (Visopan, Reichert-Jung, Vienna, Austria).

Results

Histologic

structure

_of

the

_original

_goiter

andits

_growing

trans-plant.

Outof the 300

_transplants

_examined,

250contained

well-preserved thyroid tissue whereas 50 transplants consisted pre-dominantlyof scarred connective tissue and weretherefore ex-cluded from further investigation.Thehistologicpatternof grafts from multinodular goiters grown in untreated mice was usually indistinguishablefromthatofthe mothertissueobtainedat sur-gery. In other words, solid mother tissue alsogrew in a solid patternafter transplantation, whereas transplantsderived from microfollicular goiter tissue still consistedof tiny follicles. Even-tually, large follicles predominated in transplantsfrom macro-follicular tissue (Fig. 1).

Ingrafts from normal thyroid tissue and in those fromcolloid goitersgrown in MMI-treated hosts, the majority of follicles were lined by epithelial cells with columnar shape andcontained thin, faintly PAS-stainingcolloid. Occasional follicleswere sur-rounded by flatepithelialcells andcontained strongly PAS-pos-itivecolloid,suggestingunresponsivenesstoTSH. The shell of afew follicles consisted ofamosaic of both columnarand flat epithelial cellsarrangedinclusters,indicatingthat morpholog-ically responsive andunresponsivecells may coexistinthesame follicle.

In grafts from T4-treated mice, follicles with flatepithelia containing intensely PAS-stained colloid were predominant. Thus, humangoiter tissue respondstoendogenousmouseTSH bycharacteristic structural changes (Fig. 2).

Effects

ofT4-andMMI-treatment on

proliferation offollicular

epithelia. T4 treatment in doses knowntosuppress hostmouse TSH secretion (13) did notentirely suppress cell proliferation within the transplanted follicularepithelia.Thesize ofthe FLC in transplantsderived from goiters exposed to

[3H]thymidine

for2 wkranged fromaminimum of0.5% intransplants from euthyroid nodulargoiterto 7% intransplants from a steadily growing goiter ofathyrotoxic patient (Fig. 3). Largevariations were observed between transplants takenfrom different regions ofthesamegoiter.

Extending the duration of MMI treatment before [3H]-thymidine administration increasedthe FLCwithin the follicular epithelium in atime-dependent manner. An exampleis given in Fig.4.

Regionalvariationof TSH-independent and TSH-mediated goiter

growth.

Intransplants growing in mice with T4-suppressed TSH secretion, the number of labeled cells per 100 follicular cellsvariedamongthedifferent regions ofthe sametransplant from0 to20.Similarregional variations were observed in trans-plants growing in animals with MMI-induced TSH hypersecre-tion. x2testing (P< 0.001) confirmedthe optical impression that thelabeled cells were not randomlydistributedamong the sampled regions ofagiventransplant. Very denseclustering of labeled cells in a few distinct regions strongly suggested that somegoiterareasdisplayanexceedingly high propensityto pro-liferate.

Lackofcorrelation between cell morphology andreplicating potential.Tallcuboidalorcolumnar cells may remain unlabeled, whereas entirely flat epithelial cells may readily incorporate the [3H]thymidinelabel(Fig. 5). Thus, the claim widely propagated in theliteraturethat flat epithelial cells are inactive should not onlybe abandoned for iodine metabolism (1)but also for cell proliferation.

Distribution of the

[3H]thymidine-labeled

cells within the

follicular

shell. Three-dimensional reconstruction of follicles from contiguous serial sections across entire transplants from T4-treated animals containing a FLC ranging from 0.5% to 2% revealed that up to 70% of all cells within sets of 500

Figure 1. Twodifferent specimens ofhumangoiter tissuedisplayinga ter transplantation onto a host mouseisnearlyindistinguishable from microfollicular (A) andanormofollicular growthpattern(B). The his- thatoftheoriginal tissue(Aand B).

tologicappearanceof thecorrespondingtransplant (CandD) 8wk

af-[3H]thymidine-labeled

cells werearranged in contiguous clusters comprisingbetween4and 40 cells.Five sets of 500 labeled cells infive different transplants were evaluated.

In afew individual follicles of transplants growing in T4-treatedmice,nearly allcells werelabeled whereas the large ma-jorityof allfolliclesdid not contain any labeled cells at all.

Infollicles of MMI-treated transplants, the [3H]thymidine-labeled cells were much more numerous than in the T4-treated counterparts (Fig. 3). Again, they strongly tended to form co-herentirregularly shaped patches whereas large parts of the fol-licular shell contained no labeled cells at all. A true-to-nature reconstruction of a labeled follicle is depicted in Fig. 6. The presentobservations in human goiter tissue are in full agreement with an earlier report on experimentally produced mice goiters(3).

Thefindingsindicate that all cells of the follicular epithelium do not possess the same intrinsic growth potential, but that there are subsets ofcells with a much higher than average inborn propensity toproliferate. The growth advantage appears to be conveyedfrom mother to daughter cells, in that the newly gen-erated cells closely stick together to form large families (Fig. 5-7).

Other

[3H]thymidine-labeled

organs than thethyroid gland werealsoexamined in all mice,includingesophageal and tracheal epithelium, salivary glands, liver, adrenal glands, andkidney. Noconvincingevidence forclusterformation of dividing cells

wasfound so far except perhapsfor the adrenal cortex and the renal tubularepithelium (Peter, H. J.,unpublishedobservation). In some follicles the proliferating cells formed papillary structures,protruding into the lumen. Theunderlaying connec-tive tissue also contained many[3H]thymidine-labeledcells. In otherfollicles, replicatingcell clustersformedsproutingcell buds protruding toward the interstitial space in much thesameway aspreviously describedinexperimentallyproducedmice goiters (Fig. 7) (3). Serialsections ofradioiodine-labeledorPAS-stained transplants often revealedtinylumina within thesebuds, indi-catingthattheyaretheearly stagesofnewfollicles.

Interfollicular and intercellular heterogeneityof iodine

or-ganification

in

suppressedfoicles.

Residual iodine

_{organification}

remaining after TSH suppression aswidelyvaried among dif-ferent follicles as that observed in autoradiographs of human goiters labeled invivo (3). Aregionally variable fraction ofall folliclesdisplayed highautonomousiodineuptake,whereas other follicleshadintermediateorlowuptake.

Figure2.Two transplants derived from thesame

euthyroid multinodular goiter. Transplant A, grownin athyroxinetreatedhost,isbuiltupby follicles lined byflatepithelial cells and

contain-ing abundant, stronglyPAS-positive colloid. In contrast,transplant B grown inaMMI-treated animal contains mostly follicles surrounded bya

columnarepithelium and colloid which stains only weakly for PAS. These micrographs

illus-tratethestructural response of human goiter

tis-suetovariationsof mouseTSHsecretion.

highernumberofautonomouslyfunctioning follicles than those ofcold nodules orfromnormaltissuegrown inthe sameanimal (Fig. 9).This purely quantitative characteristic was the sole and only difference that could be observed between a transplant grown outof cold mother tissue and that generated by a sample fromahotgoiternodule. Inconfirmation of previous observa-tionsin humangoiterslabeled invivo,noconsistent structural differencesweredetected between goiter tissues with genuinely highorlowiodineturnover(1-3, 15).

Stimulation ofthe host's TSH secretion byMMItreatment strikingly increased radioiodine organification not only in the transplants derived from hot nodules but also in those taken from cold goiter nodules (Fig. 10).Therefore, both tissues were responsive to TSH. There were, however, a few follicles even in TSH-stimulatedgoiters that remained almost entirely cold. These folliclesmaybe thehuman counterpart of the irreversibly cold follicles arisingin theagingmousethyroid through gradual failure

ofendocytosis (5). The large differencesof1251Iuptake between the individual follicles of hot as well asbetween those of cold transplants growing in T4-treated animals were invariably di-minished by TSH stimulation, but they were never completely abolished. This observation suggests that the rangeof TSH re-sponsiveness is similar in cold and hot follicles, but that the lowestintrinsic activity remaining after TSH suppression is in-dividually different.

Correlation offolliculargrowth andfollicular function. In goiter grafts grown in T4-suppressed hosts, [3H]thymidine-labeled cells were present in follicles with high as well as in those with low'3'Iuptake(Fig. 11).There was no correlation between ahighreplication rate of a given cell cluster and its1311uptake. Some hot follicles contained numerous labeledcells,whereasin other,identicallyhotfollicles, onlya few or noreplicating cells werefound. The lackofcorrelationbetween autonomous growth andautonomous function wasconfirmed in cold folliclesand

A;s w

t

ta.

Or

i~~~~~~~~~~~

B C

Figure 3.Effects ofT4and MMItreatmentonproliferationof the

fol-licularepithelium. This figureshows the effects of T4(v)andMMI(U)

treatmentonthe fraction of labeled cells(FLC)intransplantsderived

fromanormalthyroid (A),atoxic nodulargoiter (B),andaeuthyroid multinodular goiter (C),labeled with[3H]thymidinefor 2 wk. In all MMIgroups,theFLC ishigherthan in thecorrespondingT4-treated

counterparts.Thisprovesthat mouse-TSH doesinfactstimulate cell

proliferation of the human follicular epithelium. However, suppressive

T4treatmentdoesnotcompletelyabolish proliferation within the

fol-licular epithelium neither in normalnoringoitertissue.Rather,some

cellsproliferate autonomouslyinthe absenceof TSH.Notethatin

goiterBthe fraction ofautonomouslydividingcellsisseveral times

higherthan in the normalgland albeit with wideregionalvariations.

The fraction of autonomously replicating cellsisindependentof the function of the mother tissue. Barsrepresentmean±SEM.n=

num-ber oftransplants.

infollicles with intermediate iodine uptake. Therefore, cells with

ahigh degreeofautonomyof iodineuptakeandorganification

arenotnecessarilyidentical withthose proliferatingatthehighest

rate.On the other hand,somecells of multinodular goiters might

beautonomous inbothrespects. Autonomyofgrowthand of iodination areindependent, albeit notmutually exclusive cell properties(Fig. 11).

f

_

_<*

~~~~'

~ 0 4

.?.

20

pm

*.4

0

L_ I

Figure 5. Goiter transplantgrowninanMMI-treatedanimal labeled

with[3H]thymidinefor2 wk. Thefollicleatright, lined by columnar

epithelial cells,containsasmallfamilyof labeledcells,whereasthe large majority of follicularcellsareunlabeled. In thefollicleatleft,the

majority oftheflat epithelialcells haveincorporatedthethymidine

la-bel. Thiswasconfirmedonserial sections. Thus, there isno

correla-tion whatsoeverbetween theshape ofafollicular cellanditsresponse

togrowthstimulation. Evenextremelyflatcellsmayhaveahigh

pro-pensitytoreplicate.

fraction of labeled follicularcells

n=4 6

0 1 2 3

weeksofTSH prestimulation

Figure4.Effect of duration of MMItreatmentpreceding [3H]thymidinelabeling.The fraction ofproliferatingcells in

trans-plants of this euthyroid multinodular goiter increases with the

dura-tion of TSHstimulationprecedinginitiation of[3H]thymidine admin-istration.Transplanted samplesofthesamegoiterwerelabeledwith

[3H]thymidinefor 2 wk.Group1wastreated withT4;ingroups

2,3 and 4 MMItreatmentwasstarted 1, 2,and 3 wk,respectively,

beforeinitiation of[3H]thymidinelabeling (n=number oftransplants

per group.Bars representmeanvalues±SEM. Forcalculation of the

fraction oflabeledcells,seeMethods).Withincreasingstimulation

time,morecellsarerecruitedoutof theGI-pooltoenter themitotic

cycle.

Figure6.Model ofafollicle fromatransplant,grownfor6 wkinan

MMI-treated animal labeled with [3H]thymidine for2wk,

recon-structedfromacompletesetof 32 contiguousserialsections. The

pro-liferatingcells(darkly colored)arenotscatteredrandomly throughout the follicle but rather formlarge cohorts, whereas largeareasof the

follicular shell containonlyresting cells (lightly colored). This

distribu-tionpattern suggeststhepresenceofsubpopulations with distinctly

dif-ferentsensitivitytogrowthstimuli.

20-% fractionof labeled follicular cells

10-01

n=8

n=8 8 7 5 4 4

A

f*

..A

AkT..

ft.l

`. I

.4%

N

O.-k

i.tl-.'

:.`,

..v

4%

%

.4.

%..--r

.0

....

0:0:.:.:

ALA

-AF

,,20

_os-a

m

_-

-

_**:WF

t

.4

w

These observations were confirmed in [i3iI]- and [3H]-thymidine-labeled thyroid tissue grown in MMI-stimulated mice. Again, follicles with a high iodine uptake did not necessarily containalargefractionofdividingcells, andfollicles with a low

'I'I

uptake wereassociated with either a large, intermediate or low fraction of

[3H]thymidine

labeled cells. Findings such as those illustrated in Fig. 11 suggest that TSH stimulation does notaffect growth and function in all follicular cells to the same extent.Rather, some cells tend to respond predominantly with anincreaseofiodine uptake and iodine organification, whereas others morereadily enter the mitotic cycle. Some cells are able torespondwith bothfunctions to the same extent.

Discussion

Previousstudies inanimals and in surgically removed human goiterslabeledpreoperativelywith 125I haddisclosedtwobasically

X..

/%g's9',4',,

All'*i~t <;

4

,,, CA

,

b

h2.

i

> ,A *Db * t S ~

Figure 7. Follicle inahumangoitertransplant, grown inaMMI-treated hostmouseandlabeled with[3H]thymidinefor 2 wk. Onefamilyofheavily labeled cells forms a solid bud which protrudes out of thefollicular wall.Asinmousegoiters(3), the bud isconsideredto be anearly stage ofnewfollicle formation.

different mechanisms involved in the pathogenesis of the in-triguing heterogeneity of growth and function characterizing this thyroid disease (1, 2). One mechanism was the generation of newfolliclesfrom single cells of an epithelium that is in itself composed ofcellsfrom multiple, nonidentical subpopulations (3), and the otherwastheimpact ofenvironmental factors in-terfering with follicular function during goiter growth (4, 5). Amongthe localfactors causing heterogeneity of goiter growth are, e.g.,thefocalnecrosessubsequenttothe failureofthe cap-illary sprouts to adequately supply newly formed follicles. The deadtissueis gradually transformed into fibrousscarsforcinga nodular growth pattern upon theexpanding follicle population (6).Anotherlocalprocessisthe slow transformation ofnormally functioning follicles into irreversibly cold ones in aging mice thyroids (5). Eventually,the amountof iodineavailableduring goitrogenesisisincreasingly recognizedtohave a crucialimpact notonly on TSH secretion but also on the size and the function

b,1

Figure 8. Thisautoradiographof a is ,-VX.A goitertransplant grown inaT4-treated

of,; & X animal and labeled with

1251

forI h

9;

_;- showsthatautonomousiodine organi-* - US jji fication isnot aproperty of the follicle

as awhole but ratherofsinglecellsor

g

<

cellgroupswithin thefollicular wall.

Allhot and cold cellfamilies shownin

Of4

' so If, ', ' thefigurewerefollowedonserial

sec-*

.. tions. Some of them extendedover

En

At

* > Ilarge* areasof the follicular shell. The strikingdifferences ofautonomous io-dineorganificationamong different .,>*a;^ subpopulationsofgoiter epithelialcells

Ai * may be maskedbytherapidcolloid mixing(14)appearing,e.g., insome

v

follicles shownat_rightandatleft in thefigure.

Pathogenesis ofHeterogeneityinHumanMultinodularGoiter 1997

.-jwt__ I

_-f

F

.;"- 'A

.Awl

0

1

k

4

,4.

4

L--I

.i

I%

'k

qs

~ v~

>'s~~~tX

;;

~

Ai

¾ * '. '.;',.Ses. ....

4GiP%,rt~

4/t R'~- ~ W

4.

e F

... J~4fi.

4-¢ e

/

I'

*s

40~ ~ 41

va

t;Jj£ +*ZZ:

vi

44,~~~~~~~~~~4'

Ai v .

s 3,Xi ijjF|

F # SIF~~~~~t;8vc

0Ni

W.

,.A, 4'

0.4AV .

A.-Bulk

W. A

1%

* -,

.1w

i

.li

I

a

j

._ 4~t:' 'ow

By-

,/\N~~"'a

V.'¢"' '

Figure 9. (A) Anautoradiographed transplant derivedfrom a scintigraphi-cally hot goiter area, labeled with

1251I

forIhwhile mouse TSH was sup-pressed by T4 treatment.Follicleswith

ahigh capacity forautonomousiodine organificationarepredominant,but follicleswithlow orintermediate up-takearestill present.(B) Transplant takenfromascintigraphicallycold area of the samegoiter.Because it grew in the same animal and was ex-posedunder identicalconditions,the densityof the silvergrainsindicates therelativeuptakeof1251 byeach folli-cle.Follicleswith lowautonomous io-dineorganification are now predomi-nant, but aminority offolliclesis highlyactive. Note,incidentally,that hot and coldfolliclesare morphologi-cally indistinguishable.

of individual follicles (5, 16). Itcould, however,

altogether

not be

expected

that the

interplay

of these mechanisms could

explain

all

characteristics of heterogeneous

human

goiters,

in that this tissue

displays qualities

notfound eitherin normalthyroid tissue nor in othertypesof

goiters

(1, 2, 15, 17, 18).Asexamples,the widely variablebutfrequently unique

growth

patternofhuman nodular

goiters,

the

regionally differing

patternof cell

prolifer-ation and iodine turnover, the

dissociation

of

growth

and of function familiar from

scintigraphic investigations (19, 20),

and the

topographically

variable abnormalities of

TSH-cyclic

AMP

coupling

(7)calledfor

dynamic

studieson human

goiters

and forconfirmation and extension of

previously

obtained animal data. Such studiesbecame feasible withtheintroduction into

experimental thyroidology

of the method of

transplantation

of humantissueonto nudemice

(8-1

1).

Using

this

technique,

wedemonstrate here thata

particular

growthpattern ofthe parenttissue is

faithfully reproduced by

thegrowing transplant. Itthereforeappearsthatan individual modeof growth suchassolid,

microfollicular,

ormacrofollicular tissuestructure

of

a

goiter

doesnot

depend mainly

onthe

mi-croenvironment

of this

tissue,

butis ratheran

acquired quality

ofthe

epithelial

cells that has becomeheritableand is

passed

on to theirprogeny. Because

goiters growing

from

transplants

in miceare exact

replicas

of theparent

tissue,

the oldclaim that heterogeneity is simplydue tovariable accessofcellstoblood supplybecomesuntenable.

Kinetic studies ofthe growth of

goiter transplants

in host mice revealed-in line with

observations

on mice

thyroids

re-ported

previously (3)-that

human

goiter

tissuealso contains

subpopulations

ofcells with

widely varying growth

rates and equally

varying

TSH

dependency.

This was alsotrue for the only normal

thyroid

available for this

study.

Recent work in this laboratory has revealed that

transplanted

human fetal thyroids contain an even larger number of cell families with

*

*

I

i

W,

cpm/mgxlO4

6

4-

2-n

am

n= 3 3 3 3 8 8

cold nodule hot nodule euthyroid multinodulargoiter

of toxic goiter

Figure

10.Effects ofT4

(i)

andMMI

_(X)

treatmenton '3'Iuptakeof human

_{goiter transplants.}

MMIwas

_given

for 3 wk and

stopped

48 h before

_{131I injection.}

MMItreatmentincreases1311

uptake

in all

trans-plants

irrespective

of whether

_they

arederivedfrom

_{hot, cold,}

or

inter-mediately

active

_goiter

_tissue,

as

compared

toT4treatment.This indicates that human

_thyroid

iodine metabolism is in fact sensitiveto

mouseTSH and thatneitherhotnorcoldtissue is

refractory

toTSH. The

_only

detectabledifference between hot and cold tissuewasthe much

higher

TSH

independent,

autonomous,

i.e.,

insuppressible

io-dineturnover_{of the hot tissue. Bars represent means±SEM of}counts

perminuteper

milligram

of

transplanted tissue;

n=numberof

trans-plants.

short

_replication

time _{than any adult}tissue

(Peter,

H.

J.,

un-published observation).

Three-dimensional true-to-nature

re-construction offollicles from

_contiguous

serial sectionsreveal that the

_{rapidly replicating}

cellsare not

_randomly

distributed

throughout

the

_thyroid,

but that

_they

form colonies of

adjacent

cells within the follicular shell. These colonies

_usually

havea

large

central

_body

with

_{protrusions reaching}

far intothefollicular

shell,

while extended

adjacent regions

of thesamefollicle donot containanylabeled cellsatall

(Fig. 6).

The numberofcell col-onies with identical

_labeling

patterns,andtherefore with similar

growth

rates,

strikingly

increased in

parallel

tothetime of pre-viousTSHstimulation

(Fig. 4).

In

_{TSH-suppressed}

_mice,

_only

an occasional follicle

con-tainedaclusterof

_{[3H]thymidine}

labeled

_cells,

_comprising

any-thing

from morethan threetoupto several dozen

replicating

cells. Inafew

instances, nearly

all cellsofa

single,

lonely

follicle were labeled whereas up to 90% ofallother follicles did not

containany

replicating

cellsonserial sections.

Itiswell knownthat cellsofwhatever

origin

intissueculture have

_{widely varying}

_growth

_rates,too

_(21).

Webelieve thatthe

rapidly

multiplying

cell clones

_previously

observed in normal

mouse

thyroids

(3)

andnowin human

goiter

transplants

reflect

thesame

phenomenon

withinacell

population growing

invivo.

Very

recently,

Westermarket al.

₍₂₂₎

have demonstrated the

variability

offollicular

growth

response to

_epidermal

_growth

factor

_(EGF)

intissue culture.

The

_gradual

increase ofthefraction of labeledcellswiththe durationof

previous

TSHstimulation

(Fig.

4)

indicatesthat the

thyroid

contains a continuum ofcell subsets with individual

thresholds for

_growth

stimuli.

_Vigorous

_stimuli,

such asthose

resulting

from the

_growth

_{stimulating immunoglobulins}

of Graves' diseaseorfrom

highly

dosed

thyrostatic

_agentsin

ex-perimental

goiters

may

eventually

bring

most, ifnot all cells intothemitotic

_{cycle (1,}

_3).

Lessintense

stimuli,

suchasthose

producing slowly growing

human nodular

_{goiters (23),}

_may

in-duce

_{preferential multiplication}

of

_only

those cell clones witha

high

intrinsic

propensity

for

_replication.

They

are

responsive

to

the most subtle stimuli or may even divide in their absence. These autonomously growing cells, once generated in

large

enoughnumber,mayaccountfor theautonomousgoiter

growth

that is well knownto occurin manylong-standinghuman

goiters.

Theexceedinglyhigh sensitivityofsomethyroidcellstodivide also explainswhy growth, butnot simultaneous

morphologic

or functional stimulation, is an invariable hallmark of

slowly

developing human goiters caused by weak, chronically

acting

goitrogens(23-25). Moreover, uneventissue

growth

is but

an-other factorproducing the invariablenodularityof

long-standing

humangoiters(1,6).

Newly generated follicular cells may beintegrated into the growinggoiter inoneof three ways.First,theymayhelp

enlarging

the shell of the mother follicle(Fig. 6)orsecond,theymay form papillary protrusions into the follicular lumen (not illustrated here). Third, eventuallythey may growoutofthe mother follicle to form new daughter follicles (Fig. 7) (3). This process, first demonstrated for the mice thyroid (3), is probably the basic event ingoitrogenesis(1).

Inaddition to growth,wealso studied the function of

goiter

transplants in terms of radioiodine turnover. Many

previous

conclusions (1, 2, 4, 17, 18) drawn from observations inmouse

and ratthyroids and in humangoitersprelabeled with

1251

were

confirmed. Infact, nodifference between goitersgrowing nat-urally in patients and those transplantedontonude mice could be observed. Forexample,alow basal level of radioiodine turn-overafter TSHsuppression varied enormously among different follicles ofatransplant(Fig. 9) andeven more sobetweentwo transplants from different regions ofagoiter. Nomorphologic differences between a hot and a cold follicle could bedefined, norwasthere any consistent structural difference between trans-plants from scintigraphically hot and cold goiter areas. Trans-plants from both hot and cold nodules contained follicles with widely varying individual function justasreported for the parent tissue (1, 2, 17, 18). However, samples from areas with high autonomousiodine turnover contained a larger fraction of hot insuppressible follicles and their residual radioactivity was, on

the average, higher than in follicles from poorly functioning nodules.

Using short-term labeling with 25I,wefound that manygoiter follicles contained cell cohorts with widely differingiodinating potency. This intrafollicularheterogeneityhadpreviously been seenin mice (3, 5, 26-28) and had occasionally been described inhumangoiters(3, 15). The phenomenon has recently been observed in tissue culture by Errick et al. (29). The number of cells with intense, autonomously functioning iodination may greatlyvary fromonefollicletothe next. This observation sug-geststhat, in contrast totraditional views (15), the degree of autonomousiodineturnoveris not a quality of the follicle asa whole,but rather depends on the relative fractions of hot and cold cellsbuildingup itsepithelium.Moreover,in that new fol-liclesaregeneratedfrom one or a few dividing cells within the motherfollicle, the individual intrinsic iodine turnover of the parent cellprimarily decides whether the daughter follicle hasa highorlowiodine turnover (3). Again, the individual iodinating capacity ofepithelial cells appears to be a stable, inheritable trait.

Rather unexpected was the high responsiveness of cold as wellasofhotfolliclestoTSH, although TSH responsiveness of hot and cold nodules has been reported earlier (30, 31). A

clear-cutdifference of iodine metabolism between hot and cold tissue was,

however,

still apparentafterTSHstimulation.This suggests

1-*i i ' 8 ' '

D.o

-'

, 9

;~~~~~~~~~~~~~'t-tE.,i>

--'s~4, .vi(f ..,

AWi r Al

i

§

8

1

*

< 4

;

_Jr4

b

B

.I

4.4

*

I

"I

)

/ i!

- A

;- (

'A~~~~~~~~~~~~~&

a

A

.I

that the range ofincreased radioiodine uptake afterTSH stim-ulation is comparable in both types offollicles, but that the baseline activity is different. The observations are compatible with two alternative explanations: Eitherthe fraction of truly hot cells inagiven follicleisindeed entirely autonomousand unresponsivetoTSH,while theremainingnormal cells respond normally. As an alternative, all cells, even the hot ones, may be TSH sensitive, although their autonomous TSH-independent iodine metabolism isintrinsicallyhigher than average. The failure of cold folliclesto match the uptake of hot ones after intense TSHstimulation tends toindicatethatasimilarrange of TSH response issuperimposedonabasicallydiffering iodineturnover inindividual follicularcells.

Double-labeling experiments with

[3H]thymidine

andrapidly decaying

'3'I

permitted answeringthequestion ofwhether au-tonomous growth was linked to autonomous function or, in otherterms, whether thefollicleswith thehighest intrinsicgrowth rates werealso those with thehighestautonomousiodine

turn-'..

T-t

'St

t*.

_ev

Figure11. Transplantlabeledwith [3Hjthymidinefor 2 wk and with '31Ifor 1

hbefore sacrifice whilethehost's TSH

wassuppressedbyT4 treatment.SectionI

{.v

autoradiographed

without

delay

shows

io-* dineorganification;sectionII, exposed4

molater, i.e.,afterdecayof'3'I,shows

[3H]thymidine incorporation.

FollicleA

displaysthehighestautonomous_capacity

foriodineorganificationbut containsonly

afewproliferatingcells. In follicleB,the '31I-labeled thyroglobulin has notspread homogeneously (confirmedonserial

sec-tions). Onlyavery few of thehighly iodi-natingcellsatthe upper left

circumfer-encehaveincorporatedthethymidine la-bel whereasmostofthe less active cells (dotted line)arelabeled.Thus,autonomy ofiodine metabolism of the follicular epi-thelial cells doesnotparallel their poten-tialtoproliferate autonomously.

over. Theanswerisclearly"no." Rather,the capacitytogrow and that to metabolize iodine are two wholly unrelated phe-nomena. Highly growth-prone cells belongingto families with intense [3H]thymidine labelingmayjustas wellform follicles with high iodineturnoverthan follicles with low iodineuptake. This observation does ofcourse notruleoutthe transitory

ces-sation of function during S-phase of the cell cycle (32). Inde-pendency of growth and function fits well with the familiar clin-ical observation that cold and hot nodules donotdiffer in their growth behaviour.

Taken as acomplement toearlier studies in miceand hu-mans, the presentinvestigation ongrowing transplants allows expandingcurrent viewsonthe pathogenesis of human multi-nodulargoiter and, in particular,on thewaysits most puzzling hallmark, namely morphologic and functional heterogeneity, is brought about. Besides environmental factors, discussed above and in otherpapers(4), and besides thegenetic heterogeneity of theparenttissue from which qualitatively differentdaughter

follicles are generated during goiter growth (3), goiter cells un-dergo modifications of theirphenotypewhicharepassedon to theirprogeny.Examples fornewly acquiredtraitsarethe many individual growth patternsarising withinthesamegoiteror be-tween twodifferent goiters. Thetransplantmayperfectly repro-duce the individual morphologic structure of the parent tissue. Thisindicatesthat adistinctstructuredoesnotdepend onlyon localintrathyroidal factors,but ismorelikelyanewlyacquired quality that has become inheritable.Likewise,the high incidence of "hot"autonomously iodinatingcells intransplantsfrom "hot" nodules indicates that this functional trait is stable and inde-pendent of the environment. It

is, however,

difficult to say whether agiven aspectof goiter heterogeneity, such as the re-gionally varying TSH dependency of growth and function, is exclusively aquality inherited from thepolyclonal cells of the parent follicles and amplified through the generation ofnew daughter follicles (1),orwhether theexpandingcell population hasacquiredsomestablechangesof its

enzymatic

outfit. How-ever, for the concept of thepathogenesisofgoiterheterogeneity through amplification of individual heritable celltraits,it is of littleimportancewhetheragivenfunctional characteristic is pri-marily inborn orsecondarily incorporated into the inheritable equipmentthatdetermines thephenotypeofacell.

Although ourexperimentsdonot

provide

any information onthemuch-debated molecularmechanisms

generating

diversity among the progenyofasingle cell, afew theoretical consider-ations may nevertheless beappropriate.Itisindeedunlikelythat the ratherrare eventof somatic mutationin

growing

cell clones (33)canaccountforthe manyfacets of

heterogeneity

of human goiters. However, modern molecularbiologyhas revealed that the patternofgeneexpression is by farless stable thanthought untilrecently,and thatagenome may wellacquirenewheritable traits (34, 35).Inaddition,thereis

rapidly accumulating

evidence suggestingtheexistenceofextranuclearmechanisms modulating cellularfunction whicharepassedonfrommothertodaughter cells (36, 37). The wide variability in the regulation ofgene expression has been impressively documented by discoveries suchasthatof transposablegeneticelements(38,39), ofsomatic recombination inlymphocytes(40), andof homeotic genes which playacrucialrolein development and

cell

differentiation(41). Heterogeneity of malignanttumorsis now firmly established. Genome-boundandepigenetic mechanisms involvedincreating this heterogeneityaregradually merginginto asingleconcept. Heterogeneity amongcellswithin the same tissue also has be-come atopic of growing importance. The subjectwhich isin the focus ofactualcancerresearch has been recentlyreviewed by Heppner (42).

Itmay wellbe that heterogeneity of growth and function of humangoiter tissue,

which

offers a rather unique experimental opportunity for assessingthe functional activity of individual cellsorcellsubsets, may increasingly attract the interest of cell biologistseager to study the mechanisms that create variability within differentiated organs.

Acknowledgments

The authors would like to thank Prof. Dr. Riedwyl, Department of

Mathematics, Universityof Bern, for statistical analysis of datas;Prof.

Dr. R.Berchtoldand Dr. J. Teuscher, Department of Surgery, University

of Bern, forexcellent collaboration; PD Dr. P. Groscurth, Department

of

Anatomy,

University

of

Zurich,

for his contributiontothis_work;Mrs.

J.

_Kampf,

Mr. E.

Schurch,

and Mr. M.

_Jaberg

for technical

_assistance;

Mrs.C. vonGrunigen andMrs. L. Siebenhuner for expert secretarial assistance;and Mrs. S. Burki for thephotographs.

This workwassupported bygrantno.3.987.84 from the Swiss Na-tionalScience Foundation.

References

1.Studer, H.,and F. Ramelli. 1982.Simple goiterand its variants: euthyroidand hyperthyroid multinodulargoiters. Endocr. Rev. 3:40-61.

2.Studer, H., H. Gerber, and H. J. Peter. Multinodular goiter. In Endocrinology.Vol. 1. L. J.DeGroot,editor. Grune and Stratton New York.Secondedition. Inpress.

3.Peter, H. J.,H. Studer, R. Forster, andH. Gerber. 1982. The pathogenesis of "hot" and "cold" follicles in multinodular goiters. J. Clin. Endocrinol. Metab. 55:941-946.

4. Studer, H., H. Kohler, and H. Burgi. 1974. Iodine deficiency. Handb.Physiol. (Sect. 7).3:303-328.

5.Studer, H.,R.Forster,A.Conti,H.Kohler,A.Haeberli,and H. Engler. 1978. Transformation of normal follicles into thyrotropin-re-fractory"cold" follicles in theagingmousethyroid gland.Endocrinology. 102:1576-1586.

6. Ramelli, F., H. Studer, and D. Bruggisser. 1982. Pathogenesis of thyroidnodules in multinodular goiter. Am. J. Pathol. 109:215-223.

7. Rentsch, H. P., H. Studer, B. Frauchiger, and L. Siebenhuner. 1981.Topographical heterogeneity of basal and thyrotropin-stimulated adenosine 3',5'-monophosphate in human nodular goiter. J. Clin. En-docrinol. Metab. 53:514-521.

8.Smeds,S., B. Anderberg, B. Boeryd, L. E. Ericson, J. Gillquist, and J.Persliden. 1981. The nude mouse. A possible experimental model forinvestigation of human thyroid tissue. J. Endocrinol. Invest. 4:1

1-15.

9. Leclere, J., M. C. Bene, A. Duprez, G. Faure, J. L. Thomas, J. M.Vignaud, and C. Burlet. 1984. Behaviour of thyroid tissue from patients with Graves' disease in nude mice. J.Clin.Endocrinol. Metab. 59:175-177.

10.Schumm,P.M., K. Badenhopp, P. Kotulla, A. Luck, H. J. C. Wenisch, K.Schdffling, H. Schleusener, and K. H. Usadel. 1984. Sig-nificance of thyroid stimulating immunoglobulins in the development ofeuthyroid sporadic goiter.Ann.Endocrinol. (Paris). 45:156. (Abstr.)

11. Dralle, H., W. Bocker, K. D. Dohler, S. Schroder, H. Haindl,H.

Geerlings, R. Schwarzrock, and R. Pichlmayr. 1985. Growth and function ofthirty-four human benign and malignant thyroid xenografts in

un-treated nude mice. Cancer Res. 45:1239-1245.

12. Riedwyl, M. 1978. Angewandte Statistik in Wissenschaft, Ad-ministration und Technik. M. Riedwyl, editor. Haupt Verlag, Bern. 1-204.

13. Sharard, A., H. D. Purves, and W. S. Cague. 1970. Effect of pretreatment ofmicewith varying doses of thyroxine in the assay for LATS and TSH. Proc. Univ. Otago Med. Sch. 48:77-78.

14.Gerber, H., H. Studer, and C. von Grunigen. 1985. Paradoxical effects ofthyrotropinondiffusion of thyroglobulin in the colloid ofrat

thyroid follicles after long term thyroxine treatment. Endocrinology. 116: 303-310.

15.Miller, J. M., R. C. Horn, and M. A. Block. 1967. The autonomous functioning thyroid nodule in the evolution of nodular goiter. J. Clin. Endocrinol. Metab. 27:1264-1274.

16.Gerber, H., H.Studer,A.Conti, H. Engler, H. Kohler, and A. Haeberli. 1981. Reaccumulation of thyroglobulin and colloid in rat and

mousethyroid follicles during intense thyrotropin stimulation. A clue

tothepathogenesis of colloid goiters. J. Clin. Invest. 68:1338-1347.

17.Miller,J.M., R. C. Horn, and M. A. Block. 1964. The evaluation of toxic nodular goiter. Arch. Intern. Med. 113:72-78.

18.Miller,J. M.1975. Plummer's disease. Med. Clin. Am. 59:1203-1216.

19.Emrich,D.,and M.Bahre. 1978. Autonomy ineuthyroidgoiter: maladaptationtoiodine_deficiency.Clin. Endocrinol.

_(Oxj).

8:257-265.

20. Wiener, J.D. 1975.Asystematic approach to the diagnosis of Plummer's disease (autonomous goiter) with a review of 224 cases. Neth. J.Med. 18:218-233.

21. Pardee,A.B., W. R. Dubrow,J.L.Hamlin, and R.F.Kletzien. 1978. Animal cell cycle.Annu.Rev.Biochem.47:715-750.

22.Westermark, K., F. A. Karlson, L. E. Ericson, andB.Westermark. 1985. Epidermal growth factor: a regulator ofthyroidgrowth andfunction invitro.InThyroglobulin-TheProthyroid Hormone. Progress in

En-docrine Research and Therapy. Vol. 2. M. C. Eggo, and G. N. Burrow, editors. Raven Press, New York. 255-262.

23. Drexhage, H. A., G. F. Bottazzo, D. Doniach, L.Bitensky, and J.Chayen. 1980. Evidence for thyroid-growth-stimulating immunoglob-ulins insomegoitrousthyroiddisease. Lancet. ii:287-292.

24.Grubeck-Loebenstein, B., K. Derfler, H. Kassal, W. Knapp, K. Kirsch, K. Liszka,P. P. A. Smyth, and W. Waldhausl. 1985. Immu-nological features ofnonimmunogenic hyperthyroidism. J. Clin. En-docrinol. Metab.60:150-155.

25. Brown, R. S., I. M. D. Jackson, S. Pohl, and S. Reichlin. 1978. Dothyroidstimulating-immunoglobulinscause non-toxic multinodular goiter? Lancet. i:904-906.

26.Wollman, S. H., and I. Wodinsky. 1955. Localization of protein-bound iodine- 131 in thethyroid glandof themouse.Endocrinology. 56: 9-20.

27.Leblond, C. P., and J. Gross. 1948.Thyroglobulinformation in thethyroidfollicle visualizedbythe "coatedautograph"technique. En-docrinology. 43:306-324.

28. Wollman, S. H. 1965.Heterogeneityof thethyroid gland. Curr. Top. ThyroidRes. Proc.5thInt.ThyroidConf5:1-18.

29.Errick,J.E., M. C. Eggo, and G. N. Burrow. 1985. Factorsaffecting synthesisofthyroid-specific proteinsand iodination in culturedthyroid cells. InThyroglobulin-The ProthyroidHormone.Progressin Endo-crine Research andTherapy. Vol. 2. M. C.Eggoand G. N. Burrow, editors. Raven Press, New York. 271-282.

30. DeRubertis, F., K. Yamashita, A. Dekker, P. R. Larsen, and J. B. Field. 1972.Effectsofthyroid-stimulating hormone on adenyl cyclase activity and intermediary metabolism of "cold" thyroidnodulesand normal human thyroid tissue. J. Clin. Invest. 51:1109-1117.

31. Larsen, P. R., K. Yamashita, A. Dekker, and J. B. Field. 1973. Biochemical observations in functioning thyroid adenomas. J. Clin. En-docrinol. Metab. 36:1009-1018.

32. Roger, P. P., and J. E. Dumont. 1983. Thyrotrophin and the differential expression of proliferationanddifferentiationin dog thyroid cells in primary culture. J.Endocrinol. 96:241-249.

33.Cairns, J. 1975. Mutation selection and the natural history of cancer. Nature(Lond.).255:197-200.

34. Roeder, G. S., P. J. Farabaugh, D. T. Chaleff, and G. R. Fink. 1980. The origins of gene instability in yeast. Science (Wash. DC). 209: 1375-1380.

35. Razin, A., and H. Cedar. 1984.DNAmethylation in eukaryotic cells. Int. Rev.Cytol. 92:159-185.

36.Albrecht-Buehler,G. 1977. Daughter3T3cells. Are theymirror images of each other.J.Cell.Biol.72:595-603.

37.Aufderheide, K.J., J. Frankel, and N. E. Williams. 1977. For-mation and positioning of surface-related structures in protozoa. Micro-biol. Rev. 44:252-302.

38. McClintock, B. 1956.Controllingelements and the gene.Cold Spring Harbor Symp. Quant.Biol. 21:197-216.

39. Shapiro, J. 1983. Mobilegeneticelements. NewYork, Academic Press. 1-63.

40.Adams, J. M. 1980. Theorganisationand expressionof immu-noglobulingenes.Immunol. Today (Amst.). 1:10-17.

41.Gehring,W.J. 1985. The homeo box:akey to the understanding ofdevelopment? Cell. 40:3-5.