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JOURNAL OFVIROLOGY, Sept. 1988,p. 3103-3108 0022-538X/88/093103-06$02.00/0

Copyright © 1988, AmericanSocietyforMicrobiology

Growth Factor

Production by

Creutzfeldt-Jakob Disease Cell Lines

EMILIA L. OLESZAK,t GEOFFREY MURDOCH, LAURAMANUELIDIS, ANDELIAS E. MANUELIDIS* Yale University School ofMedicine, 333 Cedar Street, NewHaven, Connecticut06510

Received14September 1987/Accepted 19 May1988

Creutzfeldt-Jakob disease(CJD),aprogressivedementia ofhumans,iscausedbyaninfectious agent thatis closely relatedtothe scrapieagentof sheep.Although the molecularnatureof these"unconventional"agents isstillamatterof speculationandcontroversy,evenless is knownconcerning themechanism(s) of theireffects onthe centralnervoussystem.Togain insight intothe cellulareffectsof theseagents,wehave examinedaseries ofcell linesderiveddirectly from CJD-infected hamster brainorproduced fromnontransformed rodent lines byexposuretoCJDinfectiousfractions in vitro. These cell linesappeartransformedbyavarietyof criteria and secrete growth factorsinto the culture medium. All CJD lines produce a factor that is likea-transforming growth factor (a-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glial fibrillary acidic protein innormal astrocytic cells in vitro. This effect is mimicked by purified a-TGF and platelet-derived growth factors. Further study of CJD-induced growth factor production may elucidate fundamentalpropertiesof these unconventional agents.

The molecularnatureof theagents(7) causing the

uncon-ventional viral diseases (Creutzfeldt-Jakob disease [CJD], scrapie, kuru) is still a matter of intense speculation and controversy (16, 18, 25, 26). Little attention has been given

to the observation that cells from scrapie-infected brains

readilyproducepermanentorimmortalized lines in vitro (3,

4, 8, 12). Furthermore, in more than 20 independent

at-tempts, cells fromalmost all CJD-infectedmouse and ham-ster brains yielded permanent cell lines, while cells from control brainsgenerallygaveriseto senescentcultures(12). We have also recently shown that in vitro exposure of BALB/c 3T3 cells and other normal early-passage primary rodent celllinestoCJD infectious preparationsreproducibly inducestransformation, whereastreatmentof thesamelines

withnormal brain preparations doesnot(24). This and other biological properties are reminiscent of retroviruses (17).

However, up to the present time no retroviruses or other

conventional viruses knownto elicit either central nervous systemdiseaseortransformation have beendemonstrated in normalorCJD-infected braintissue, despiteacareful search by manyinvestigators.

In eachoftwocultures assayed, inoculations of homoge-nizedlong-term (multiplypassaged) CJD cell lines produced the characteristic incubation features, clinical symptoms,

andhistologicbrainlesions of CJD(12). These data indicated thatthe agentwas obviously replicating in these lines. The

titer of infectivity was significantly lower than in CJD-infected brain tissue and thus the cell lines arenotanideal

source for agent purification. We considered that detailed characterization of the transformedphenotype of these cell lines could provide insight into fundamental biologic

prop-ertiesof theagentwhich relatetoits centralnervoussystem effects. This initial study demonstrates that all CJD-trans-formed cell lines, whether derived from brain ortreated in vitro with infectious material,appeartoproduceafactor that is like a-transforming growth factor (a-TGF). This result suggeststhat theCJDagenthasinducedtransformationbya similar mechanism in each cell line. The potential in vivo significance of this phenomenon is illustrated by the ability

* Corresponding author.

tPresent address: Department of Pathology and Laboratory Medicine,Universityof TexasMedicalSchool, Houston, TX77030.

of conditioned media from CJD-transformed cell lines (as well as purified a-TGF) to increase glial fibrillary acidic protein(GFAP) production in vitro. This effect mimics the increasedastroglial GFAP productionseenduring thecourse

of the disease in vivo(13, 19).

MATERIALS ANDMETHODS

Cell lines. Immortalized cell lines from CJD-infected

mouse(TC 740 and TC 746) and hamster brains(TC 724 and

TC728)wereestablishedfrom explant culturesaspreviously

described (12). The passages utilized in these studies are

capable of formingtumorsinnude mice andcantherefore be

considered transformed (11, 14, 17; unpublished data).

Cul-tures ofnormal mouse brain (TC 760) and normal hamster brain (TC 744A and TC 763) were established in parallel.

With serial passage, these control lines generally became

senescent. Low passages of these lines contain approxi-mately 50% astrocytes by morphologic and immunocyto-chemical examination. Similarpreparations have previously been usedby othergroups to studyastrocyte growth

prop-erties(31).

Invitro CJD-transformed cell lineswerederived from 3T3

cells and the control hamster line (TC 744A)by treatment

with CJD brain homogenates (TC 744A/CJ, 3T3/CJ-H) or

crude CJD synaptosomal preparations (3T3/CJ-SM) as de-scribed previously (24). 3T3 cells treated with the corre-sponding fractions of normal brainweredesignated 3T3/N-H and 3T3/N-SM. A highly malignant methylcholanthrene-induced mouse glioblastoma cell line (TC 509), which pro-duces numerous type C retroviralparticles wasused as an additional control (15).

Preparation of conditioned medium. Confluent cultures

were thoroughly rinsed in serum-free Delbecco minimal

essentialmedium(DMEM) followed bya16-h incubationat

37°C in DMEM to remove residual serum proteins. The cultureswerethenincubated infresh serum-free DMEM for 48h, andthe resultantconditioned mediumwasadjustedto

1 mMphenylmethylsulfonyl fluoride, clarified by centrifuga-tion (20,000 x gfor 20min), and storedat -20°C. Insome

cases, conditioned mediumwasconcentrated 20- to70-fold by using Diaflow YM-10membranes (Amicon). Foreaseof

comparisonbetweensamplesinfigures,allvolumes of added

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conditioned media are expressed as the original volume beforeconcentration.

Assays of DNA synthesis and colony formation. Indicator BALB/c3T3cells(original stockgenerouslyprovidedbyP. Besmer, Sloan-Kettering CancerCenter) were seededonto 24-well plates (2 x 105cells per well) in DMEM with 10% fetalcalfserum. After attachment, the cells were rinsed in serum-free medium and arrested in

Go

by a48-h incubation in serum-free medium. Various amounts of conditioned medium and [3H]thymidine (1 piCi/ml, 45 Ci/mM; New England Nuclear)were thenadded.Acid-precipitable radio-activitywas measured 24 hlater forassessment of reinitia-tion ofDNAsynthesis (24).

Anchorage-independent growth in soft agar was deter-mined essentially as described by McPherson and Mon-tagnier (20). BALB/c 3T3 cells (1 x 103 to 5 x 103) were suspended in 1.2% methylcellulose F4M (Dow Chemical Co.) withDMEMand10% fetal calfserumcontaining300,ul of conditioned medium per mlandplatedon abase layer of 0.9%agar(Bacto; DifcoLaboratories). At 1 weeklater,cells were refed with fresh medium and 150

RI

ofconditioned medium. Colonies of >20 cells were scored 2 weeks after initial plating, as previously depicted (24). Tests with Sea-Plaque agarose(FMCCorp.)generated comparable results. EGFradioreceptor assay.Epidermal growthfactor (EGF)-like factorswerequantitated byacommercialradioreceptor assay on the basis of A431 cell membranes (Biomedical Technologies), following the protocol of the manufacturer. Theability of portions of concentrated conditionedmedium to compete with

1251I-labeled

EGF binding was compared with purified EGF standards.

GFAP immunocytochemistry.

Early-passage

normal mouse brain cells were seeded onto eight chamber slides (Miles Scientific) at 1 x 104 to 2 x 104 cells per chamber. After attachment, concentrated conditioned mediumor pu-rified growth factors were added for an additional 3 to 4 days. After being rinsed in serum-free DMEM, cells were fixed for5 minwith buffered4% paraformaldehydeandthey were

permeabilized

for 1 min with cold methanol. GFAP

immunoreactivity

wasdetermined witha non-species-specif-ic polyclonal anti-GFAP kit (Miles Scientific), using the peroxidase-antiperoxidase method. This antibody

selec-tively

delineates GFAP fibrils in reactiveastrocytesin brain aspreviously depicted (19).

RESULTS

Figure

1 (top) shows theeffect of unconcentrated serum-free conditioned media fromthevarious cells lineson DNA synthesis in

GO-arrested

normal 3T3 indicatorcells. Condi-tioned medium of cell lines from CJD-infected brains (TC 728,TC 724, TC 746, TC 740) and lines exposed toCJD in vitro(TC 744A/CJ, 3T3/CJ-H,

3T3/CJ-SM)

typically stimu-lated

[3H]thymidine

incorporation in the indicator cell line up to 25-fold, equal to orgreater than the effect of fresh serum-containingmedium. Incontrast, conditioned medium from control lines (TC 760, TC 509) andfrom lines treated withfractionsofnormal brain(TC 744A/N,

3T31N-H,

3T3/ N-SM) showed onlyminimalstimulationofDNAsynthesis. Notably, conditioned medium fromcontrolTC509,derived fromamethylcholanthrene-induced glioblastomawhich pro-duces type C virus, had nocomparable stimulatory effect.

In order to quantitate growth factor production, condi-tioned medium was concentrated 20- to 70-fold by ultrafil-trationand the effectsof serialdilutions of this concentrate on 3T3 cell DNA synthesis were assayed. The resultant

w

<

a.

w z 0

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z

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-I

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TC SOURCE OF SUPERNATANTS

EFFECT OF CM ON 3T3 CELL DNA SYNTHESIS

0. 1

ml equivalent. CM added

FIG. 1. Mitogenic effect of conditioned medium from repre-sentative CJD and control cell lines.(Top) Conditioned medium of cells derived from CJD-infected hamster(TC 728 and TC 724) or

mouse (TC 746 and TC 740) brain and from nontransformed cells exposedtoCJD in vitro(TC744A/CJ, 3T3/CJ-H, and 3T3/CJ-SM) stimulate DNA synthesis in GO-arrested cells, whereas media of control lines donot(e.g., normal mousebrain line TC760,mouse

glioblastoma TC 509, and control cell lines exposedtononinfectious brain fractions [TC 744A/N, 3T3/N-H,3T3/N-SM]). Unconcentra-tedconditioned mediumwasaddedat0.1(hatched bars)or0.4(open bars) ml. Values shownare averages oftriplicate determinations. (Bottom) Concentrated conditioned media (CM) wereused to de-termine relative amounts of growth factor production by repre-sentative CJD lines (open symbols) and control cultures (solid symbols). Pool-N is apool of normal supernatants from normal hamster brainline TC763, normalmousebrain line TC760, and in vitro normal-treated TC744A/N.Barrepresents standard deviation. Concentrated conditioned medium, expressedasmilliliter

equiva-lentsoforiginal conditioned medium,i.e., 0.1mlofa1Ox

concen-trate, isequalto a1-mlequivalent of conditionedmedium.Cultures

treatedwith brainhomogenatesaredesignated withanHand those treated with synaptosome-enriched fractionsaredesignated SM.

dose-responsecurves(Fig. 1 [bottom]) show that theamount ofgrowth factor produced by the series ofCJD cell lines varies overnearlyatwo-logrange. Ingeneral,3T3celllines exposedtoCJDinvitroproducethe leastamountofgrowth factor. However, this low production of growth factor ap-pears to relate to the underlying cellular phenotype rather than to theprocess of in vitro exposure, because anormal hamsterbrain celllinesimilarly exposedtoCJDinvitro(TC 744A/CJ) produced large amounts of growth factor. Its

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CREUTZFELDT-JAKOB DISEASE GROWTH FACTOR TRANSFORMATION 3105

TABLE 1. Stimulation of3T3colony formation by conditioned medium

Supematantsource Colonies/disha Cloning

Medium (species) (mean+ SD) efficiency(%)b

Normal brain culture TC 760 (mouse) 4 ± 1 0.2

TC763 (hamster) 3 ± 1 0.2

CJD brainculture TC 746 (mouse) 195± 14 5.1

TC740 (mouse) 177 +33 8.8

TC724 (hamster) 487± 9 48.7

TC 728 (hamster) 308 ± 80 30.8

Control treated in vitro 3T3/N-H (mouse) 5 ± 1 0.5

3T3/N-SM (mouse) 15± 3 0.3

CJD treatedinvitro 3T3/CJ-H(mouse) 173 ± 25 17.3

3T3/CJ-SM (mouse) 188± 5 3.8

TC744A/CJ (hamster) 80± 5 8.0

a>20 cells per colony.

bOnly CJD lines showed a significant effect on colony formation (actual colonies formed) and cloning efficiency (percentage of colonies formed per cells plated).

controlcounterpartexposed touninfectiousbrain(TC744A/ N) produced nosignificant comparable growthfactor. Con-ditioned medium from CJD cultures also stimulated anchor-age-independentgrowthof indicatorcells. Table 1 shows the effect of culturesupematantsof CJDlineson3T3 cell colony formation in soft agar compared with that ofthe superna-tants of various control lines. Again, all CJD lines,whether derived from brain tissueorby in vitrotreatment,stimulated colonyformation, whereas control lines did not.

Table 2 summarizes the biochemical properties of the growth factor(s) produced by CJD lines. The mitogenic factor is trypsin labile but heat and acid stable. These properties are consistent with a low-molecular-weight pep-tide factor. The sensitivity to treatment with dithiothreitol suggests that thefactor consists ofpeptide chains joined by disulfide bonds (like PDGF or

P-TGF)

or has functionally important intrachain disulfide bonds (like EGF and a-TGF) (9, 21).

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Theabove-mentioned biologic activities and biochemical properties of the CJD-induced growth factorareallfulfilled by a-TGF. Because o-TGF binds withhigh affinityto EGF

TABLE 2. Percentstimulation ofDNAsynthesis inGo3T3

indicatorcellsbysupernatantsof CJD cell lines

% Stimulation in indicated cell line Treatmenta

3T3/CJ-H 3T3/CJ-SM TC744A/CJ TC724 TC728 TC740

None 100 100 100 100 100 100

Heatb

89 NDC 97 94 114 118

Acidd 108 101 101 100 104 100

Trypsin ND 107 106 111 ND ND

controle

Trypsinf 15 11 25 19 31 32

DTT8 26 15 9 19 11 22

aSecretedgrowth factorsfromallCJD lineswereheat and acidstable,but inactivated by trypsin and dithiothreitol. Inactivation of growth factors in conditioned medium was measured by [3H]thymidine incorporation in

Go-arrested cells. b56°C for30min. cND,Notdetermined.

dDialysis against0.17 Macetic acid for16hat4°Cfollowedby dialysis against saline.

e100,ug oftrypsin inhibitor perml in thepresence of 50,ug oftrypsin (Sigma) permlfor2 h at37°C.

f50pg/mlat37C for2 h.

8Dithiothreitol(DIT)treatment was at0.065Mfor2 h at22°C,followedby dialysis against saline.

receptors,concentratedconditioned mediafrom control and CJD cell lines were therefore evaluated in an EGF radio-receptor assay (Fig. 2). The conditioned medium from the CJDcelllines containedvaryingamountsofEGF-like activ-ity equivalentto60to600pg permlofEGF

(10-10

to 10-11 M). This concentration of ot-TGF may be sufficient to accountforthe observedstimulations ofDNAsynthesisand anchorage-independent growthin theindicator BALB/c3T3 cells (1, 9). However, a potentiation of ot-TGF activity by

3-TGForothergrowth factors has notbeen ruled out. Because a-TGFproduction appearedtobe afundamental featureofcelllines derivedfrom CJD-infected brains,it was of interestto determine whether a-TGF could account for any ofthe observed in vivo effects of CJD infection. The mostconsistent morphologicfeatureof CJDis hypertrophy and possibly hyperplasia ofastrocytes (gliosis). Biochemi-cally, this hypertrophy is the consequence of a markedly increased production of the astroglial-specific intermediate filament GFAP. To date, most studies of the effects of peptide growth factor effectsonastrocytes havefocusedon

ml equivalents CM added (or ng EGF) FIG. 2. Conditioned media fromrepresentativeCJD lines(open symbols) and from control lines (solid symbols) were tested at

variousconcentrationsforbindingtotheEGF receptorbyusinga

commercial radioreceptor assay (Biomedical Technologies). Only

conditioned media from CJDlines effectivelycompetedwithEGF for the EGF receptorandwerecomparedwithknownEGF

concen-trations(+). VOL.62, 1988

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3106 OLESZAK ET AL.

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FIG. 3. EffectsonGFAPexpressioninindicator cells by conditioned media from representativecontrol TC 760 (A) and CJD cultures TC

728(B). Only the latter shows numerous brown(peroxidase)-stained GFAP cellbodies andprocesses. Nuclei werecounterstained with

hematoxylin. Magnification, x100.

thestimulation of cellular proliferation(27, 32),althoughone

preliminary studysuggested that fibroblastgrowthfactorcan

regulate GFAP expression (5). Therefore, the effects of conditioned medium from the CJD cell lines and purified oa-TGFonGFAPproduction in low-passagemousebraincell cultures were examined. After a 3-day incubation, these cells showed a modest increase in saturation density, a

prominent increase inbranching astrocyticprocesses, anda

markedly increased amount ofimmunocytochemically de-tectable GFAP (Fig. 3). Conditioned media from all CJD lines induced this effect while that from control cultures (including the glioblastoma line TC 509) did not. EGF and platelet-derived growth factor produced similar effects,

while

P-TGF

alone had significantly less effect. Thepotency

ofconditioned medium in inducing these effects compared with thatofthepurified growth factors appeared somewhat

greater than expected from its a-TGF-like factor content

measuredbytheradioreceptorassay.Therefore, production ofadditionalsynergisticfactors by theCJDlinescannotbe ruledout.

DISCUSSION

The marked gliosis seen in brains infected with CJD is morethan canbe accounted foras asecondaryresponseto

tissuedestruction(13).Infact,thestimulation ofGFAPgene

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CREUTZFELDT-JAKOB DISEASE GROWTH FACTOR TRANSFORMATION 3107

expression (assessed as GFAP mRNA accumulation)

tem-porally

precedes the neuronal damage (19),

especially

when selected brainregionsareexamined. The near-global activa-tion ofastrocytes is a fundamental part of the pathophysiol-ogyof thisclassof diseases. Itcould be the consequenceof a direct interaction between the infectious agent and astrocytes, orit could be caused byanagent-induced factor which influences astrocytic geneexpression throughout the brain.

The production of a-TGF activity by CJD-derived cell lines and the ability of conditioned medium (or purified growth factor) to stimulate GFAP expression in cultured astrocytes suggest that the second hypothesis deserves further consideration. Growth factor production is fre-quently a component ofthetransformed phenotype. How-ever, many tumorlines, includingthe malignant glioma line used here as a control (TC 509), do not produce a-TGF activity. The relatively high initial growth rate of cells in primary cultures of CJD-infected brains suggests that the resultantestablished linesmayhaveproducedana-TGF-like factor from theirinception and

prior

to the

acquisition

ofa complete transformed phenotype. These lines may be de-rived from cells induced in vivo to produce a-TGF

by

the CJD agent. The

mechanism(s)

ofthe stimulation of

growth

factorproduction by the unconventional CJD agentis pres-ently unclear. Even in the limited sphere of viral transfor-mation, there are clearly multiple distinct potential mecha-nisms involved. Both DNA and RNA viruses can directly encode growth factors (2, 30).

Additionally,

oncogenic

vi-ruses canactivatea

variety

ofoncogenes

by

transductionor insertional

mutagenesis,

with consequent stimulation of growth factorproduction (21). Boththe

underlying

host cell phenotype andthe

specific

oncogene(s) activatedmay deter-mine which specific growth factor is

produced. Notably

however, all CJD lines, regardless of

lineage, produce

an a-TGF-like factor. Some glial cell lineshavebeenshownto produce factors that are like

platelet-derived growth

factor (23) andcontain v-sis-related mRNA

(6).

The exact

lineage

ofthe transformed CJD brain lines is

uncertain,

because of theirpoorly differentiated state.

On a broader

level,

the as yet undefined

relationship

between the transformed

phenotype

of CJD cells and the neurophathology of infectious dementias may provetobeof critical

importance. Although

the

gliosis

present in CJD-infected brains approaches levels reminiscent of

low-grade

astrocytomas(13),hamstersinfected with CJDor

scrapie

do not develop obvious brain tumors.

However,

it is now apparent that

tumorigenesis typically

requires

theactivation ofmorethana

single

oncogene

(10).

In

addition, oncogenic

potential

need notbe

expressed

within the central nervous

system asobvioustumorformation.Thecellsinfected

by

the transforming JCpapovavirus havea

cytologically

malignant

phenotype, yet the gross

pathologic

lesion in adult brain is typically

demyelination (33).

Similarly,

transgenic

mice car-ryinganactivated simianvirus 40

T-antigen

gene

develop

a

demyelination syndrome (28),

although

tumors maybe pro-duced in visceralorgans

(22).

Inthiscontext, the

investiga-tion ofoncogenic expression in infectious dementias is a novel andpotentiallyfruitful avenueof research.

ACKNOWLEDGMENTS

WeareindebtedtoW.Fritchforhelpwith tissue cultures andto R.Chesney for typingthemanuscript.

Supported by Public Health Service grants AG 03106 and NS 12674from the NationalInstitutes of Health andbythe

Common-wealth Fund.

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Figure

FIG.1.exposedglioblastomacellsmousecontrolbrainbars)sentativeConcentratedstimulatevitrolentstrate,treatedtreatedhamster(Bottom)tedterminesymbols).sentative Mitogenic effect of conditioned medium from repre- CJD and control cell lines
TABLE 1. Stimulation of 3T3 colony formation by conditioned medium
FIG. 3.728hematoxylin. Effects on GFAP expression in indicator cells by conditioned media from representative control TC 760 (A) and CJD cultures TC (B)

References

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