JOURNAL OF VIROLOGY, Sept. 1988, p. 3103-31080022-538X/88/093103-06$02.00/0Copyright © 1988, American Society for Microbiology

Growth Factor Production by Creutzfeldt-Jakob Disease Cell LinesEMILIA L. OLESZAK,t GEOFFREY MURDOCH, LAURA MANUELIDIS, AND ELIAS E. MANUELIDIS*

Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510

Received 14 September 1987/Accepted 19 May 1988

Creutzfeldt-Jakob disease (CJD), a progressive dementia of humans, is caused by an infectious agent that isclosely related to the scrapie agent of sheep. Although the molecular nature of these "unconventional" agentsis still a matter of speculation and controversy, even less is known concerning the mechanism(s) of their effectson the central nervous system. To gain insight into the cellular effects of these agents, we have examined a seriesof cell lines derived directly from CJD-infected hamster brain or produced from nontransformed rodent linesby exposure to CJD infectious fractions in vitro. These cell lines appear transformed by a variety of criteria andsecrete growth factors into the culture medium. All CJD lines produce a factor that is like a-transforminggrowth factor (a-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glialfibrillary acidic protein in normal astrocytic cells in vitro. This effect is mimicked by purified a-TGF andplatelet-derived growth factors. Further study of CJD-induced growth factor production may elucidatefundamental properties of these unconventional agents.

The molecular nature of the agents (7) causing the uncon-ventional viral diseases (Creutzfeldt-Jakob disease [CJD],scrapie, kuru) is still a matter of intense speculation andcontroversy (16, 18, 25, 26). Little attention has been givento the observation that cells from scrapie-infected brainsreadily produce permanent or immortalized lines in vitro (3,4, 8, 12). Furthermore, in more than 20 independent at-tempts, cells from almost all CJD-infected mouse and ham-ster brains yielded permanent cell lines, while cells fromcontrol brains generally gave rise to senescent cultures (12).We have also recently shown that in vitro exposure ofBALB/c 3T3 cells and other normal early-passage primaryrodent cell lines to CJD infectious preparations reproduciblyinduces transformation, whereas treatment of the same lineswith normal brain preparations does not (24). This and otherbiological properties are reminiscent of retroviruses (17).However, up to the present time no retroviruses or otherconventional viruses known to elicit either central nervoussystem disease or transformation have been demonstrated innormal or CJD-infected brain tissue, despite a careful searchby many investigators.

In each of two cultures assayed, inoculations of homoge-nized long-term (multiply passaged) CJD cell lines producedthe characteristic incubation features, clinical symptoms,and histologic brain lesions of CJD (12). These data indicatedthat the agent was obviously replicating in these lines. Thetiter of infectivity was significantly lower than in CJD-infected brain tissue and thus the cell lines are not an idealsource for agent purification. We considered that detailedcharacterization of the transformed phenotype of these celllines could provide insight into fundamental biologic prop-erties of the agent which relate to its central nervous systemeffects. This initial study demonstrates that all CJD-trans-formed cell lines, whether derived from brain or treated invitro with infectious material, appear to produce a factor thatis like a-transforming growth factor (a-TGF). This resultsuggests that the CJD agent has induced transformation by asimilar mechanism in each cell line. The potential in vivosignificance of this phenomenon is illustrated by the ability

* Corresponding author.t Present address: Department of Pathology and Laboratory

Medicine, University of Texas Medical School, Houston, TX 77030.

of conditioned media from CJD-transformed cell lines (aswell as purified a-TGF) to increase glial fibrillary acidicprotein (GFAP) production in vitro. This effect mimics theincreased astroglial GFAP production seen during the courseof the disease in vivo (13, 19).

MATERIALS AND METHODS

Cell lines. Immortalized cell lines from CJD-infectedmouse (TC 740 and TC 746) and hamster brains (TC 724 andTC 728) were established from explant cultures as previouslydescribed (12). The passages utilized in these studies arecapable of forming tumors in nude mice and can therefore beconsidered transformed (11, 14, 17; unpublished data). Cul-tures of normal mouse brain (TC 760) and normal hamsterbrain (TC 744A and TC 763) were established in parallel.With serial passage, these control lines generally becamesenescent. Low passages of these lines contain approxi-mately 50% astrocytes by morphologic and immunocyto-chemical examination. Similar preparations have previouslybeen used by other groups to study astrocyte growth prop-erties (31).

In vitro CJD-transformed cell lines were derived from 3T3cells and the control hamster line (TC 744A) by treatmentwith CJD brain homogenates (TC 744A/CJ, 3T3/CJ-H) orcrude CJD synaptosomal preparations (3T3/CJ-SM) as de-scribed previously (24). 3T3 cells treated with the corre-sponding fractions of normal brain were designated 3T3/N-Hand 3T3/N-SM. A highly malignant methylcholanthrene-induced mouse glioblastoma cell line (TC 509), which pro-duces numerous type C retroviral particles was used as anadditional control (15).

Preparation of conditioned medium. Confluent cultureswere thoroughly rinsed in serum-free Delbecco minimalessential medium (DMEM) followed by a 16-h incubation at37°C in DMEM to remove residual serum proteins. Thecultures were then incubated in fresh serum-free DMEM for48 h, and the resultant conditioned medium was adjusted to1 mM phenylmethylsulfonyl fluoride, clarified by centrifuga-tion (20,000 x g for 20 min), and stored at -20°C. In somecases, conditioned medium was concentrated 20- to 70-foldby using Diaflow YM-10 membranes (Amicon). For ease ofcomparison between samples in figures, all volumes of added

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3104 OLESZAK ET AL.

conditioned media are expressed as the original volumebefore concentration.

Assays of DNA synthesis and colony formation. IndicatorBALB/c 3T3 cells (original stock generously provided by P.Besmer, Sloan-Kettering Cancer Center) were seeded onto24-well plates (2 x 105 cells per well) in DMEM with 10%fetal calf serum. After attachment, the cells were rinsed inserum-free medium and arrested in Go by a 48-h incubationin serum-free medium. Various amounts of conditionedmedium and [3H]thymidine (1 piCi/ml, 45 Ci/mM; NewEngland Nuclear) were then added. Acid-precipitable radio-activity was measured 24 h later for assessment of reinitia-tion of DNA synthesis (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) weresuspended in 1.2% methylcellulose F4M (Dow ChemicalCo.) with DMEM and 10% fetal calf serum containing 300 ,ulof conditioned medium per ml and plated on a base layer of0.9% agar (Bacto; Difco Laboratories). At 1 week later, cellswere refed with fresh medium and 150 RI of conditionedmedium. Colonies of >20 cells were scored 2 weeks afterinitial plating, as previously depicted (24). Tests with Sea-Plaque agarose (FMC Corp.) generated comparable results.EGF radioreceptor assay. Epidermal growth factor (EGF)-

like factors were quantitated by a commercial radioreceptorassay on the basis of A431 cell membranes (BiomedicalTechnologies), following the protocol of the manufacturer.The ability of portions of concentrated conditioned mediumto compete with 1251I-labeled EGF binding was comparedwith 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 medium or pu-rified growth factors were added for an additional 3 to 4days. After being rinsed in serum-free DMEM, cells werefixed for 5 min with buffered 4% paraformaldehyde and theywere permeabilized for 1 min with cold methanol. GFAPimmunoreactivity was determined with a non-species-specif-ic polyclonal anti-GFAP kit (Miles Scientific), using theperoxidase-antiperoxidase method. This antibody selec-tively delineates GFAP fibrils in reactive astrocytes in brainas previously depicted (19).

RESULTS

Figure 1 (top) shows the effect of unconcentrated serum-free conditioned media from the various cells lines on DNAsynthesis in GO-arrested normal 3T3 indicator cells. Condi-tioned medium of cell lines from CJD-infected brains (TC728, TC 724, TC 746, TC 740) and lines exposed to CJD invitro (TC 744A/CJ, 3T3/CJ-H, 3T3/CJ-SM) typically stimu-lated [3H]thymidine incorporation in the indicator cell lineup to 25-fold, equal to or greater than the effect of freshserum-containing medium. In contrast, conditioned mediumfrom control lines (TC 760, TC 509) and from lines treatedwith fractions of normal brain (TC 744A/N, 3T31N-H, 3T3/N-SM) showed only minimal stimulation of DNA synthesis.Notably, conditioned medium from control TC 509, derivedfrom a methylcholanthrene-induced glioblastoma which pro-duces type C virus, had no comparable stimulatory effect.

In order to quantitate growth factor production, condi-tioned medium was concentrated 20- to 70-fold by ultrafil-tration and the effects of serial dilutions of this concentrateon 3T3 cell DNA synthesis were assayed. The resultant

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FIG. 1. Mitogenic effect of conditioned medium from repre-sentative CJD and control cell lines. (Top) Conditioned medium ofcells derived from CJD-infected hamster (TC 728 and TC 724) ormouse (TC 746 and TC 740) brain and from nontransformed cellsexposed to CJD in vitro (TC 744A/CJ, 3T3/CJ-H, and 3T3/CJ-SM)stimulate DNA synthesis in GO-arrested cells, whereas media ofcontrol lines do not (e.g., normal mouse brain line TC 760, mouseglioblastoma TC 509, and control cell lines exposed to noninfectiousbrain fractions [TC 744A/N, 3T3/N-H, 3T3/N-SM]). Unconcentra-ted conditioned medium was added at 0.1 (hatched bars) or 0.4 (openbars) ml. Values shown are averages of triplicate determinations.(Bottom) Concentrated conditioned media (CM) were used to de-termine relative amounts of growth factor production by repre-sentative CJD lines (open symbols) and control cultures (solidsymbols). Pool-N is a pool of normal supernatants from normalhamster brain line TC 763, normal mouse brain line TC 760, and invitro normal-treated TC 744A/N. Bar represents standard deviation.Concentrated conditioned medium, expressed as milliliter equiva-lents of original conditioned medium, i.e., 0.1 ml of a 1Ox concen-trate, is equal to a 1-ml equivalent of conditioned medium. Culturestreated with brain homogenates are designated with an H and thosetreated with synaptosome-enriched fractions are designated SM.

dose-response curves (Fig. 1 [bottom]) show that the amountof growth factor produced by the series of CJD cell linesvaries over nearly a two-log range. In general, 3T3 cell linesexposed to CJD in vitro produce the least amount of growthfactor. However, this low production of growth factor ap-pears to relate to the underlying cellular phenotype ratherthan to the process of in vitro exposure, because a normalhamster brain cell line similarly exposed to CJD in vitro (TC744A/CJ) produced large amounts of growth factor. Its

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CREUTZFELDT-JAKOB DISEASE GROWTH FACTOR TRANSFORMATION 3105

TABLE 1. Stimulation of 3T3 colony formation by conditioned medium

Supematant source Colonies/disha CloningMedium (species) (mean + SD) efficiency (%)b

Normal brain culture TC 760 (mouse) 4 ± 1 0.2TC 763 (hamster) 3 ± 1 0.2

CJD brain culture TC 746 (mouse) 195 ± 14 5.1TC 740 (mouse) 177 + 33 8.8TC 724 (hamster) 487 ± 9 48.7TC 728 (hamster) 308 ± 80 30.8

Control treated in vitro 3T3/N-H (mouse) 5 ± 1 0.53T3/N-SM (mouse) 15 ± 3 0.3

CJD treated in vitro 3T3/CJ-H (mouse) 173 ± 25 17.33T3/CJ-SM (mouse) 188 ± 5 3.8TC 744A/CJ (hamster) 80 ± 5 8.0

a>20 cells per colony.b Only CJD lines showed a significant effect on colony formation (actual colonies formed) and cloning efficiency (percentage of colonies formed per cells plated).

control counterpart exposed to uninfectious brain (TC 744A/N) produced no significant comparable growth factor. Con-ditioned medium from CJD cultures also stimulated anchor-age-independent growth of indicator cells. Table 1 shows theeffect of culture supematants of CJD lines on 3T3 cell colonyformation in soft agar compared with that of the superna-tants of various control lines. Again, all CJD lines, whetherderived from brain tissue or by in vitro treatment, stimulatedcolony formation, whereas control lines did not.Table 2 summarizes the biochemical properties of the

growth factor(s) produced by CJD lines. The mitogenicfactor is trypsin labile but heat and acid stable. Theseproperties are consistent with a low-molecular-weight pep-tide factor. The sensitivity to treatment with dithiothreitolsuggests that the factor consists of peptide chains joined bydisulfide bonds (like PDGF or P-TGF) or has functionallyimportant intrachain disulfide bonds (like EGF and a-TGF)(9, 21).The above-mentioned biologic activities and biochemical

properties of the CJD-induced growth factor are all fulfilledby a-TGF. Because o-TGF binds with high affinity to EGF

TABLE 2. Percent stimulation of DNA synthesis in Go 3T3indicator cells by supernatants of CJD cell lines

% Stimulation in indicated cell lineTreatmenta

3T3/CJ-H 3T3/CJ-SM TC 744A/CJ TC 724 TC 728 TC 740

None 100 100 100 100 100 100Heatb 89 NDC 97 94 114 118Acidd 108 101 101 100 104 100Trypsin ND 107 106 111 ND ND

controleTrypsinf 15 11 25 19 31 32DTT8 26 15 9 19 11 22

a Secreted growth factors from all CJD lines were heat and acid stable, butinactivated by trypsin and dithiothreitol. Inactivation of growth factors inconditioned medium was measured by [3H]thymidine incorporation in Go-arrested cells.

b 56°C for 30 min.c ND, Not determined.d Dialysis against 0.17 M acetic acid for 16 h at 4°C followed by dialysis

against saline.e 100 ,ug of trypsin inhibitor per ml in the presence of 50 ,ug of trypsin

(Sigma) per ml for 2 h at 37°C.f 50 pg/ml at 37C for 2 h.8 Dithiothreitol (DIT) treatment was at 0.065 M for 2 h at 22°C, followed by

dialysis against saline.

receptors, concentrated conditioned media from control andCJD cell lines were therefore evaluated in an EGF radio-receptor assay (Fig. 2). The conditioned medium from theCJD cell lines contained varying amounts of EGF-like activ-ity equivalent to 60 to 600 pg per ml of EGF (10-10 to 10-11M). This concentration of ot-TGF may be sufficient toaccount for the observed stimulations ofDNA synthesis andanchorage-independent growth in the indicator BALB/c 3T3cells (1, 9). However, a potentiation of ot-TGF activity by3-TGF or other growth factors has not been ruled out.Because a-TGF production appeared to be a fundamental

feature of cell lines derived from CJD-infected brains, it wasof interest to determine whether a-TGF could account forany of the observed in vivo effects of CJD infection. Themost consistent morphologic feature of CJD is hypertrophyand possibly hyperplasia of astrocytes (gliosis). Biochemi-cally, this hypertrophy is the consequence of a markedlyincreased production of the astroglial-specific intermediatefilament GFAP. To date, most studies of the effects ofpeptide growth factor effects on astrocytes have focused on

ml equivalents CM added (or ng EGF)

FIG. 2. Conditioned media from representative CJD lines (opensymbols) and from control lines (solid symbols) were tested atvarious concentrations for binding to the EGF receptor by using acommercial radioreceptor assay (Biomedical Technologies). Onlyconditioned media from CJD lines effectively competed with EGFfor the EGF receptor and were compared with known EGF concen-trations (+).

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FIG. 3. Effects on GFAP expression in indicator cells by conditioned media from representative control TC 760 (A) and CJD cultures TC728 (B). Only the latter shows numerous brown (peroxidase)-stained GFAP cell bodies and processes. Nuclei were counterstained withhematoxylin. Magnification, x 100.

the stimulation of cellular proliferation (27, 32), although onepreliminary study suggested that fibroblast growth factor canregulate GFAP expression (5). Therefore, the effects ofconditioned medium from the CJD cell lines and purifiedoa-TGF on GFAP production in low-passage mouse brain cellcultures were examined. After a 3-day incubation, thesecells showed a modest increase in saturation density, a

prominent increase in branching astrocytic processes, and a

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

while P-TGF alone had significantly less effect. The potencyof conditioned medium in inducing these effects comparedwith that of the purified growth factors appeared somewhatgreater than expected from its a-TGF-like factor contentmeasured by the radioreceptor assay. Therefore, productionof additional synergistic factors by the CJD lines cannot beruled out.

DISCUSSION

The marked gliosis seen in brains infected with CJD ismore than can be accounted for as a secondary response totissue destruction (13). In fact, the stimulation ofGFAP gene

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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 whenselected brain regions are examined. The near-global activa-tion of astrocytes is a fundamental part of the pathophysiol-ogy of this class of diseases. It could be the consequence ofa direct interaction between the infectious agent andastrocytes, or it could be caused by an agent-induced factorwhich influences astrocytic gene expression throughout thebrain.The production of a-TGF activity by CJD-derived cell

lines and the ability of conditioned medium (or purifiedgrowth factor) to stimulate GFAP expression in culturedastrocytes suggest that the second hypothesis deservesfurther consideration. Growth factor production is fre-quently a component of the transformed phenotype. How-ever, many tumor lines, including the malignant glioma lineused here as a control (TC 509), do not produce a-TGFactivity. The relatively high initial growth rate of cells inprimary cultures of CJD-infected brains suggests that theresultant established lines may have produced an a-TGF-likefactor from their inception and prior to the acquisition of acomplete transformed phenotype. These lines may be de-rived from cells induced in vivo to produce a-TGF by theCJD agent. The mechanism(s) of the stimulation of growthfactor production by the unconventional CJD agent is 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 directlyencode growth factors (2, 30). Additionally, oncogenic vi-ruses can activate a variety of oncogenes by transduction orinsertional mutagenesis, with consequent stimulation ofgrowth factor production (21). Both the underlying host cellphenotype and the specific oncogene(s) activated may deter-mine which specific growth factor is produced. Notablyhowever, all CJD lines, regardless of lineage, produce ana-TGF-like factor. Some glial cell lines have been shown toproduce factors that are like platelet-derived growth factor(23) and contain v-sis-related mRNA (6). The exact lineageof the transformed CJD brain lines is uncertain, because oftheir poorly differentiated state.On a broader level, the as yet undefined relationship

between the transformed phenotype of CJD cells and theneurophathology of infectious dementias may prove to be ofcritical importance. Although the gliosis present in CJD-infected brains approaches levels reminiscent of low-gradeastrocytomas (13), hamsters infected with CJD or scrapie donot develop obvious brain tumors. However, it is nowapparent that tumorigenesis typically requires the activationof more than a single oncogene (10). In addition, oncogenicpotential need not be expressed within the central nervoussystem as obvious tumor formation. The cells infected by thetransforming JC papovavirus have a cytologically malignantphenotype, yet the gross pathologic lesion in adult brain istypically demyelination (33). Similarly, transgenic mice car-rying an activated simian virus 40 T-antigen gene develop ademyelination syndrome (28), although tumors may be pro-duced in visceral organs (22). In this context, the investiga-tion of oncogenic expression in infectious dementias is anovel and potentially fruitful avenue of research.

ACKNOWLEDGMENTS

We are indebted to W. Fritch for help with tissue cultures and toR. Chesney for typing the manuscript.

Supported by Public Health Service grants AG 03106 and NS12674 from the National Institutes of Health and by the Common-wealth Fund.

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