Distinguishable transformation-defective phenotypes among temperature-sensitive mutants of Rous sarcoma virus.

JOURNAL OF VIROLOGY, Mar. 1977,p.1042-1055 Copyright©D 1977 AmericanSociety for Microbiology

Vol.21,No. 3 Printed inU.S.A.

Distinguishable

Transformation-Defective Phenotypes

Among

Temperature-Sensitive Mutants of Rous Sarcoma

Virus

DOROTHEA BECKER,* REINHARD KURTH,' DAVID CRITCHLEY,2 ROBERTFRIIS, AND HEINZ

BAUER

InstitutfarVirologie,FachbereichHumanmedizin, 6300 Giessen,FederalRepublicof Germany,*and

Imperial

Laboratories,

Received for publication28October 1976

Eight transformation-defective, temperature-sensitive (ts) mutants of the

Prague strain of Rous sarcoma virus, subgroup A, have been isolated after

mutagenesis with 5-bromodeoxyuridine followed by selection on the basis of

focus tests. Five of these mutants, ts GI201, GI202, G1203, G1204, and G1205,

exhibit properties like most previously reported isolates in that they show a

temperature-sensitive response to each of a variety of transformation-specific

parameters tested. Interestingly, GI201, in addition to the

temperature-sensi-tive defect, carries a lesion that was observed as a nonconditional loss of

expression ofplasminogen activator protease. Three mutants, ts GI251, GI252,

and GI253, have been designated partial transformation-defective (PTD)

mu-tants sincethey behave as ts mutants accordingto sometestsfortransformation

andaswild typeaccordingtoothers. These threemutantsfail to form foci at the

nonpermissivetemperature (4100) andarenontumorigenicin3-week-old

chick-ens (body temperature, 420C). The agglutinability by concanavalin A ofcells

infected with thesemutants shows a definite temperature sensitivity, as do the

rate of 2-deoxyglucose uptake and the disappearance of the 250,000-dalton

normal cell glycoprotein (large, external, transformation sensitive [LETS]).

Although the PTD mutant-infected cells, unlike cells infected with other

trans-formationmutants, exhibit a cell-bound plasminogen activator protease atthe

nonpermissivetemperature, this activator is not detectable as afree proteasein

the medium, as it is with wild-type, virus-infected cells. The PTD mutants

behavelike the wild-typeparent intheir abilityto inducetransformed growth

properties in the infected cells, i.e., growth beyond normal cell saturation

density with or without serum-supplemented medium and growth leading to

colony formationinsoft-agar- or methyl cellulose-containing suspensionmedia.

A great variety ofchanges in cell behavior,

form, and metabolic activity are characteristic

of fibroblasts that have undergone

virus-in-duced transformation. Transformed cells fail to

respondto conditions thatinhibitthe growth of

normal cells, such as high population density (39),absence ofserumfrom themedium (13), or

absence ofa substrate for anchorage (23, 37).

Formation of characteristic foci makes

trans-formed cells apparentinamonolayer of normal

cells (38). Striking changes in the rate of trans-portofcertainsugars,which greatly exceed the range of values obtainedwith logarithmically growing or stationary normal fibroblasts (11,

IPresent address: Friedrich Miescher Laboratorium der MaxPlanck-Gesellschaft,74Thbingen,Federal Republic of Germany.

2Present address: Department of Biochemistry, Univer-sity of Leicester, Leicester LE1 7RH, England.

43), have been observed in transformed cells.

The increased mobility or altered distribution

of appropriatereceptorsresultsinsignificantly

enhanced agglutinability of transformed cells

by concanavalin A and other lectins (5, 32).

Increasedexpression ofaproteasemeasured as

aplasminogen activator (27, 30, 40, 41)occurs

ascells becometransformed. Itisprobable that

some proteolytic activity, in turn, influences

the structure of the cell surface by degrading

cellsurface proteins, among them the

250,000-dalton large, external,

transformation-sensi-tive (LETS) glycoprotein (12, 14, 34, 37, 45).

Various temperature-sensitive (ts) mutants of avian sarcoma viruses have been studied

with respect to certain ofthese parameters of

transformation (2, 4, 6, 9, 20, 22, 24, 47). In

general, mutants isolated by selection, based

on a focustest, have been temperature

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS 1043

tive for all otherparameters tested. Two

nota-ble exceptions have beenreportedinthe

litera-ture: ts LA334 bears a transformation defect, which, although it preventsfocus formation at thenonpermissive temperature, does not inter-dict formation of colonies in soft-agar

suspen-sion (9); andts LA25, which, though defective

for focus and colony formation at the

nonper-missive temperature, does stimulate cells to

grow todensities much higher than those ob-tained by normal cells (22, 47).

This paper will document the isolation of

eight transformation-defective (TD)avian

sar-comavirus mutants, three of which exhibit an interesting dissociation of transformation pa-rameters, i.e., respond as temperature-sensi-tive mutants accordingto certain testsbut

be-have like wild-type virus according to other

tests. Hence,twophenotypically different cate-gories emerged as analyses of these mutants

werecarried out, and theprofiles of these two

categories will bedescribed.

MATERIALS AND METHODS

Cells and viruses. Chicken embryo cell cultures were prepared from fertile white leghorn embryos after 9 to10daysof incubation. Eggs for this purpose weregenerously supplied from a leukosis virus-free flock by E. Vielitz, Lohmann Tierzucht GmbH, Cux-haven, West Germany. Tissue cultures of normal and infected chicken embryo cells were grown in Dulbecco-modified Eagle medium prepared from a

powder(L and S Labor Service, Munich, West Ger-many)supplemented with antibiotics, 5% heat-inac-tivated calf serum, and 10% tryptose phosphate broth (Difco Corp., Ann Arbor, Mich.).

The parent virus used for the mutagenization and isolation of mutants was the Prague strain of Rous sarcomavirus,subgroupA.The original parent had beenrepeatedlyclonedbyisolation, with an aspirat-ing pipette,of single foci occurring at high dilution inafocus test (33), and mutants subsequently ob-tained were routinely cloned at eachstock prepara-tion step. The mutant designations (ts GI201

through205;ts GI251 through253)conformto

sug-gestionsputforthby Vogtetal. (42).

The procedures for preparation of soft-agar

sus-pensioncultures(8)andmethylcellulosesuspension

cultures (31, 46) usedin tests forcolonyformation have been describedpreviously. The compositions

andprotocolsfor the agar caseinplaqueoverlaytest

and the soft-agar casein suspension culture have been reported by Goldberg (10) and Balduzzi and Murphy (3), respectively. In allcases Dulbecco-mod-ified Eagle medium wasused withtheappropriate supplements.

Testforin vivotumorigenicity ofmutants.

Chick-ens wereinoculatedinthewing webs of bothwings

at 21days of age and examinedbiweeklythereafter for appearance oftumors atthe siteof inoculation. Based on the method of Bauer(unpublished obser-vation), it was found that both wings could beused, using differentdoses to givetwoindependent

titra-tions of the inoculated sample in a single animal. Concanavalin A agglutination test. The concana-valin A agglutination test used was a modification ofthat of Pollack and Burger (28), which has been described in detail elsewhere (21). It was found nec-essary to use plastic pipettesand test tubes for all manipulations to minimize damage to the cell plasma membranes. Briefly, subconfluent chicken embryo cells were washed twice for 5 min each with phosphate-buffered saline (PBS), pH 7.2, on a rock-ing platform. Cells were suspended by 15 min of incubation at 37°C in PBS containing 0.02% (wt/vol) EDTA and, subsequently, were washed once in warm serum-free medium followed by centrifuga-tionfor 5 min at 1,200 x g. The cell number was determined with an electronic cell counter and ad-justed to 2 x 106/ml in PBS. Lyophilized concana-valin A (Sigma, St. Louis, Mo.) wasreconstituted in distilled water, dilutedinPBS tothe desired concen-trations, and predispensed in 5-,ul quantities into wells of a Microtest plate (type I, Falcon Plastics). Each well then received 104 cells in a

5-,gl

Determination of LETS glycoprotein. Chicken

embryo cells grown in90-mmdishes were washed three times with PBS and labeled by adding1mlof PBS containing 5 mM glucose, 0.1 U of glucose

oxidase, 20 ,Ag of lactoperoxidase, and 400 ,uCi of carrier-free sodium[251]iodide.After 10 min at room temperature, the reaction was stopped by adding8

mlof PBS containing 50 mM sodium iodide, and the cells were washed three times with thesamebuffer. Cells were scraped into PBS containing50 mM sodium iodide and2 mM phenyl sulfonyl fluoride, pelleted, and dissolvedat100°Cin 0.4mlofsample

buffer (0.1MTris, pH 6.8, 15% glycerol, 2%sodium dodecyl sulfate). Prior to electrophoresis, samples weremade 0.1 Mwith respect todithiothreitol and reheated to 100°C. The procedure has been described

indetail elsewhere (17).

Proteins were separated by using discontinuous sodiumdodecylsulfate-polyacrylamideslabgel

elec-trophoresis (17). Gelswerefixed and stained in 30%

methanol-10% acetic acid containing Coomassie

blue, dried ontopaper, andexposedtoKoderex X-ray film.

Determination of plasminogen activator. Insolu-ble ['25I]fibrinplates werepreparedasdescribedby

Unkeless et al. (41). Harvest fluidsweretaken from cellcultures that hadbeen washedoncewith

serum-freemedium,incubated for2hwith serum-free me-dium,andfinallywashed athirdtime with serum-free medium prior to incubation for12h inmedium

supplemented with 2.5% chicken serum (for cell-bound plasminogen activator determination) or in

unsupplementedmedium(for supernatant

plasmin-ogen activator determination). After collection of harvest fluids, the cell numberwasdetermined for eachplate, and the harvest fluidswerediluted with the appropriate unincubated medium so that the harvestunitcould be definedastheactivityin 1 ml

of medium incubatedover106 cellsfor12h. Harvest

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1044 BECKER ET AL.

fluidswerecentrifuged for15minat1,200 x gprior

to storage at -80'C until tested on insoluble

[1251]fibrin plates. The fibrinolytic reactionwas al-waysperformed at 370C, as previous experiments

(Friis, unpublished observations) had shown that

neither supernatant plasminogen activator nor

plasmin frommutantpreparations was more tem-perature sensitive than that fromwild-type

virus-infectedpreparations. The fibrinolyticreactionwas

performedwith 1 ml of harvest fluidperplate

con-taining,orsupplementedwith(accordingtothe type

of harvest fluid),2.5% chickenserum as a sourceof

plasminogen. The fibrinolytic reaction was

moni-tored over a period of 12 h, with 0.1-ml samples takenatintervals and countedinaliquid

scintilla-tioncounter. Forevaluationofplasminogen activa-toractivities, reaction timeswere chosen atwhich notmorethan 30 to50% ofthe available insoluble

[1251]fibrin (accordingto trypsindigestion controls) had beenreleased with the wild-type virus-infected preparationsormutantvirus-infectedpreparations from cellsgrownatthepermissivetemperature.

RESULTS

Isolation of mutants after

5-bromodeoxy-uridine mutagenesis. Cells infected with a

stock of cloned Prague strain Rous sarcoma

virus, subgroup A, were treated according to

the method of Bader andBrown (1) inthe dark

from the time of infectionuntil 12 h

postinfec-tion with100 jigof5-bromodeoxyuridineperml

in medium lacking the usual

tryptose-phos-phate broth supplement. At40hpostinfection,

thesupernatantswereharvested, andthe titer

of infectious virus in treated and untreated

sampleswasdeterminedinafocus test. Itwas

found that 5-bromodeoxyuridine treatment

re-ducedthe titerof treatedsamples byafactor of

about20. The 5-bromodeoxyuridine-treated

vi-rus wasplatedathigh dilutionsinafocustest,

and single foci were isolated as presumptive

mutagenized virus clones, whichweregrownto

small stocks in aMicrotestplate. Cloneswere

screenedfor theabilitytoreplicate andtoform

foci at permissive (350C) and nonpermissive

(41°C) temperatures in Microtest plate wells

usingaTiter-tecsemiautomatic dilutorsystem

(Flow Laboratories, Bonn) to facilitate

screen-ing. Out of440 clones examined, 8werefound

tobe temperaturesensitivefor focusformation.

None ofthe 440 clones exhibited temperature

sensitivity for replication ofprogeny; rather,

the titers produced at 41°C were consistently

higher byafactor of2or3than those produced

at35°C. The eighttemperature-sensitiveclones

observedwereserially clonedthreetimes,after

which their properties in various assays for

transformation were examined. Table 1shows

asummaryof results withsomeof themutants,

showing their relative abilities to form fociat

permissive and nonpermissive temperatures. Growth-related parameters of

transforma-tion. The ability of transformed cells to form

coloniesinasuspension medium of soft agar or

methyl cellulose is a measure of

anchorage-independent growth capacity, a property not

exhibited by normal fibroblast cells (23, 36).

Table 2 shows theresults of a test for the ability

ofseveral temperature-sensitivemutants to in-duceanchorage-independent growthininfected

cells at permissive and nonpermissive tempera-tures. Prague strain Rous sarcoma virus

(sub-group A) wild-type-infected cells formed

colo-nies with aboutthe same efficiency at either 35

or41'C,whereas normal cells failed todosoat

both temperatures. Significantly, the group of

ts mutants, GI251, G1252, and GI253,

desig-nated partial transformation-defective (PTD)

mutants, produced colonies like the wild-type

virus at both temperatures, in either a

soft-agar or methyl cellulose suspension culture.

The conventional TD ts mutants, GI201 and

GI202,aswell asGI203, GI204, and GI205 (data

not shown), failed to form colonies at the

non-permissive temperature.

The ability of a virus to induce

anchorage-independent growth is usually considered a

useful measure of the oncogenicity of a virus

strain (35). Therefore, several

temperature-sensitive mutantswere testeddirectly for their

ability to induce tumor formation in chickens,

which have a body temperature of 420C. The

birds were each inoculated with two dilutions,

oneineach wing web. The results of the

experi-ment aresummarized in Table 3, which shows

that although 60% of the animals formed

tu-mors with tsGI251, none formed tumors with ts

GI252, and only 10% formed tumors with ts

GI253 and with tsGI202, even though the doses

TABLE 1. Focus-formingtitersof differentmutantsa

Focus titer(FFU/ml) Focus-forming

Virus efficiency

350C 410C

(410C/350C)

WTPrA 8 x 105 8 x 105 1

ts GI201 8 x 105 <101 <10-4 tsGI202 1 X 106 1 X 102 1 X 10-4

ts GI251 2 x 106 <101 <10-5 ts GI252 7 x 105 <101 <1O-4

tsGI253 8 x 105 <101 <10-4

a Cloned virus prepared insmall stocks at

350C

wastitrated in a focus test performed in duplicate at

35 and 41'C. The number of foci appearing was

countedonday8after plating. FFU, Focus-forming units;WTPrA, wild-type Rous sarcoma virus, Pra-gue strain, subgroup A.

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS 1045

TABLE 2. Efficiency of colony formation in soft-agar and methyl cellulose suspension cultures

Fraction of inoculated cells formingcoloniesin:

Virus Soft agar Soft methyl cellulose

35°C 410C Efficiencyb 35°C 410C Efficiency

None <0.001 <0.001 <0.0001 <0.0001

WT PrAc 0.09 0.08 0.9 NDd ND

ts GI201 0.1 <0.001 <0.01 0.02 <0.0001 <0.005

ts GI202 0.3 <0.001 <0.003 0.02 <0.0001 <0.005

ts GI251 0.04 0.1 2.5 0.08 0.2 2.5

ts GI252 0.3 0.2 0.7 0.02 0.002 0.1

ts GI253 0.2 0.2 1.0 0.07 0.2 2.9

a Suspension culture mediawerepreparedaspreviously described (8, 31, 46). For the soft-agar colony test, 103cells infected at amultiplicity of infection of approximately 2 focus-forming units (FFU)/cell were plated on a35-mmplate. For the soft-methyl cellulose colony test,104cells, alsoinfected at2FFU/cell,were

usedon50-mmplates.

bThe efficiency of colony formation expresses the ratio of the number of colonies obtained at the nonpermissive temperature to that obtained at the permissive temperature. The values are underlined to

stresstheir derivative nature.

e WTPrA, Wild-type Rous sarcoma virus, Prague strain, subgroupA.

dND, Not done.

used with the mutant viruses were 100-fold

greaterthan thoseneededtoproducetumorsin

100%of the animals with the wild-type parent. Hence, asignificant degree of defectiveness for

tumorinductionwasobserved with allmutants

tested; thus the focus test, according towhich all mutants studied were temperature

sensi-tive, was abetter measure ofoncogenicity in

this case than the suspension culture test,

which had indicated that PTDmutantswere as

capable of inducing anchorage-independent

growth at the nonpermissive temperature as

the wild-type virus.

Anothermeasureof growth characteristic for

transformed cells, butnotnormal cells, is the

absence of, or increased, saturation density

(39).Table4shows that after 96 h, normal cells cultured inmedium containing5% calfserum

attain a maximum density much lower than

that ofmutant-transformed cellsatthe permis-sive temperature. Thisdensity, about 1.5 x 105 cells/cm2, remains relativelyconstant with

nor-mal cellsfor severaldaysmoreinculture,after

which the cells detachspontaneouslyas a

mon-olayer. Transformed cells, on the otherhand,

continuetoincreaseindensityuntilmedium is

exhausted or cellsspontaneously detach. It is

apparent from these results (Table 4) that

whereasts GI202-infected cellsgrowlike

trans-formed cells at 35°C and like normal cells at

41°C, ts GI253-infected cells behave like the

wild-typevirus-infected cells atboth

tempera-tures. Likewise, an experiment (Table 4)

con-TABLE 3. In vivo tumor formation in chickens (body temperature,420C)

Tumor-positive animals/total

ani-Dose malsinoculated after:

Vlrus(FFU)b 14 21 28 35

days days days days WT PrAc 102 8/10 10/10 3/3d 3/3d

103 10/10 10/10 2/2 2/2d

tsG1202 103 0/9 0/9 0/9 0/9

104 1/9 1/9 1/9 1/9

tsGI251 103 0/10 0/10 0/10 0/10 104 5/10 6/10 4/8d 4/8d

ts G1252 103 0/9 0/9 0/8e 0/8e

104 0/9 0/9 /8Me 0/8e tsG1253 103 0/12 0/12 0/12 0/12 104 1/12 1/12 1/12 1/12

a Groups of9 to 12 3-week-old white leghorn chickens fromaleukosis-free flockwereinoculatedinthewingwebs, eachwith 0.05 mlofvirusdilutedto contain theindicated dose. The right wing webs received a 10-fold-lower dose than the left, sothat the titrationcould be done at two dilutionswith aminimal number ofanimals. Independent titration of inoculainseparatewings ofasingle animal has been demonstrated (Bauer, unpublished observation). Ex-aminationsfortumorformationweremadebiweekly.

bFFU, Focus-formingunits.

cWTPrA,Wild-typeRous sarcoma virus,Prague strain,

subgroupA.

dSelected animals weresacrificed after21days because largenecrotictumorshad developed, renderingtheanimals moribund.

eOne animal died of causesunrelatedtovirus inocula-tion.

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TABLE 4. Cell densitiesobserved under variouscultural conditions

Cellscounted/cm2 (x 10-5)after 96 ha

Virus GM + 5%CSb GM -5%CSC GM agaroverlay+5%CSd

35°C 410C 41C/35aCe 355C 410C 41C/35aCe 355C 410C 415C/35Ce

None 1.7 1.2 0.7 0.4 0.6 1.5 1.1 1.2 1.1

ts G1201 ND ND ND ND 4.0 1.3 0.3

ts G1202 3.0 1.6 0.5 1.3 0.7 0.5 ND ND

ts GI251 ND ND ND ND 1.7 4.4 2.6

ts GI253 3.0 3.7 1.2 1.4 1.6 1.1 3.7 6.0 1.6

WTPrA 3.0 3.1 1.0 1.6 1.7 1.0 3.0 3.7 1.2

a Chickenembryo cellswereinfected with the various viruses at amultiplicity of infection of approxi-mately 2focus-forming units/cell, and after4days of incubation at 350C thecellswereseededintoduplicate

30-mmplates at a density of0.5 x 105cells/cm2 inmedia and attemperatures as indicated. After96 h, without medium change during the dayssinceseeding,liquid mediumculturesupernatantswerecollected

andpooled with cells suspended by trypsin treatment (0.25% trypsinfor 10 min at roomtemperature)from

the corresponding plate.Cells that had been overlayedwithagar-containingmedia werecollected after agar

had beengently aspirated using suction throughaPasteur pipette,followedby trypsintreatment tosuspend the cells. Thecell numbers weredetermined by an electronic cell counter,and the results areexpressedas

the cell number per squarecentimeterofplate surfacearea.

b Growthmedium (GM) wasDulbecco-modifiedEaglemediumcontaining10%tryptosephosphatebroth

and5%calfserum (CS).

c GMwasmade for footnoteb exceptthat CSwasabsent.

d GMwasmadeasfor footnote b except that24hafterseeding, plates received GMcontainingafinal

concentration of0.5%agar (Difco Corp.).

eThe ratio of the cell number per square centimeterobserved at 410Ctothatobserved at 35°Cispresented

tosummarize thedifferences betweenTDandPTDmutant-infected cells and betweennormal cells and

wild-type Rous sarcoma virus strain Prague,subgroupA (WTPrA)-infected cells. These derivative valuesare

underlined.

fND,Notdone.

ducted without serum in the medium showed

thatthe characteristic inhibition ofgrowth

ex-pected with normal cells (13)wasseen withts

GI202 only atthe nonpermissivetemperature,

and not at all with ts G1253. Finally, Table 4

showsanexperimentinwhichanagaroverlay,

inhibitory to normal cell growth, perhaps

ow-ing tothehigh concentration ofanionicgroups

containedinthe crudeagar, was usedto

dem-onstrate that whereas ts GI201-infected cell

growth is temperature sensitive under these

inhibitory conditions, ts GI251- and

G1253-in-fected cells exhibited no growth inhibition at the nonpermissive temperature. ts G1201, ts

GI202,ts GI203,tsG1204,andts GI205 behaved

alike in such experiments (data not shown),

whereas the PTD mutants behaved like the

wild type.

Figure 1 shows agrowth kineticexperiment

similar to that described in Table 4, which

clearlyshows thetemperatureindependence of

ts G1251-infected cellsfor growth inliquid

me-diumon asolid substrate and thetemperature

sensitivity ofts GI201-infectedcells.

ConcanavalinAagglutinability and hexose

transport as measuresof transformed cell

al-tered membrane functions. Lectin

agglutina-bility of cells infected by the variousmutants,

atpermissiveandnonpermissive temperatures,

was studied using concanavalin A. Table 5

shows thataccordingtothiscriterionof altered

membrane function in transformed cells,

pre-sumably resulting from increased mobility of

the lectin binding sites (32), the PTD and TD

mutants do not differ. A definite temperature

sensitivitywas apparentforall mutants.

Greatly increased transport of certain hex-osesandaminoacidsisratheruniquely charac-teristicoftransformedfibroblasts, sincenormal

cells, underany state of growth, never attain

transport levels equivalent to those of

trans-formed cells (11, 43). Therefore, the mutants

were examinedfor changes in the

2-deoxyglu-cose transport rate,which might occurafter a

shift ofinfectedcells from the nonpermissive to

the permissive temperature. Figure 2 shows

that the increase in rate of 2-deoxyglucose transport seen with ts GI201 is precisely

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS

05L

0 24 48 72

Hours post-infection

96

FIG. 1. Growth curves with ts GI201 (U)-and ts GI251 (A)-infectedcells, as well as with normal cells (0), is shown at 359C (open symbols) and 419C (closed symbols). Cells were infected with a multi-plicity of infection of2 focus-forming units/cell at time 0 and plated after absorption for 30 min at roomtemperature in suspension on 35-mm plates at

an initial density of 3.5 x 105 cells/plate in liquid medium. Cell numbers per plate were determined at intervals as shown.

TABLE 5. Agglutinationofmutant-infectedcells with concanavalinAa

Growth Degree of agglutination at concan-Virus temp avalin Aconcn(gg/ml)b of:

mutant tOC_

('C) 0 62.5 125 250 500

None 35 - - - - +

41 - - - - +

tsGI201 35 - ++ ++ +++ ++++

41 - - - - +

tsGI202 35 - +++ +++ ++++ ++++

41 - - - - +

tsGI251 35 - +++ ++++ ++++ ++++

41 - - -

-tsGI252 35 - +++ +++ ++++ ++++

41 - - - + +

tsG1253 35 - +++ ++++ ++++ ++++

41 - - - -

-aTheprocedureused is describedinMaterials and Meth-ods.

bAgglutinationwasscoredmicroscopicallyasfollows:-, 90 to100%; +,70 to90%; ++,50 to70%; + ++, 30to50%; + + ++,0 to 30%singlecells.

flected inasimilarincreasedrate seenwithts

GI251after shifttothe permissivetemperature.

Table 6 shows the normalized values obtained

withseveral mutant-infected celltypes,normal

cells, and wild-type virus-infected cells. A

sig-nificanttemperaturesensitivitywas seenwith

allmutantsfor thistransportfunction(datafor

ts G1203,tsG1204, andtsGI205notshown). The

values obtained for the three PTDmutants at

the nonpermissive temperature were,

regu-larly, slightly higher thanthoseseen with the TD mutants or normal cells. This is perhaps

explainable inthat PTD-infected cells are not

subject, atthe nonpermissive temperature, to

the growth inhibition that, even though the

cultures were still subconfluent, may already

have begun to retard mutant-infected or

nor-mal cell growth. That the state of cell growth

influences hexose transport rates to some

de-gree (two- to threefold) is recognized (24, 43).

The verylargedifferential between uptake by

normal phenotype and transformedphenotype

cells is probably attributable to the compound

influences of transformation and density

inhi-bition ofnormal cellgrowth.

Examination of expressionof LETSprotein

inmutant-infected cells.Transformed cells

ex-hibit reduced amounts or a total absence ofa

250,000-daltonglycoprotein,the so-called LETS

glycoprotein (12, 14, 34, 37, 45). Inthepresent

study the level of LETSglycoproteinwasfound

to be temperature sensitive in cells infected

with ts GI202, ts GI251, ts GI252, andts GI253

as detectedeitherbylactoperoxidase-catalyzed

cell surface iodination (Fig. 3) or Coomassie

blue stainingoftotalcell protein(Fig.4).

Inter-estingly, cells infectedby ts GI201 retained a

considerable amount of LETS glycoprotein

35-A

? 30

-u

25-in 0

:o

20-0o 15

-'a 5 Wa

0 2 6

Hours after shift ('2'C-351C) 18

FIG. 2. Uptake of 2-f3H]deoxyglucose bytsGI201 (i)-andtsGI251 (A)-infectedcellsatvarious inter-valsafterashift from the nonpermissive tothe

per-missivetemperatureis shown.Chicken embryocells

were infected as described in footnote a, Table 6,

transferred into 30-mmdishes, and maintained for 18 hat41'C.Atintervals,plateswereshiftedto35'C;

at36 hafter transferallplates werewashed,and

2-PH]deoxyglucose uptake was determined as

de-scribed in Table 6. The results are expressed as

countsperminuteper106rcells.

obL

CL;

aB

co

I~~~~~~~~~~~~~~~~~~~

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1048 BECKER ET AL.

TABLE 6. 2-Deoxyglucoseuptake bynormal and virus-infected cells

2-Deoxyglucoseuptake(cpm/101cells)a Virus

350C 410C 410C/350Cb

None 1,700 1,700 1.0

WTPrAc 28,000 25,000 0.9

tsGI201 19,000 1,400 0.07

ts G1202 23,000 1,800 0.08

tsGI251 26,000 2,100 0.08

ts GI252 28,000 2,400 0.09

ts G1253 29,000 3,600

0.12-a Chicken embryo cells were infected at a

multi-plicity of approximately 2 focus-forming units/cell

and, after3days of incubation at350C,were trans-ferred to 30-mm plates at the temperatures indi-cated, with an initial density of 0.5 x 105cells/cm2.

Two days later, when the cells were nearly confluent with approximately 1.2 x 105 cells/cm2, the plates weregently washed twice with glucose-free Hanks balanced salt solution (H minus) at410C,incubated with 0.5,uCi of2-[3Hldeoxyglucose-supplemented H minussolution for 10 min at410C,andwashed three times withice-cold H minus solution. The cells were suspended using trypsin (0.05%trypsinfor 10 min at roomtemperature), asample was counted with an electronic cell counter, and 80% of the total was lysed with 1% Triton X-100 followed by liquid scin-tillation counting in a water-compatible mixture. The results obtained are expressed as counts per minuteobserved per 106 cells present in the sample. bThe ratio of the counts per minute uptake mea-sured withcells grown at410Ctothatobserved with cells grown at 350C is presented to illustrate the differences observed with the different mutant-in-fected andwild-type virus-infected cells. The values

areunderlined to stress their derivative nature. e WTPrA, Wild-type Rous sarcoma virus, Prague strain, subgroupA.

even when grown at 35°C (50% of the level

observedat 41°C), although accordingto other

parameters, asshown above,ts GI201-infected

cellsat35°C arefully transformed. This result

wouldsuggest that ts GI201 isnonconditionally

defective foranactivity thatisresponsible for

the loss of LETS glycoprotein in transformed

cells.

Measures of plasminogen activator

pro-teasewith mutant-infected cells. The finding

that loss of LETS glycoproteinwastemperature

sensitive with all mutants tested suggested

thatexpression ofplasminogen activator pro-tease (40) or other protease activities (7, 44)

might be defective at the nonpermissive

tem-perature,sinceproteaseshavebeen proposedas

onepossible mechanism by whichLETSprotein

might be lost during transformation (15, 18). Experimentswerefirstperformed usingafocus test modified to include opaque casein in the overlay as a substrate for protease activities, especially plasmin (10). It was found that whereas TD mutant-infected cells failed to

pro-duce clear areasorplaques in the overlay at the nonpermissive temperature, the cells infected with PTDmutantsdid, though somewhat later than wild-type virus-infected cells (data not shown). At the permissive temperature, all

mu-tants tested, as well as wild-type virus, could induce plaques over cells, whichwerelater ap-parent as foci of transformed cells. Thedelayed positive plaque formation with PTD mutant-infected cells at the nonpermissive temperature suggested some protease activity, though prob-ably less than that induced by wild-type virus.

A second type of caseinolytic test was

per-formed basedonthe suspension cultureplaque

testof Balduzzi and Murphy (3). Figure5shows

the results withtsGI202- andts GI253-infected

cells in suspension culture at 35 and 41°C. Cleared areas orplaquesare seen with tsG1202 only at the permissive temperature, whereas with ts GI253 plaques are numerous at both temperatures,being ofevenlargerdiameterat the nonpermissive temperature. These tests

had been incubated for only3days,sothe

abil-ityofts GI253toform coloniesinsuspensionat

the nonpermissive temperatureplayedno

ma-jor role. Single transformed cells are

appar-ently capableofactivatingsufficientplasminto

produceaplaque. Theplaques observedinthis

test with wild-type virus-infected cells (data

not shown) were similar to those seen with cells

infectedwith PTD mutants;hence,accordingto

thisassay, thePTD mutants, in contrast to TD mutants, were notdefective atthe nonpermis-sive temperature for a plasminogen activator protease.

Quantitative measurements of plasminogen

activator wereperformed using the fibrinolytic assay of Unkelessetal. (41). This fibrinolytic

activity has been identifiedasarisingfrom the

actionofurokinase-likeprotease (plasminogen

activator;40) onthezymogenplasminogen,

re-sulting in activation of plasmin, the protease

actually measured with the insoluble

[125I]fibrin test under the conditions used. To

meetthe requirements ofa testfor temperature

sensitivity, it was necessary toobtainharvests

of plasminogen activator from cells grown at

either 35 or41°Cand tobeabletomake compar-isons of their activities. Hence, separate

har-vestfluidsweretaken from cellsatthese

tem-peratures and reacted on cell-free plates

con-taining insoluble [125I]fibrinat asingle

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS

-OK

41

0c

350

c

a

b

c

d

e

g

h

- -~~~-- wo

FIG. 3. Iodinated cell surface proteins of mutant-virus-infected cells grown at 35 and410C. The LETS band is prominent in cells infected with all mutants at 41'Cand with normal cells, but is significantly reduced with mutantsat350C and wild-type virus-infected cells. Cells were labeled using the lactoperoxidase system when nearing confluence. Gel tracks contain equal amounts of cell protein. (a-e) Cells grown at 41°C: a, ts GI201; b, ts GL202; c, tsG1251;d, tsGI252; e, ts G1253.(f-j) Cells, in the same order, grown at350C. (k) Nor-malcells. (1) Wild-type-infected cells grown at350C.The kand 1 LETS bands are displaced relative to others because these samples were subjected to electrophoresis on a separate slab gel.

-

41

0c

a

b

c

d

ON

-44

350

c

e

f

g

h

I

V"O

fflK * ~ 4_01

1. -I.

lt

:;3;

z~~lo4O

FIG. 4. Total cellular proteins of mutant-virus-infected cells grown at 35 and 41°C, as detected by

Coomassieblue stain. The tracksarelabeledasinFig.3.Tracks handjreceivedmarkerprotein of 130,000 (E. coli /3galactosidase) and 68,000 (bovine serum albumin) molecular weight, respectively. The band

correspondingtothemajoriodinatedprotein (LETS; molecular weight, 250,000) is also indicated.

*i

i

k

I

:.-j

4250

-v130

1049

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1050 BECKER ET AL.

a

FIG. 5. Suspension culture system containing casein is presented to indicate the plasminogen activator expressionoftsGI202-infected cells (a and b) andtsGI253-infected cells (c and d)atpermissive (a and c) and nonpermissive (b andd) temperatures.

ature,

370C,

activator was no more temperature sensitive

than such plasminogen activator derived from

wild-typevirus-infected cells.

Based onthe experimentswith the

caseinoly-tic assay, a method was used to distinguish

betweencell-boundand supernatant activator.

This was possible because harvest fluids

ob-tained from well-washed cells, incubated in a

medium lackingserum asplasminogensource,

couldonlycontainplasminogenactivator. Such

plasminogen activatorwasmixed withchicken

serum directly on the insoluble [1251]fibrin

plate, so that the final measurement was

ac-tually plasmin-digested [1251]fibrin. On the

other hand, cell-bound plasminogen activator

could bemeasuredby obtaining harvest fluids

from cells incubatedinthe presence of chicken

serumas aplasminogen source. Of course,this

assay ofcell-bound activity must include also

the supernatant plasminogen activator, which

will have simultaneously activated

plasmino-genduring the incubation; however, the actual

problem with the mutantsstudiedhas beento

recognizecell-bound plasminogen activator

un-der conditions where supernatantplasminogen

activator is absent. Furthermore, supernatant

plasminogen activator, perhaps because its

half-life isshorter thanthat of plasminduring

the 12-h incubation on cells, always gives a

reaction of lower activity on the insoluble

[I25I]fibrin plates than is seen with the

cell-bound activatortest.

Results of a typical experiment using these

procedures to measure cell-bound (including

supernatant) plasminogen activator, compared

with those measuring supernatant

plasmino-gen activator alone, are shown in Table 7. ts

GI201 is ofparticular interest because it

ex-hibits almost no detectable supernatant

plas-minogen activator at either temperature, and

the cell-boundactivity is alsoextremelylow. ts

GI202 is simply temperaturesensitive for both

types of plasminogen activator. The PTD

mu-tants, on the other hand, although strikingly

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS 1051 TABLE 7. Temperaturedependence ofplasminogen

activator protease expression byvirus-infectedcells [1251]fibrin solubilized/harvest unita Virus Cell-bound

plasmino-

Supernatant

plas-gen activator" minogenactivatore

35aCd

None 630 260 330 380

WTPrAe 31,800 27,600 13,500 10,900

ts GI201 2,200 900 690 360

ts GI202 24,000 460 11,100 640

ts GI251 29,100 24,800 15,100 660

ts GI252 27,200 21,800 13,000 550

ts GI253 27,900 23,600 12,500 400

a Thirty-millimeter (7.06-cm2) plates prepared

with 8,000cpmof insoluble [1251]fibrin and 10 gg of

cold fibrinpercm2,accordingtothe method of

Unke-lessetal.(41),wereusedtomeasurethe proteolytic

activityoftwotypesofplasmin-containing

prepara-tions. One milliliter of harvest fluidwas incubated onthe fibrinplatesat370C. Atintervals, asample wastaken and countedin anaqueousmixture ina

liquid scintillation counter. The values shown are

for 8-h digestion of the fibrin plates corrected to

reflect the counts per minute 125I solubilized per

plateperharvestunit. One harvest unitwasdefined

as1ml ofharvest fluid obtained after12 hof incuba-tion of the appropriate medium at the indicated temperature with 106 cells. Cell counts were

per-formedonplatesfrom which harvest fluids had been

obtained, and,basedontheresults,the fluidsample

wasdiluted with unincubated mediumtonormalize for celldensity.

bFormeasurementofcell-boundplasminogen

ac-tivator, harvest fluids were prepared as described

aboveusing Dulbecco-modified Eaglemedium

sup-plemented with 2.5% chicken serum as asource of

plasminogen. Fluidswerefrozenat -80°C. e For measurement ofsupernatant plasminogen activator, harvest fluids were obtained using Dul-becco-modifiedEagle medium withoutsupplement. Fluidswerefrozenat-80°C.At the time ofassayfor

proteolytic activityon[125I]fibrin plates,chicken se-rum wasaddedtoafinal concentration of2.5% to the fluids as a source ofplasminogen. Controls done

without this source ofplasminogen showedonly a

background activity (approximately 600 cpm/plate

per harvest unit), which was subtracted individ-ually from the valuespresented.

d Chickenembryocellswereinfected ata

multi-plicity ofapproximately 2 focus-forming units/cell and,after 3daysofincubationat35°C,were trans-ferred to 30-mm plates at the temperatures indi-cated. Two days later, cells were washed and

pre-paredasdescribed abovefor harvest fluid harvests.

e WTPrA, Wild-typeRoussarcomavirus, Prague

strain, subgroupA.

temperature sensitive for expression of

plas-minogen activator in harvested supernatants,

display virtually undiminished activity ofthe

cell-bound type. Hence, the finding that foci

and colonies ofPTD mutant-infected cells

ex-hibited temperature-independent caseinolysis

maybe understood. The casein-containing

me-dia also contained serum as a matter of course. This serum made plasminogenavailableto the cell-bound plasminogen activator, and the re-sulting plasmin was responsible for caseino-lysis.

DISCUSSION

Table 8 presents a summary of the response

of cells infected with the various mutants in

separate parameters of transformed behavior.

For purposes of this discussion it is of central

importance todistinguish between the

parame-tersof cell behavior thatcanbemeasured and

the functions of the virus-coded transformation

proteinthat indirectly, after an unknown

num-ber of intervening processes, induces the

pheno-typic parameters we recognize. The TD

mu-tants, like most previously studied

tempera-ture-sensitive mutants of Rous sarcoma virus,

induce consistently temperature-sensitive

be-haviorinthe host cells. In contrast, the results

withthe PTDmutantsdelineateadissociation

betweencertain parametersof transformation.

PTD mutant-infected cells behave atthe

non-permissive temperature like wild-type

virus-infected cells according to three measures of

altered social behavior: loss ofanchorage,

se-rum, and density-dependent growth control.

Concomitant with, and perhaps causally

re-lated to, this altered growth control, the PTD

mutantsinduce intheir hostcells, atthe

non-permissive temperature, expression of

plas-minogen activator. Thisplasminogen activator

is indeed not really similar to that found with

wild-type virus-transformedcells, since it

can-notbe detected freeinthe supernatant butonly

withprocedures thatexposeplasminogentothe

cell surface. This mutant-inducedplasminogen

activator is, however, in terms of enzymatic

activity, comparable to that ofthe wild-type

virus-infected cells, and,mostsignificantly, the

cell-boundactivity found with thePTD mutant

systemislackingwith allotherTD mutants at

the nonpermissive temperature.

In reporting the existence of mutants like

those in the PTD category, the question of

whether these mutants may not be simply

"leaky" inthe conventionalgenetic sense (i.e.,

fractionally functional owing perhaps to a

merely statistical inactivation of the altered

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TABLE 8. Summary of parameters and responsesobserved with different mutants at41'C

Anchor-Turor for- age-, den- Concana- Increased Disap- Bound Free plas-Virus Category Focus for- mation in sity-,se- valinA hexose up- pearance

plasmino-

mation vivo rum-inde- agglutina- take of LETS genacti- activator

pendent bility vator

growth None Uninfected

-WTPrAa Wild type + + + + + + + +

tsG1201 _

tsG1202 - -

-tsGI203 TD- NDb ND - ND ND ND

tsGI204 mutants _ ND - ND ND ND ND

tsGI205 - ND ND ND ND ND

tsGI251 PTD - - + - - +

-tsGI253 mutants - - +

aWT PrA,Wild-type Rous sarcomavirus,Prague strain,subgroupA.

bND, Notdone.

protein at the nonpermissive temperature) is immediately encountered. Admittedly, the PTD mutants might be loosely described as

leakyaccordingtosomeparametersof

transfor-mation, although according to other

parame-terstheyarenot moreleaky than manyother

conventional TD mutants. The questions of sensitivity of measurement with the various parameters and oflinearity of the assays

be-come problematic for arrivingataconclusion.

Theconceptof"leakiness"resolves into the first

oftwo hypothetical models that may be

con-structedtoexplain the existence ofPTDaswell as TDmutants. (i)Onemay assumethatas a

result of the nature of the lesion in certain

mutants, asmall fraction of the altered

trans-formationprotein retainsfunction.Then,ifone assumesthat thisfunctionalproteinacts inthe

sameway (withasingle active site)onseveral

discrete cellular sites, some of which have higherthresholds than others in ordertoexert

an effect on the total cellular phenotype, one

couldexpect toobtain results like those found with PTDmutants. TDmutantswouldsimply be mutants in which the altered protein

re-tainedno function. (ii) Avirus-coded

transfor-mation protein maynot be limited to asingle

typeoffunctioninitsinteraction with the cellu-lar machinery. Ifseveral transformation func-tions, presumably carriedoutby a single

pro-tein molecule withmultiple active sites, have independent effects on the host cell, one may assume thatundercertain conditions a

muta-tionin thisprotein would lameone functional

site without injuryto theother(s). This would also result in aphenotypic dissociation of

var-iousparameters measuring theindirect effects of the transformation functions. SoaPTD

mu-tant would be amutant inwhich aparticular

functionisselectively destroyed, andaTD

mu-tant would not necessarily be amultiple

mu-tant but one that might also have suffered a

lesion, which would alter the conformation of

the entire protein.

Adissociation of transformation parameters

has been reported with linesofmouse3T3cells

infected with athermosensitive mutant

poly-oma virus (25); however, in this case, though

transformed growth properties were

tempera-turesensitive, agglutinability byconcanavalin

A was enhanced at nonpermissive as well as

permissive temperatures. Hence, these cell

lines, assuming this dissociation is directly

brought about by a mutation in the viral ge-nome, exhibit not the same properties, but

rather properties complementary to those

shown by cells infected with the PTD mutants

discussed above.

Extensive analysis of the whole range of

transformationparametersexhibited bya

vari-etyof random clones ofmouse3T3cellsinfected

by simianvirus 40has beenreportedby Risser

and Pollack (31). In this work the viruswasnot

mutagenized; rather, the variability in

proper-ties detected among the different clonesis

as-sumed to derive fromthe nature ofthe

virus-cellinteraction. Thisapproach ledtothe recog-nitionof distinctcategories inthe alteration of

host growth controlafter infection; it was

possi-ble to identify clones that showed

anchorage-dependent growthbutretainedserum- and

den-sity-dependent growth properties.

Dissociation of transformation parameters like thatseenbetweenthedifferent simian

vi-rus40-infectedclones has beenrecognizedwith

cellsinfectedwithtwodifferent aviansarcoma

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DIFFERENT TRANSFORMATION-DEFECTIVE MUTANTS

virusts mutants. tsLA334-infectedcellsexhibit

adegree of anchorage-independent growth,

al-though density-independent growth was not noted (9, 47). ts LA25-infected cells, although

almost completely anchorage dependent, show

a striking density- (22, 47) and

serum-inde-pendentgrowth pattern (Friis and Weber, un-published observations).

The involvement of a protease activity, now recognized as a urokinase-like plasminogen

ac-tivator(41) acting toproduce plasminin

serum-containing media (30), indetermining the

mor-phological (27) and many of the growth-related

properties of transformed cells (26) has been

further emphasized by the work of Pollack et

al. (29) and Shin etal. (35), which has shown

strong correlation among

anchorage-independ-ent growth, plasminogen activator protease,

and ability of cellsto inducetumors invivo in

"nude" mice.

Jones et al. (19) have further shown that

tumorsresulting from inoculation of low-level

plasminogen activator-producing cell lines are

invariably made up of high-level plasminogen activator-producing cells.

The results reported above with the PTD

mu-tantsdonotfundamentally contradict the

exis-tenceofacorrelation betweenplasminogen

ac-tivator production and oncogenicity. The PTD

mutants seem tobeasortof intermediate cate-gory in which the dissociation of

transforma-tion parameters isplaceddifferently. The

dem-onstrated anchorage-independent growth with

the PTD mutant-infected cells isparalleled by

protease activity, asthe caseinoverlay plaque

testshowed (Fig. 5).Furthermore, experiments

inour laboratory have shown that

anchorage-independent growth of transformed cells

re-quiresthe presenceof plasminogeninthe

soft-agar suspension culture medium (Becker, un-published observation). The fact that in vivo tumorformation is notefficient withthe PTD mutantsmayindeed reflectanimportant

func-tional difference between cell-bound and

super-natant plasminogen activator in contributing

totumorigenicity.

The presence of LETS glycoprotein on the

surfaces of PTD mutant-infected cells at the

nonpermissive temperature is an important

finding,because itarguesagainst the

hypothe-sis that LETS may play a regulatory role in

establishing density inhibition ofgrowth (15,

16).Ourfindings are, however, consistent with

the report that restoration of LETS

glycopro-tein to transformed cells results in increased

adhesion to substrate and more normal cell

morphology but failstoimpose

density-depend-entgrowth regulation (48), sincePTDmutants

at the nonpermissive temperature do exhibit

normal or near-normal morphology and

rela-tively high adhesion to substrate (Friis and

Weber, unpublished observations) under condi-tions approximating transformed cell growth.

The TD mutant tsGI201, which is unusual in

being non-conditionally defective for cell-bound and supernatant plasminogen activator pro-tease, brought useful insight into the question

ofthe mechanism by which LETS glycoprotein

is removed from transformed cells. ts GI201, which is transformed according to almost all

parameters at the permissive temperature,

shows only partial removal of LETS

glycopro-tein. Since the PTD mutant-infected cells at the

nonpermissive temperature, under conditions

where significant cell-bound plasminogen acti-vator is expressed and in medium containing

high plasmin activities, nonetheless exhibit

LETSglycoproteininnormal amounts, one can

speculate thatadifferent protease, onethat is

notplasmin and is perhaps not even involvedin

plasminogen activation, is responsible for

LETS removal.

The study of the transformation events

occur-ringafter avian sarcoma virus infection is very

complex because of the variety of parameters to

be monitored andthe unknown relationships of

the parameters to each other. Studies such as

that reported above may help to clarify the

relationships of the parameters, for example, the possible dependence on particular protease

activitiesfor loss of the LETS glycoprotein. Itis

to be hoped that examination of mutants such

as thesePTD mutantsandothers ofa

comple-mentary nature, i.e., mutantsdefective for

in-ducing growth parameters of transformation butlikethewild-typevirusfor inducingcertain

biochemical changes in infected cells (such as

increased hexose transport), could eventually

lead to recognition of distinct transformation

functions directly coded for by the sarcoma viral genome.

ACKNOWLEDGMENTS

Wewouldlike to express our thanks to Sigrun Bingel for veryable technicalassistance.

This work was supported by Sonderforschungsbereich47 of the DeutscheForschungsgemeinschaft.

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