Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

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Transformation

of

Rat

Liver

Cells

with

Chicken

Sarcoma

Virus B77

and

Murine

Sarcoma

Virus

C. ALTANER Anm E. HLAVAYOVA

Cancer ResearchInstitute, Slovak Academy of Science,Bratislava, Czechoslovakia

Received for publication 18August1972

Rat liver cells in vitro were transformed with chicken sarcoma virus B77,

giving RL(B77) cells, and with murinesarcomavirus(Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL

cells. Inaddition, the RL(MSV) cell linewasadapted for growth in culture fluid

containing 25ggof5-bromodeoxyuridineperml. All celllinesweretumorigenic

in 1-wk-oldrats. The numberof cells needed for induction oftumorgrowthwas

1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV)

cells and RL cells. No production of viral particles from any ofthe cell lines investigated was detected by plating concentrated supernatant fluid of the cultureson differentsecondary embryo cells with and without fusion by Sendai

virus, bylabeling with uridine-5-9H, orbyassayfordeoxyribonucleic acid

polym-erase activity. The viral genome was rescued by fusion ofRL(B77) cells with

chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in platingefficiency on duck cells from B77 virus rescued from transformed rat

embryo cells. No virus wasrescued after fusion ofRL(MSV) and RL cells with mouse,rat, orchickenembryo cells. Infectious murinesarcomaviruscanbe

in-ducedby5-bromodeoxyuridine from RL(MSV) cells.

Transformation of heterologous cells with chicken sarcoma viruses

usually

leads to viro-genic cells with complete viral genome, butno

virus

production.

However, it is known that B77 virus is able to induce a stable

virus-pro-ductive state of virus integration in rat tumor

cells(4). Theratcells transformed withchicken sarcoma virus B77 contain the whole virus

genome, and thevirus rescued from such cells by fusion with chicken cells has a higher plat-ing efficiency for rat cells than the virus used for the

original

infection (5). It was suggested

by

us that this "host modification" ofvirus is a consequence of the integration ofthe virus genome into host cell genome leading to the virushaving morehomologyto ratcellgenome.

Virus B77 was originally isolated from a tu-morofchicken liver (21). Itwastherefore of in-terest to seewhetherit is capableof

transform-ing heterologous liver cells and how the virus will be changed after passage through these

cells. The transformation ofrat livercells with

murine sarcoma virus

(MSV)

(Harvey) was

in-vestigated.

In this report we describe the

properties

of four rat liver lines which were obtained

by (i)

transformation withchickensarcomavirus B77

in vitro, (ii) transformation with murine sar-coma virus (Harvey) in vitro, (iii)

"spontane-ous" transformation of rat liver cells in vitro, and (iv) adaptation ofRL(MSV) cells togrow in the presence of 25 ,ug of bromodeoxyuri-dine(BUdR) per ml.

MATERIALS AND METHODS

Tissue culture and media. General procedures

werethe same as usedpreviously (5). Tissue culture

cellswerecultivatedat38C inahumidified CO,

in-cubator. The standard culture fluid consisted of

Eagle minimum essential medium (E), supplemented with 20% tryptose phosphate broth (ET), calf serum (ETC), and antibiotics.

Rat livercells. Livers from 18-day-old embryos

of Sprague-Dawley rats were used. The cells

pre-pared by trypsinization were kept in Eagle HeLa

medium (Difco) supplemented with 10% tryptose

phosphate broth, 20% calf serum, and 1% chicken

embryoextract. The 32nd passage ofliver cells was

used for infection. After transformation the cell

lineswerekeptinE mediumsupplementedwith10%

tryptosephosphate broth and5%calfserum.

Viruses. Stock of B77 virus concentrated by

ul-tracentrifugation by the procedure earlierdescribed

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ALTANER AND HLAVAYOVA

(5) contained 5 x 101focus-formingunits(FFU)/ml.

MSV strain of Harvey was obtained through the

courtesy of J. Zavada, Institute ofVirology,

Brati-slava, Czechoslovakia. It was multiplied in mouse

embryo cells(C57B1) and hadatiter of104FFU/ml.

Determination oftumorigenicity of cells.

Rap-idly growing tissue culture cells were harvested by

trypsinization, and the number of cells was

deter-mined. The cells werediluted in

tris(hydroxymethyl)-aminomethane (Tris)-buffered saline and injected

into the back of 1-wk-old rats of Sprague Dawley

strain. Animals were inspected for tumors every

otherday bypalpation. Metastasestothelunginfew

animalswereobserved.

The number of cells needed for induction of

tu-mor growth in 50% of the inoculated animals was

calculated bythe method of Reed and Muench(16).

The tumors were determined by histological

exam-ination to be sarcomas.

Labeling of viral particles. The medium for

cellsinlogarithmic phaseofgrowthwaschangedfor

a medium containing 5

gCi

ofuridine-5-3H per ml.

On the nextday the hot mediumwasharvested and

the supernatant fluid of the cultures was

concen-tratedby ultracentrifugationandlayeredonalinear

sucrose gradient prepared from 70% (w/v) through

15% sucrose in 0.01 M

Tris-ethylenediaminetetra-acetic acid (EDTA) buffer. Centrifugation was for

1hr at 45,000rev/mininaSpincoSW50.1rotor.

Incorporation of thymidine-6_3H. The culture

fluid waschanged for medium containing 10jsCi of

thymidine-6-3H per ml. Three hours later the

me-dium was removed, and the cells were treated on

plates with 10% trichloroacetic acid, washed with

ethanol, and dried. Dried cells were dissolved in

0.2 M NaOH, and samples were taken for

radio-activity determinations. Deoxyribonucleic acid

(DNA) was determined by the diphenylamine

re-action.

Radioactivity determinations. Sucrose gradient

samples were diluted with water and taken in

dioxane-based scintillation fluid. All counting was

done inaTri-Carbliquidscintillationcounter.

Polymerase assays. The standard DNA

polym-erase assay of Temin and Mizutani (20) was used

throughout.

Cell fusion. Cells of secondary chicken cultures

made from RIF-free embryos were seeded on the

day before infection in petri dishesinmedium

with-out serum. The following day, the attached cells

were rinsedtwice withcold, serum-free medium. The

cultures were overlaid with 0.2 ml of

ultraviolet-in-activated Sendai virus (500hemagglutinating units)

and incubated at 3 C for 10 min. The cells were

rinsedoncewith cold E medium, and different

dilu-tionsofX-ray-irradiated (6,000 R) cells were plated

in 0.2 ml of E medium. The cultures were rocked

and incubated at 38 C for 40 min. Finally, the

cul-tureswere overlaid with ET mediumcontaining 5%

calf serum. The following day, the medium was

changed for medium containing 0.5% agar.Thesame

procedure was used with mouse and rat embryo

cellsaswell.

The procedure for infection of cells treated with

Sendai virus with viral preparations was the sameas

forthe infectious centers, exceptthatthe cells were

overlaid after virus adsorption with medium

con-taining0.5%agar.

RESULTS

Transformation of rat liver cells byB77

virus and MSV. Cultures of rat liver cells

(4 x 105 cells perplate) wereinfected withB77 virus at a multiplicity ofinfection of 0.5 FFU

per cell. Similar cultures were infected with

MSV by using the same multiplicity of

infec-tion. Cultures of rat livercells consisted of epi-thelioid cells which grew very slowly.

Twenty-one days later, a colony of small fusiform cells appeared in the culture infected with MSV.

The cells in the focus were growing very

rap-idly, andit was possible topick them upfrom

the growing focus and transfer them to a new

plate. Cells were grown up and kept as cell

line RL(MSV).

Rat liver cells infected with B77 virus and

control cells were kept by serial transfers of cells. The medium was changed every 3 days.

Sixty-eight days

after infection of rat liver

cells with B77 virus(one cell transferwas made

3 wk after infection), a focus of transformed cells appeared, and the cells from it were

picked up by suction and grown up, leading to the cell line designated RL(B77). The morphology of RL(B77) cells was epithelioid

and did not differ from the original

untrans-formedratlivercells.

After further cell passages (nine) of

unin-fected cells, the epithelioid character of the

liver cells and the mode of growth were

changed. The term "RLcells" designates cells

morphologically

transformed spontaneously to

small fusiform cells.

Adaptation ofRL(MSV) cells for growth

in medium containing 5-BUdR. The

RL(MSV)

cell line was adapted for growth in

medium containing 25

Ag

of 5-BUdR per

ml

leading

to a cell line RL(MSV)BU-25.

RL(MSV) cells were adapted for growth in

5-BUdR-containing medium by the usual tech-nique used for this purpose (12). The concen-tration of 5-BUdR in culture fluid during the

adaptation was changed according to the ac-tual state of cultured cells. After 5 mo, the

cells were adapted for growth in 25 ,ug of 5-BUdR per ml containingculture fluid (Fig. 1).

Similar results were obtained with RL(B77)

cells. The RL(MSV)BU-25 cells were able to grow in medium supplemented with

amino-methopterin (0.4

AM),

glycine (3

uM),

thymi-178 J. VIROL.

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6

fl-ch.ch

5.106

0

I. . .c . . . .

0-- 1 2 3

-7

24'

0

FIG.1GrwhcreofRRL(MSV)BU25 rL(MSV)

10~~

0 a-@ ETC 1O

and Methods); medium containing 25 gof 5-BUdR perml, and HATG medium. All media were changed the third day after seeding of cells (fl.ch.).

dine (16

yM),

and hypoxanthine (0.1 mM) (HATG medium, reference 14).

There was no significant difference between

RL(MSV) and RL(MSV)BU-25 cells in the

ability to incorporate labeled thymidine (Ta-ble 1). Thus RL(MSV)BU-25 cells, despite theirabilitytogrowin5-BUdR,arenotlacking

thymidine kinase. The RL(MSV)BU-25 and

RL(B77)BU-25 growing in 5-BUdR medium

werelight sensitive.

Tumorigenicity of cell lines. All four cell

lines were tested for their abilitytoinduce tu-mors in 1-wk-old rats. RL cells and RL(MSV) cells were highly oncogenic for 1-wk-old rats.

It isstriking that the injection ofaslittleas40

cells into rats gave rise to progressively

grow-ing tumors. On the other hand, the RL(B77)

cells required a thousand times more cells for

tumor induction in 50% of the inoculated

ani-mals (Table 2).

Virus production and presence of DNA

polymerases. To determine whether the in-vestigated cell lines are producing viral

par-ticles, the following experiments were done.

Tissue culture medium harvested from these

cells was 100-fold concentrated by

ultracen-TABLE 1. Incorporation ofthymidine-6-3HintoDNA

Cell line

Counts/min/

Cellline p~,gofDNA

RL.11.500a

RL(B77) ... 8.840

RL(B77)BU-25.7.500

RL(MSV). 5.400

RL(MSV)BU-25.4.150

Ha(SR). 6.190

Ha(SR)BU-100C.23

aThe values represent the averages of

determina-tions on duplicate cultures oftwo different

experi-ments.The rangewas notgreater than10%.

b Hamster cells transformed with the

Schmidt-Ruppin strain of Roussarcomavirus.

cThe Schmidt-Ruppin Rous sarcoma virus

trans-formed hamster cells resistant to 5-BUdR and

de-ficient in thymidine kinase activity (C. Altaner,

preparedforpublication).

trifugation,andthe material was platedon dif-ferentsecondary embryocells(Table3).

To testthe

possibility

that viralparticlesare

produced with different viral envelopes, the

techniqueof fusion

by

inactivated Sendaivirus wasused. Underthese conditions weobserved

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[image:3.493.100.390.67.337.2] [image:3.493.251.442.400.492.2]

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production of virus infectious for mouse cells fromRL(MSV)BU-25 cellsonly.

Tissue culture fluid from RL, RL(B77), RL(MSV), and RL(MSV)BU-25 cells was

con-centrated 100-fold by the procedure used for concentration of viruses. The search for

DNA polymerase activity in the sedimented

material obtained from RL, RL(B77), and RL(MSV) cells was negative. A small activity

wasfoundinthematerialfromRL(MSV)BU-25 cells.

The uridine-5-3H labeling technique with

subsequent bandingofthe viral material in su-crose gradient centrifugation gave negative

re-sultsinthecaseofRL,RL(MSV), and RL(B77) cell lines. These cell lines were also negative

in theultraviolet-XC plaque assay forthe leu-kemia viruses (18).

Attempts to recover the viral genome

from transformed cells by fusion with

diff-erent kinds of embryo cells. To determine whether the transformed cells contained viral

[image:4.493.61.449.286.604.2]

genome,

they

wereirradiated with X ray(5,000 R) and fused with the aidofinactivatedSendai virus with secondary embryo cells. From

TABLE 2. Tumorigenicity ofRL,RL(MSV),and

RL(MSV)BU-25 cell lines in rats

Number of Average Cell line cellsfor latentperiod

ED50a (days)

RL <103 14

RL(MSV) 4 x 10 17

RL(B77) 2 x 105 15

RL(MSV)BU-25 102 15

aED50,

Mean effective dose.

RL(B77) cells, infectious viruswasreadily

res-cuedbyfusionwith chickenembryocells.

Cells of the RL(MSV)BU-25linegave riseto

typical MSV foci after fusion with mouse

em-bryo cells, whereas the RL cells and the RL(MSV) cells did not induce focus formation after fusion of 106 cells with mouse, rat, and chicken embryo cells

(Table

4).

Also,

culture fluid fromthese mixedcultures didnotcontain anyinfectiousvirusformouseembryo cells.

The virus rescued from RL(B77) cells was

characterized by its ability to transform chickenand duck cells in comparison with the

virus which was used for their transformation and with the viruses rescued from the three

different clones ofratembryo cells transformed with B77 virus (Table 5). The virus rescued

from rat livercellswas more than 1,000 times

less effective in transformation of duck cells than the virus obtained from transformed rat

embryo cells.

TABLE4. Experimentsto rescuethevirus genome

by cellfusion

Assay cells Cell line

Chicken Mouse Rat

RL >lo6a >106 >106

RL(B77) 68 >10f >106

(Nofusion 2 x 104)

RL(MSV) >106 >106 >106

RL(MSV)BU-25 >106 2 x 103

NDW

aNumberofcells needed for the induction of one

focus oftransformed cells by using the fusion

tech-nique.

"ND, Not done.

TABLE 3. Test for virusproduction and detection of DNA polymerase

Assay cells

~~~~~~Exogenous

Cell line Chicken Duck Mouse Rat DNA

polymerase

NTa Sendai' NTa Sendai' NTa Sendai' NT| Sendai'

activityc

RL Od ° 0 T 0 0 0 0 0

RL(B77) 0 0 0 0 0 0 0 0 0

RL(MSV) 0 0 0 0 0 0 0 0

RL(MSV)BU-25 0 0 0 0 2 2

0

0 112

aNT, Not treated.

ISendai,fusionby inactivated Sendai virus (see Results for further explanation).

cThenumberofcountsper minute of3H-thymidine triphosphate

(3H-TTP)

incorporated per 0.0125 ml of

virussampleincubated at 40 C for 60min by using calf thymus DNA as a template. Under the same conditions

ofthe assayapreparation ofchicken sarcoma virus B77 of 106 FFU/ml gave 18.580 counts/min of 3H-TTP

incorporation.

dThe number representstheaveragenumber of foci on plates infected with 0.2 ml of 100-fold concentrated

tissueculture fluid harvested from confluent cultures.

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TABLE 5. Relative plating efficiency of viruses rescued from RL(B77) and R(B77) cells

Relative plating Viruses rescuedfrom: efficiency

(duck/chicken)

RL(B77).9.1 x 10-,a

R(B77) no. 1.1.1 x 10-2

R(B77) no. 2.1.4 x 10-2

R(B77) no. 3.7. 1 x 10- 2

Original B77 8.0 x 10-2

aViruses were rescued from rat liver-transformed

cells RL(B77) and from different clones of

trans-formed rat embryo cells [R(B77) no. 1; R(B77)no. 2;

R(B77) no. 3] by fusion with chicken embryo cells.

These viruses and virus B77 originally used for

infec-tion of rat liver cells were assayed on duck and

chicken embryo cells. Relative plating efficiency

(duck/chicken) was calculated. Titers of all viruses

used werearound 106FFU/mlonchicken cells.

DISCUSSION

The chicken sarcoma virus B77 is known as a virus with broad host range. It is able to

in-duce sarcomas in rats (8) and to transform some mammalian cells in vitro (5). For the study ofcell transformation, primarily embryo fibroblasts were used.

Under certain conditions it is possible to

cultivate mammalian liver cells in tissue

cul-ture (6, 19). Rat liver cells kept in tissue

cul-ture are of

epithelioid morphology.

It was

shownby Coon (6) that such cells are

produc-ing some serum proteins, and several

enzy-matic activities typical for liver tissue have

beenfound.

We have proved that rat liver cells are

sus-ceptible

to transformation with chicken

sar-coma virus B77 and murine sarcoma virus. In

both cases the transformed cells are

tumori-genic and contain the whole viral genome. The

greatdifference inthe

ability

toinducetumors in ratsbetween RL(MSV) cellsorRLcells and RL(B77) cells could be

explained by

their abil-ity to grow in vitro, their

morphology,

and

by

the presence oftumor-specific

transplantation

antigens, or

by

a combination of these prop-erties. RL(MSV) cells and RL cells are

small,

rapidly growing cells, whereas RL(B77) cells

are epithelioid with slower growth rate. The

higher doses of RL(B77) cells needed to

in-duce tumors in rats could be in connection with the presence oftumor-specific

transplan-tation antigen on the surface of cells

trans-formedwithchickensarcomaviruses.

RL(B77) cells did not differ from other rat

embryo cells transformed with B77 virus.

They

are virogenic without production of an

infec-tious virus or viral particles. After fusion of

these cells with chicken embryo cells, infec-tious virus could be rescued. This virus

diff-ered from the original one and from viruses rescued from transformed rat embryo cells in its property to transform duck cells. Its ability totransform duck cells is much lower, showing

that host modification of virus occurred. This is in agreement with earlier observations of

B77 virus, which we interpreted as a conse-quence of virus genome integration into the

hostcell genetic material(5).

A number of tissue culture mouse cell lines after long-term cultivation appeared to be transformed in vitro. Some lines produce viral

particles

and some do not. The presence of

virus particles does notseems tobeassociated with malignancy(7).

Our line of rat liver cells transformed

spon-taneouslyin vitro ishighly tumorigenicin rats. It did not produce any detectable infectious

virus or viral particles. Also, preliminary ex-periments to try to induce some virus by

5-BUdR by using the uridine-5-3H technique

gave negative results. Therefore, it seemsthat this neoplastic transformation is not a conse-quence of some virus action, but more

experi-mentsmustbe doneinthis direction.

There are several cell lines transformed by MSV with no detectable virus

production

(9, 2). Such nonproducer cells release MSV when super-infected with a helper leukemia virus in vitro (9, 17, 23) or in vivo (13). Infectious

virus canbe rescuedafter cell fusion with cells sheddingamurineleukemiavirus(10).

The RL(MSV) cells behaved as

nonproduc-ers. These cells did not produce infectious

virus, virus particles, or particle-bound DNA

polymerase

activity as detected by different

techniques. The RL(MSV) cell line did not

produce any viral particles which could have the character of MSV(O) described by Ting

(23). The RL(MSV) cell line was adapted for growth in a medium containing 5-BUdR. These cells incorporated labeled thymidine and did grow inHATG

medium,

which means

thatthey are not

deficient

inthymidine kinase

activity. The ability to grow in medium con-taining 5-BUdR could be in connection with the presence of ahigh level ofrepair enzymes.

Recently it was found that murine leukemia viruses can be activated in vitro by treatment

of cells with 5-BUdR or 5-iododeoxyuridine

(3, 15).Focus-formingvirus canbe also induced

from

nonproducer

cells transformed by MSV

(1, 11).

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It was found that the RL(MSV)BU-25 cell line is producing infectious virus which

in-ducedtypical MSV focion mouseembryo cells. The induced sarcoma virus is able to trans-form mouse cells, but these cells did not pro-duce infectious virus showing that this virusis unable to replicate in the absence of helper leukemia virus (C. Altaner, unpublished data).

ACKNOWLEDGMENTS

Wewishtothank H. M. Temin forhelpfulcommentsand

S. Bodocka fortechnical assistance. Theinvestigation was partially supportedby PublicHealth Service research grant CA 07175 from theNational CancerInstitute. A part ofthe workreported in this paper was undertaken during the ten-ureof aTravel Fellowshipawardedto onefromus(C. A.)

by theIntemationalAgency for Research on Cancer in Mc-Ardle Laboratory for CancerResearch,University of Wiscon-sin,Madison, Wisconsin.

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182 J. VIROL.