Acta path. rnicrobiol. scand. 77, 57-65, 1969

Institutc of Pathology, University of Lund, Lund, Swedcn.

FURTHEX STUDIES ON THE INTERACTION I N VITRQ BETWEEN MAMMALIAN ROUS

SARCOMA CELLS AND CHICKEN FIBROBLASTS

BU

NILS .JONSSON Received 15.iv.69

The transformation of chicken embryonic fibroblasts (CEF) and initia- tion of RSV-production by co-cultivation of tumour cells f rom RSV- induced mammalian sarconias and CEF is generally assumcd to involve a transmission of the HSV-genome from the mammalian sarcoma cell to the chicken cell withoul the participation of coniplcle biologically active virus particles. The mechanism of this transmission has been studied very intensely durins lhc last few years (SimkouiE e t al. 1964, Suoboda 1964, Vigier & Suoboda 1965, Jonsson 1966, Lindberg &? Jons- son 1968). Three possibilities have been taken into consideration:

Although most mammalian Rous turnours do not release any dc- monstrable infectious virus, neithcr in uitro nor in uiuo (for review see Ahlsfr6in 1964, Suoboda et al. 1963, Jonsson 1966) evidence has becn prescntcd that some rodent cells arc able to synthesize mature virus (Suoboda 1964) or a t least viral antigen (Sheulyaghin &? Marti- rosyan 1966). I1 thus cannot he excluded that the process of Iransmis- sion involvcs the release of virus from the mammalian tumour cells in such %mall amounts or in such a labile state that it cannot bc dcmon- stratccl by commonly used techniques. A virus production by thc rodent- turnour cells after transfer of some hypothclical substance from thc CEF must also be taken into consideration.

Secondly, the viral gcnonie could be transferred l o the chicken cells by the passage of some subviral determinant, presumably by means of some type of cell-to-cell contact. Support to this view has been given by Suoboda e t a2. (1968), and the possible morphological basis of such contacts has becn discussed by Lindberg & Jonsson (1968) in uitro and by Ahlstriinz d? Lindberg (1969) on the chorioallantoic membrane of cmbryonatecl eggs, inoculated with Rous hamster sarcoma cells.

A third possibility is the complete fusion of two types of cells with a formation of heterokaryons. This mechanism would require a virus

This work mas supported by grants from the Swedish Concer Society and Leonie Deshaljes f o n d .

synthcsis by these heterokaryons, infecting new CEF with a formation of transforrncd foci, unless fused cells could scgregntc again, as the hcteroliaryons would soon be eleniinaled from thc mixed culturcs. Evidence of this mechanism has been prcsentcd by Vigier & Montagnier (1966) .

A s it secmcd of intcrest to study the possiblc mcchanism, attcmpts wcre made to initiatc RSV-production by mcans of co-cultivating irra- diatcd CEF and rodent Rous sarcoma cells as well as attempts to dc- monslratc viral antigen in rodent tumour ccll prcparalions, capahlc of absorbing antiviral activity from antiviral immune scrum. Further- more, altcnipts wcre inadc to interfere with the infection of CEF in mixcd culture by incans of antiviral irnmunc serum that would be cxpcclcd to inhibit infcction 17ia free virus particlcs.

M A T E R I A L A N D M E T H O D S

Chick embrr~o cultures. Primary cultures were prepared from 9-11-day-old chick cmbryos in 100 m m petri dishcs (Falcon) essentially according to the technique of Teriiin & Rubin (1958). Tests for viral activity were performed on secondary culturec, prcpared 4-5 days latcr. Only embryos highly susceptible to SR-RSV were used. During the later part of the euperimcnt embryos could he obtained from an isolated, RIF-free stock of white 1,eghorn (Vinterleds g B r d , Laggcsta, Sweden). These embryos proved highly and almost uniformly snsccptible to SR-RSV.

Virus. The same SR-RSV pool was used in all experiments. I t was prepared by using a simplified mcthod of BrUan et ul. (1954) as described previously (Jonsson h Sjdgren 1965). The titrc was 2 x 104 FFU/ml.

Antiuiral immune seru against SR-HSV werc collected from chickens after an immunization procedure according to Fink S Rauscher (1961), using hcat-in- activated virns and subsequently repcatcd challengcs with live virus. Sera from individual birds or pooled sera wcrc stored at -20" C. The antiviral activity was assayed according to the technique of Rubin ct nl. (1960, 1961). After inactivation at 56" C for 30 minutes serum dilutions were incubated for 30 minutes a t 37" C with the appropriate virus dilution. A 90 per cent reduction of the number of foci w a s reqnircd for neutrali7ation. The highest serum dilution giving this neutraliza- tion was used in the absorption tests. Control serum derived from untreated chickens.

Tumour cells derived from sarcomas, induced by suhcutancous inoculation of a snspcnsion of SR-RSV chicken sarcoma into newborn mice or rats. The mouse tumours RSC, RYC, and RS57D, induced in the inbred mouse strains A/Sn, A.BY, and C57B1/K1 respectively, have been used and describcd in previons papers ( J o n s - son & Sjdgren 1965, Jonsson 19GG). They all contain stable tumour specific trans- plantation antigens and have hecn repeatedly checltcd jn mixed cultures for the presence of the RSV-gcnomc. Thc tnmour RR6690 was originally induced in a commercial white ra t and has also been repeatedly tested for the presence of thc viral genome. All the tomours werc kept in serial passage in uitro. Tnmour cell suspensions were prepared by trypsinization, and the number of trypan blue un- stained tumour cells was estimated (Bo!]se ef at . 1962). Irradiation procedure. Trimour cell suspensions were irradiated at 0" C by X-rays,

gcnerated at 200 kV, 15 mA and filtered hy 1 mm Al. The dose was 8000 r. Disruption of the cells was performed by sonication (MSE ultrasonic power unit,

18-20 ItC/sec.; 10 x ?h min. at 0" C) or by freczing and thawing repeated three times.

Miser2 cultures were preparcd as described previously (Jonsson 1966). Known num- bers of mammalian turnour eclls werc added to 1.2 X 100 CEIJ in 100 mm petri dishes. In the antiserum cxperimcnts antiviral chicken immune serum or control chicken serum was added in the appropriate conccntrations. 4-6 days later cultures were passed to G O mm petri dishes with the same antiscrum conccntrations in the medium and on the following day covered with agar medium. The cultures were stained

with Gicmsa 6-9 days later, when virus infected control cultures showed evident foci. A s previously reported, the system requires a certain number of tumour cells (about 5 x l o 4 cells) to give registrable foci of transformed CEF, and increase in th i s cell number results in a roughly l inear increase of the number of foci.

R E S U L T S

Effect of Irradiation or Sonicaliorz of the Chicken Fibroblasts

Tests for viral activity were performed on cell-free media from cul- tures of Rous mouse turnour cells ( 5 x 10' cells per culture) , cultivated alone or after the addition of CEF, untreated, X-irradiated or sonicatcd ( 2 x 106 cells per culture). The results of the tests, performed at various agcs of the cullnres are collcctcd in Table 1. i t is apparent that virus synthesis can only he denionstrated in the presence of intact CIZF and that virus synthesis can be induced neither in the mouse tumour cells after the (even repeated) addition of X-ray damaged CEF or sonicatcd fragments of CEF, nor in hypothetically formed hctcro- karyons from the mammalian tumoiir cells and irradiated CEF. This is further confirmed by the negative finding in tests for viral activity in sonicatcd cell preparations from mixed cultures containing X-irra- diatcd or sonicatcd CEF.

TABLE 1 Results o f Tests f o r RSV-Actiui t!) in the Media f r o m Cultures of Mnmmalian Roils Snrconia Cells ( f r o i n the Three Mouse Tanlours RS57D, RSC and R Y C ) nf ter the

Addit ion o f CEF, Untreated, X-Irradiated or Sonicuted.

Type of culture Amount of FPU/ml' 'medium on day number

6-8 10112 14-16 20-22 ~

HS57D HS57D + CEF (untreated) RS57D + CEF (X-irradiated) RS57D 4- CEF (sonicatcd)

RSC HSC + CEF (untreated) RSC + CEF (X-irradiated) RSC + CEF (sonicated)

RYC HYC + CEF (untrcated) RYC + CEF (X-irradiated) RYC + CEF (sonicatcd)

0 0 0 0

0 0 0 0

0 0 0. 0

0 7 60

0 0 0

740 0 0 0 0 0 0

n.t. n.t. n.t. n.t.

0 520

0 0

0 0 0 0

0 360

0 0

n.t. n.t. n.t. n.t. 0

170 0 0

~

n.t. = not tested. Determined f rom the mean number of foci on 3-4 Pctri dishes.

Attempts fo Absorb Antiviral Serum Activity by Cel l Preparafions

Like the majority of mammalian Rous tuniours cell-free preparations from the tumours used in this investigation have given no evidence of viral activity (Jonsson 1966). A s it could be objected that filtration or sonication of the cell preparations to obtain a cell-free material might

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61

rcinove or destroy small amounts of virus, espccially if thesc were in a labile form, attempts wcrc madc to dcmonstrate viral activity by mcans of absorption tcchniquc. Antiviral ininiunc sera in different dilutions, including the highcst dilution giving clear-cut virus ncutra- lization, wcrc tested for their rcmaining antiviral activity aftcr absorp- tion for 30 min. a t 37” C with cell matcrial from diffcrcnt mousc and rat tumours, frozcn and thawed rcpcatedly threc tinics. Thc rcsults are presented in Table 2, from which it is apparent that therc is no demon- strable dccreasc in thc ncutralizing capacily, i.e. the turnour cell ma- terial cannot absorb any antiviral activity from the immunc scrum. Negative results werc also obtained with similarly treated material from niixcd cultnrcs in which Lhc CEF wcre X-ray daniagcd.

The scnsitivity of thc procedurc was tested by including SR-KSV virus in small aniounts or chickcn tumour material, Lreatcd as abovc, as control. I t is apparent from Table 2 that a reduction of the virus- neutralizing capacity of Ihc scrum dilution can he oblaincd with thc chicken tumour material as wcll as with a virus dilution containing about 10 biologically aclivc virus particlcs.

Effec t of Antiviral lmniune Serum on Mixed Cultirre.7

In vicw of the possibilities that the number of foci of transfornicd CEF in thc mixecl culture systcm might be dcpcndent upon or at lcast influenccd by a formation by thc mammalian tumour cells of small amounts of virus, alternatively vcry lahilc virus particles, it sccmcd of intcrcst to invcstigatc the cffect of antiviral immunc scrum of the focus formation in the systcm uscd. This would bc cxpccted to prevent the sprcad of virus during thc cultivation in liquid medium and thus also help to clarify the qucslion whcthcr virus is forincd during the carly phasc ( u p to 4-6 days) of co-cultivation eilhcr by “infcctcd” CEF (aftcr contact) or by teniporarily formcd hctcrokaryons.

Table 3 illustratcs thc cffect on mixed cultures (CISF + irradiated RR 6690 cells) of antiviral antiscruni at conccnlrations that give clcar-cut virus neutralization. Thc scrum was presciit as long as the cultures were kcpl in fluid medium (up to thc day after passage), but was omitted from thc agar mcdium. I t is apparcnt from thc table that the nuinber of ioci of transformed chicken cclls in the culturcs arc of about thc same magnitude, irrcspcctively of whcthcr antiscrum is present or not. Thus, thcre is no indication of any free virus, neutra- lizahlc by thc imrnunc scrum, participating in this early phase of the expcriment.

D I S C U S S I O N

Thc present results confirm and cxtcnd those obtained in previous sludics. Thus, it is not only impossible to dcmonstrale any viral activity in ccll-free material from the diffcrcnt mammalian tumours inves-

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63

tigatcd, but also any viral anligcns, capable of absorbing antiviral activity from immunc sera. Nor can any viral activity or such viral antigcns be demonstrated after the addition to thc tuniour cclls of irra- diated or sonicated CEF. Thus, there is no evidence of any transfer from the irradiated chickcn cell to the mammalian tuniour cell of some hypothctical substance, ahscnt in thc mammalian cell but neces- sary for the virus synthesis, ncithcr sonic kind of metabolic tool, nor sonic type of helper virus.

The ncgativc results of the expcrimcnts with mixcd cultures con- taining irradiated chicken cells furthermore indicatc that X-irradiation of the CEF before mixing the cell typcs prcvcnts virus rcleasc from the CEF. This is in good accordance with lhc findings by Rrrbin C Teinin (1959) that the HSV-producing capacity of chick embryo cells is very sensitivc to X-irradialion pcrformed bcforc virus infection, probably due to the inlcrference with cell division that scems ncccssary for virus production (I’eniin 1966). Anolher explanation has, howcvcr, also to be takcn into considcration, uiz. that thc X-ray inhibited proliferation of the CEF greatly diminishes the chance of cell-to-cell contacts be- tween thc two typcs of cells, ncccssary for the transfcr of viral genelic matcrial.

Thc results of co-cultivation of the two cell typcs in the prcscnce of a high concentration of antiviral antibodies can throw somc light upon thc possibilitics of a relcase of small virus amounts or labile virus from thc rodent cells or an early virus rclcasc from tcniporarily formed non-scgrcgable hctcrokaryons with a secondary infcclion of other CEF, as proposed by Vigier h Monfagnier (1966). Both these possibilities seem less probahlc, as the antiviral serum would, with thcsc mechanisms, considerably rcducc the number of foci of transformed CEF, which could not bc dciiionstratcd in thc cxpcrinients. An objcclion to this intcrprctatioii is, howcver, that such rclcased virus particles could differ in their antigenic coat from the SR-RSV uscd for immunization.

It scems morc probablc that the foci of transformed CEF, registered after passage, reflect the nunibcr of chicken fibroblasts that have originally rcceivcd the viral information from the turnour cells by means of a subviral dctcrminant via sonic type of (temporary?) cell- to-cell contact, not accessihlc for Ihc action of antiviral antibodies. This is in good accordancc wilh the findings by Suoboda et al. (1968) that cven a short treatnienl of a cell mixture with Sendai virus, probably crcating cytoplasmic bridges, increases the frequency of RSV genome transfer grcatly. Possible morphological bases for such a transfer have been discussed by Lindberg & .lonsson (1968) and Ahlstrijm C Lind- berg (1969). This also fits well with the approximately linear incrcasc of foci of transformed CEF in mixcd cultures with increasing tumour cell number (Jonsson 1966). In the casc of participation of virus particles such a n incrcasc would have bcen expcctcd to be logarithmic. These fisures as well as the rather large number of tumour cells rcquired to

64

demonstrate a transfer of thc viral gcnoinc indicate that such a cell- to-cell contact is a very rare spontaneous event in uitro (once per 104- 10’ cclls) but that probably one “contact-making” tuniour cell niight form coiitacls with iiiorc than onc chicken cell.

S U A1 hI A R Y

The nicchanism of the induclion of virus production in mixed cullurcs of Rous mouse or rat sarcoma cclls ancl chicken embryonic fibroblasts was studied by iiieans of irradiation of the chicken cells, attcrnpls to absorb anliviral activity with cell prcparalions and co-cultivation in the presence of antiviral immune serum. Irradiation of the chicken cells in mixed cullurcs prcventcd the prodaction of virus. KO decrease of antiviral activity of the immune serum could be demonslraled by means of prcparatioiis from mammalian tumour cells in spite of the presence of specific transplantation antigens. The presence of anti- scruin in the mixed cultures did not significantly affect the nnmbcr of foci of transformed chickcn cells formed after passage.

The results indicate that RSV synlhcsis cannot be indiicccl in the rodent tumour cclls by mere contact with chicken cclls, and that the rodent liimour cells are not capable of synthesizing any viral antigen, demonstrable by absorption technique. It seems improbable that thc foci registered after passage of the mixed cultures within 4-6 days derive from virns rclcased from rodent tumour cclls or temporarily surviving hetcrokaryons; the number of foci reflects probably the num- ber of chicken cclls that havc directly received the viral information via some type of cell-to-cell conlacl.

H E F E I3 E N C E S

1. Ahls trom, C . G . : Keoplasms in mammals induced hy Rous chicken sarcoma material. Xat. Cancer Inst . Monogr. 1 7 : 299-319, 1964.

2. ..lhlsfriiin, C. G. & Lindberg, I , . G . : Growth of Rous hamster sarcoma on the chorioallantoic membrane. Acta path. microbiol. scand. 77 : 49-66, 1969.

3. Doyse, E. A. , O[d, L. J. & Thomas, G . : A report on some ohscrvations wi th a simplified cytotoxic test. Trauspl. Bull. 29: 63-67, 1962.

4. Or{jan, W. R., Mo[one!r, J . 13. h Calnan, Zl.: Stable standard preparations of t he 13011s sarcoma virus preserved by frcezing and storing a t low temperatures. J. Nat. Cancer Inst. 15: 315-329, 1954.

5. F i n k , M . A . h Rauscher, F . J . : A simple method fo r the preparation of potent chicken anti-Rous sarcoma virus scrum. J. Nat. Cancer Inst. 26: 519-522, 1961.

6. Jonsson,N.: Studies on the presence of thc viral genome in Rous virus- induced mouse tumonrs. Acta Univ. Lund. Sectio 11, No. 23, 1966.

7. Jonsson, N . & Sjogren, H. 0.: Fur ther studies on specific transplantation antigens in Rous sarcoma of mice. .J. Exp. med. 122: 403-421, 1965.

8. Lindberg, L. G . h Jonsson, iV.: Types of contacts in uitro betwcen Rous mouse tumour cells and chicken fibroblasts as studied wi th the electron micro- scope. Acta path. microbiol. scand. 73: 359-368, 1968.

9. Rubin, H . : .4 virus in chick embryos which induces resistance in uifro to infec- tion wi th ROLIS sarcoma virus. Proc. Nat. Acad. Sci. 4 0 : 110.5-1119, 1960.

10. Rubin, H., Cornelius, A . h Fanshier , L.: The pattern of congenital transmission of a n avian leiicosis virus. Proc. Nat. Acad. Sci. 4 7 : 1058-1069, 19c1.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Rubin, H . ds Temin, H . M.: A radiological study of cell-virus interaction in the Hous sarcoma. Virology 7 : 75-91,1959.

ShevZ?jaghin, V . 1’. h Martiros!jan, D. M.: Reproduction of Rous virus in mouse embryonic tissue . in oitro. Fol. Riol. 22: 184-188, 1966.

SirnlcoviE, D. , Valenfoua, N . ds Thurzo, V . : An in uitro tcm for the detection of the Kous sarcoma virus in the cells of the r a t tumour SC. Neoplasma 9 :

Suoboda, J.: Malignant interaction of Rous virus wi th mammal ian cclls in oiuo and in uitro. Nat. Cancer Inst. Rlonogr. 17: 277-298,1964.

Suoboda, J. , Chijle, P., Simkovic*, D. ds Ili lyerf, I . : Demonstration of t he absence of infectious Hous virus in r a t tumour XC, whose structurally intact cells produce Rous sarcoma when transformed t o chicbs. Folia Biol. 9 : 77-81, 1963.

Suoboda, J . , Hlojdnelc, I . & Machalu, 0.: Quantitative assay systcm for the trans- mission of HSV genome f rom virogenic mammalian cells into the chicken cell with the help of Sendai virus. Fol. Biol. 14: 26-33, 1968.

Ternin, I f . M.: Studies on Carcinogenesis b y avian sarcoma viruses. V. Requirc- ment for new DNA synthesis and for cell division. J. Cell Physiol. 69:

Ternin, H . Af. & Rubin, H . : Characteristics of a n assay fo r Rous sarcoma virus and Rous sarcoma cells i n tissue culture. Virology 6 : 669-688, 1958.

Viyier, Ph. ds Monfagnier, L.: Pcrsistencc and action of the Hous sarcoma virus genome in transformed rodent cells. In “Subviral carcinogenesis” p. 156- 175 Ed. Y. Ito, Kyoto 1966.

Vigier, Ph. h Suoboda, J . : e tude , en culture, de l a production du virus de Rous p a r contact cntrc lcs cellules du sarcome XC du r a t et lcs cellules d’em- bryon de poule. Compt. Rend. Acad. Sci. (Par i s ) 261: 4278-4281, 1965.

104-106, 1962.

53-64, 1966.

6 ACTA PATII. 77, 1