Transformation In Vitro of Glial Hamster Cells by Rous Sarcoma Virus1. 2 G. F. Habottl," J. Gogusev,3 B. Teutsch," F. Mongiat-Lardemer,3 and F. Haquenau'':

ABSTRACT-Cell Iines from the brains of inbred CF hamster embryos were established in vitro. The morphology of the cells in the light and electron microscopes was that of glial cells, and the cells contained the nervous system-specific protein 5-100. Infection with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup B, resulted in foci of transformation. The transformed cells were virogenic and upon intracerebral and sc inoculations into young hamsters, they developed into histologically typical gliomas.-J Natl Cancer Inst 60: 113-124, 1978.

Malignant transformation in vitro of normal glial cells by RSV has not been demonstrated. Since RSV after ic inoculation is known to propagate in the brain of chicks (1) and large amounts of this virus can be easily extracted from infected brain tissues even in the absence of gross lesions (2), investigators have speculated that cells other than the typical Rous sarcoma cells seen in the meninges (1, 3, 4) could be responsible for virus replication. However, no one has been able to locate the exact cellular site of virus synthesis within the brain (2). In chickens infected ic with RSV, meningeal sarcomas were readily induced (5); electron microscope studies showed that only the typical RSV-transformed meningeal cells produced virus, and no virus was ever observed in the brain tissue per se (6). Nevertheless, Adams (7) showed that normal chicken glial cells in vitro may support growth of RSV without any evidence of transformation. In Adams' work, however, no electron microscopy was done to show that typical glial cells were effectively producing virus. However, studies have shown that RSV inoculated ic can ind uce typical gliomas in aseries of mammals (8, 9), and other studies have shown that one can infect cells ?erived from gliomas with RSV in vitro (10-12); but smce these cells are tumor cells, one cannot draw from these studies any conclusions as to malignant transformation. The present work shows that RSV can transform normal hamster glial cells cultivated in vitro. This conclusion is based on the characteristic glial cell structure in the conventional and electron microscopes, the presence of S-100 protein, and the development of typical gliomas after ic or sc inoculation of transformed cells into young hamster.

MATERIALS AND METHODS Brains from inbred CF hamster embryos (10-12 days) were dissected and minced. The pieces were vigorously dispersed by pipette into Eagle's basal medium containing 5% calf serum and antibiotics. After 2-3 weeks the VOL. 60,

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cultures were trypsinized and transferred to and maintained in secondary medium where the serum was reduced to 2%. Subsequent transfers were made every 15-20 days, during which time cultures were not fed. This caused intense granulation of the fibroblasts present in the cultures during the first few months. The low serum content and the infrequent change of medium resulted in complete disappearance of fibroblasts in some of the cultures after 3-4 months. Four or five days after transfer, cultures between generations 7 and 10 were infected with SR-RSV-2 at a multiplicity of infection of 1-10 and were maintained in secondary medium. Clones of transforrned cells that appeared 3-4 weeks later were pooled and propagated in the same medium. The transformed cells were cocultivated with chick embryo C IE fibrob1asts [Edinburgh Leghorn Brown (ELB); obtained from Dr. F. Lacour, Villejuif, France] (10 6 transformed cells and 2 x 106 chicken cells) in 75cm" Falcon bott1es. After two passages, the culture medium was titrated in chicken CIE and CIBE and quai1 QIBCD embryo fibroblasts. Neutralization tests were done with secondary chick embryo fibroblasts of phenotype CIE and CIO. Chicken antisera with specificity against subgroup A, B, and D viruses were used. For the tests, 0.8 ml of each dilution of antiserum was mixed with 0.2 m1 of virus suspension containing about 103 FFU of the different viruses and incubated at 37° C for 40 minutes. The surviving fraction was assayed by focus formation. The nervous system-specific acid protein S-100 (13) was tested in glial cells with the use of the indirect fluorescent antibody method (14). The globulins from rabbit anti-S-100 from cow brain (prepared by Dr. R. Herschman, U niversity of Copenhagen, Copenhagen, Denmark) were obtained through the courtesy of Dr. F. De Vitry (College de France, Paris). ABBREVIATIONS USED: RSV = Rous sarcoma virus; ic intracerebral(ly); SR-RSV-2 = Schmidt-Ruppin strain of RSV, subgroup B; FFU = focus-forming units; SV40 = simian virus 40; PBS = phosphate-buffered saline; RSV(RAV 1) and RSV(RAV 2 ) = Bryan strain of RSV, subgroups A and B, respectively; SR-RSV-H = virus rescued after cocultivation of SR-RSV-2 transformed hamster cells with susceptible chicken fibroblast cultures. 1 Received J anuary 10, 1977; revised J uly 6, 1977; accepted J uly 21, 1977. 2 Supported in part by a subsidy from the Fondation pour la Recherche Medicale Francalse. 3 Laboratoire de Medecine Experimentale, College de France, et U nite 112, Institut National de la Sante et de la Recherche Medicale , 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France , 4 We are indebted to Professor G. Lyon (Departement de Neurologie Pediatrique , Clinique St. Luc, Brussels, Belgium) for reviewing the pathology slides.

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The cells to be tested were grown on cover slips and fixed in cold acetone. They included normal and RS Vtransformed hamster glial cells, SV40-transformed mouse glial cells used as positive controls, rat cells from a tumor of the hypo physis , and hamster normal fibroblasts used as negative controls. The fixed cells were incubated with different dilutions of normal 01' anti-S100 rabbit globulins for 1 hour in a humidified chamber at 22° C. The slides were extensively rinsed in PBS, dried, and stained with labeled goat anti-rabbit globulin at a dilution of 1:10. The slides were rinsed in PBS, mounted in 10% glycerol in PBS, and viewed in a Leitz fluorescence microscope. Young CF hamsters were inoculated ic 01' sc with various concentrations of normal 01' transformed cells. For electron microscopy, monolayers of normal and transformed glial cells were fixed in situ with 5% glutaraldehyde for 2 hours at 4° C. They were rinsed three times in Sörensen's phosphate buffer (pH 7.4). Cell pellets were obtained after cen trifugation at 3,000 r pm/ minute for 10 minutes followed by post-fixation with 1% OS04 for 1 ho ur . They were then dehydrated in graded ethanol solutions and embedded in Araldite according to the procedure of Barnes (15). Cells were also embedded in situ in Epon (16). Sections were studied with a Siemens Elmiskop 102.

RESULTS Cultures of normal cells from fetal hamster brain grew slowly. Separated by wide spaces to form a lacelike net, they never reached confluence. In the light microscope, these cells were seen to be of different sizes. They were large, multipolar (often triangular 01' bipolar), ovoid bodies. At each end of the cells were long, fine, protoplasmic extensions (figs. 1, 2). In some cultures, foci of transformation appeared 3-4 weeks after RSV infection. In contrast to the normal cells, the transformed cells grew in a crisscross pattern leaving little or no space between them. Contact inhibition was lost, the growth rate was increased, ce11s rounded up, and both the nuclei and cytoplasm of the cells appeared hypertrophied (figs. 3,4). In the electron microscope, both normal and transformed cells were seen to have flat, large, and regularly shaped nuclei and a remarkably clear nucleoplasm. As a rule, the perinuclear chromatin ring was strikingly absent or extremely thin. The nucleoli were compact and round (figs. 5, 7). In the cytoplasm, the ergastoplasm was tenuous, undilated, and truncated. Ribosomes were extremely osmiophilic (figs. 8, 10-13). Mitochondria sometimes showed characteristic longitudinal orientation of the cristae (figs. 8, 10, 11). Fibrils with the characteristics of gliofibrils were present in a11 parts of the cytoplasm (figs. 12, 13). Dense, homogeneous lysosomes typical of glial cells were also present (fig. 8). The fibrils were 8-9 nm in diameter and not always completely smooth. At times, wisps arising from their surface formed bridges between adjacent fibrils. The general aspect of these preparations was quite J

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different from that of fibroblasts (cf. fig. 5 with fig. 6, and fig. 8 with fig. 9). Virus partic1es were never observed in the transformed cells. Cultures of normal and RSV-transformed harnster fibroblasts studied in parallel exhibited different morphologies: The nuclei of these cells were irregular in shape , and their r irns were thick. The chromatin was conspicuously marginated. The nucleoplasms were not homogeneous. The nuc1eoli were irregular in contour (fig. 6). In the cytoplasm, the ergastoplasrn was hig hly developed and rarely tru ncated: its cisternae were often dilated. The typical appearance of the ergastoplasm is shown in figure 9. When present , lysosomes varieel in appearance and structure. Microfilamen ts wer e sornetimes abundant. With the fluorescent antibody method for S-100 proteins, staining in the normal cells was scattered in t.he cytoplasm, mostly in the perinuclear region (fig. 14); in some cells the staining also appeared as fluorescent spots on the plasma membrane. The sa me type of fluorescence was present in mouse glial cells tr ansformed by SV40. The markedly reduced fluorescence of the negative control cells anel the absence of fluorescence in glial cells treated with normal rabbit globulin strongly suggested that the S-100 protein was being stained by the anti-S-100 antibody. The intensity of S100 staining appeareel reduced in transforrned glial cells as compareel to normal glial cells. Cocultivation of the RSV-transformeel glial ce11s with fibroblasts from ELB e mbryos of phenotype C/E resulteel in the rescue of the virus. The host range properties of the rescued virus were indistinguishable from those of the virus used in the initial transformation of glial cells. Both viruses hael a com parable titer of 10ö -10 7 FFU/ml when titrateel in C/D or C/E chick e mbryo fibroblasts and of approximately 10 FFU/ml when titrated in the C/BE ehiek embryo fibroblast 01' Q/BC]) quail embryo fibroblast. The serologie properties of the virus reseued from RSV-transformed glial eells are presented in table 1. One can see that the reseued virus was neutralized by ehieken antiserum specifie for subgroup B virus [RSV(RAV 2 ) anel SR-RSV-2] anel th at an tiser urn against SR-RSV-2H was again capable of neutralizing a11 subgroup B viruses. The partial cross-reaction of antisubgroup D antiboelies with subgroup B viruses as shown in table 1 was alreaely observeel by Weiss (17). Antiserum against subgroup A virus was ineffeetive. Transformed eells were inoculated ie 01' sc at varying eoneentrations into young CF hamsters. The resulis (table 2) showed that 100% of the animals inoculated with s x 104 eells elevelopeel tumors within 1 month. The histologie features of the tumors were those of gliomas (fig. 15).

DISCUSSION The major problem encou nrered in the effort to obtain a population of glial cells from hamster brain eultures has been fibroblast eon tamination. To obviai e VOL. 60, NO.

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HAMSTER GUAL CELLS TRANSFORMED IN VITRO BY

TABLE l.-Neutralization tests" of sarcoma virus subgroups A and B with the use of chicken anti-A, anti-B, and anti-D antisera Surviving fractions of"

Antisera and final dilution of antisera b

RSV (RAV 1 )

Anti-RSV(RAV 1 ) Control 1:800 Anti-RSV(RAV 2 ) Control 1:800 Anti-SR-RSV-2 Control 1:400 Anti-SR-RSV-2H Control 1:400 Anti-SR-RSV-D Control 1:800

RSV (RAV 2 )

1 0.005

SRRSV-2

SRRSV-2H

1 0.9

1 1

1 0.01

1 0.01

1 0.01

1 1

1 0.01

1 0.005

1 0.01

1 1

1 0.01

1 0.01

1 0.01

1 1

1 0.1

1 0.1

1 0.1

"See "Materials and Methods." b Anti-RSV(RAV 1)=chicken sera neutralizing subgroup A of Bryan strain of RSV. Anti-RSV(RAV2)=chicken sera neutralizing subgroup B of Bryan strain of RSV. Anti-SR-RSV-2=chicken sera neutralizing subgroup B of Schmidt-Ruppin strain of RSV. Anti-SRRSV-D=chicken sera neutralizing subgroup D of Schmidt-Ruppin strain of RSV C The surviving fractions of neutralized virus are calculated as percent of FFU of the control made equal to 1. TABLE 2.-Tumors in young hamsters inoculated with RSV-transformed glial cells"

Cells Normal Transformed

No. of cells inoculated xl0 4

Results" after: Intracerebral inoculation

Subcutaneous inoculation

0/5 0/6 80/91 60/60 75/75

0/10 0/15 75/101 126/126 51/51

5 7 2.5 5 7

a Inbred hamsters were inoculated between 3 and 6 wk of age. These results were obtained 1 mo post inoculation for hamsters inoculated with transformed cells and 8 mo post inoculation for hamsters receiving normal glial cells. b No. ofhamsters with tumors/total No. of hamsters inoculated.

the predominant growth and invasion by fibroblasts that always occur, we grew the hamster glial cells in Eagle's basal medium containing 2% calf serum. Hamster fibroblasts were previously observed to grow poorly in this medium and to degenerate after a few passages if the cultures are fed every 15-20 days. Under these conditions, fibroblasts disappeared completely in about 2 months, and the remaining population of cells grew slowly, never became confluent, and could be transferred every 2-3 weeks. These cells are believed to be of glial nature on the basis of the following criteria: a) morphology characteristic of glial cells, b) presence of the nervous systemspecific acidic protein S-100 (13), and c) histopathology of the tumors that develop after inoculation of the RSV-transformed cells into young hamsters. The morphology of the cells clearly differed from VaL. 60, NO. 1, JANUARY 1978

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that of fibroblasts, as evidenced in the phase-contrast and the electron microscopes. Ir did eorresponel to that of the glial cells in normal brain tissue from different mammals, as deseribed by other authors (18, 19), anel that of the glial cells in experimental glial tumors, described by us (20-22). It also corresponded to that deseribed in short-term eultures (23, 24) and long-term cultures of normal or tumor glial cells (10-12,25). The identifieation of these glial cells in the electron mieroseope was aided by the study of cultures eonsisting of transformed glial eells eoeultivated with normal ehieken fibroblasts, sinee RSV-transformed nonpermissive hamster glial eells are virogenie. This enabled us to traee, in the same preparation, the permissive ehieken cells by the presence of budding viruses and the hamster cells that contained no virus. Another feature also served as a marker of the hamster eelJs: the nuclear bodies that are abundant in these eells but whieh are not observed, as a rule, in ehieken nuclei (unpublished observation) . The cells contain the S-100 protein, one of the several nervous system-specific cytoplasmic antigen cornponents. The reagent used in the test for this antigen gave a positive result also with mouse SV40-transformed glial cells. It was negative with cultured hamster fibroblasts. In our material, normal glial cells contained more S-100 than did transformed cells. Even so, the transformed cells were unquestionably positive. The presence of S-100 protein in cultured rat glial tumor was previously reported (26). The transformation of hamster glial cells in vitro by SR-RSV is not surprising in view of previous results on the induction of gliomas in vivo (9). The malignaney of the cells could be easily demonstrated by inoculation of as few as 2.5 X 104 eells ie and 5 x 104 cclls sc into young isogenie hamsters. The morphology of the tumors thus induced in the inoeulated hamsters was that of gliomas. No virus particles were seen in the transformed eells, but the capacity of these eells for yielding infeetious virus eould be easily demonstrated after coeultivation with ehieken fibroblasts. Ir had already been shown that cells from glial tumors induced in mammals by RSV were virogenie (9). In those previous data , however, one eould not exclude the possibility that the virus may have been rescued from transformed fibroblasts present in the glial tumors. The demonstration that typical glial eells transformed by RSV in vitro after eoeultivation ean express the mature virus particles with its original serologie and host-range markers clearly proves that mammalian neuroeetodermic eells ean enter the virogenic state after transformation by RS V .

REFERENCES (l) VAZQUEZ-LoPEZ E: On the growth of Raus sareoma inoculated

into the brain. Am J Cancer 26:29-55, 1936 (2) RAUSCHER FJ, GROUPE V: Im portanee of the infecting dose on growth patterns of Raus sarcorna virus (RSV) in ehick brain. J Natl Cancer Inst 25:1391-1404,1960 (3) DURAN-REYNALS F: The signifieanee of nonneoplastic lesions

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(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

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induced in the central nervous system of ducklings by the virus of a duck variant of the Rous sarcoma. Yale J Biol Med 22:555-564, 1950 GRouPE V, RAuscHER FJ: Growth curve of Rous sarcoma virus and relationship of infecting dose to yield of virus in chick brain. J Natl Cancer lnst 18:507-514, 1957 VIGIER P: Passage intracerebral du virus du sarcome de Rous chez le poussin nouveau-ne . Bull Cancer (Paris) 74:409-421, 1957 RYTER A: Transformations morphologies du es aux injections intracerebrales de virus du sarcome de Rous chez le poussin nouveau-ne , Etude au microscope electronique , Bull Cancer (Paris) 7:237-252, 1960 ADAMS EV: Response of cultured chicken brain neuroglial ceIls to infection with Rous sarcoma virus. J Natl Cancer Inst 37:347-352, 1966 RABOTTI GF, RAINE WA: Brain tumours induced in hamsters inoculated intracerebrally at birth with Rous sarcoma virus. Nature 204:898-899, 1964 RABOTTI GF: Experimental intracranial tumours ofviral etiology. In The Experimental Biology of Brain Tumours (Kirsch WM, Grossi E, Paoletti P, eds). Springfield, Ill.: Charles C Thomas, 1972, pp 148-180 PONTEN J, MAcINTYRE EH: Long term culture of normal and neoplastic human glia. Acta Pathol Mierobiol Scand 74:465486, 1968 MAcINTYRE EH, GRIMES RA, VATTER AE: Cytology and growth characteristics of human tumour astrocytes transformed by Rous sarcoma virus. J Cell Sei 5:583-602, 1969 MAcINTYRE EH, PONTEN J, VATTER AE: The ultrastructure of human and murine astrocytes and of human fibroblasts in culture. Acta Pathol Microbiol Scand [A] 80:267-283, 1972 MOORE BW: A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 19:739-744, 1965

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(14) HYDEN H, McEwEN B: A glial protein specific for the nervous system. Proc Natl Acad Sei USA 55:354-358, 1966 (15) BARNES BG: The fine structure of the mouse adenohypophysis in various physiological states. In Cytologic dc l'Adcnohypophyse (Benoit J, Dalage C, ecls). Paris: Editions clu CNRS, 1963, pp 91-109 (16) BRINKLEY PR, MURPHY P, RIcHARDsoN LC: Proceclurc for embeclding in situ selectecl cells culturecl in vitro. J Cell Biol 35:279-283, 1967 (17) WEISS RA: lnterference ancl neutralization studies with Bryan strain Rous sarcoma virus synthesized in the absence of helper virus. J Gen Virol 5:529-539, 1969 (18) PETERS A, PALAY SL, WEBSTER HF: The fine structure of the Nervous System. New York: Harper & Row, 1970 (19) RAINE es, PODUSLO SE, NORTON WT: The ultrastructure of purified preparations of neurons ancl glial cells. Brain Res 27:11-24,1971 (20) BUCCIARELLI E, RABOTTI GF, DALTON AJ: Ultrastructure of gliomas induced in hamsters with Rous sarcoma virus. J Natl Cancer Inst 38:865-889, 1967 (21) HAGUENAU F, RABOTTI GF, LYON G, et al: Gliomas inclucecl by Rous sareoma virus in the clog-an ultrastructural stucly. J Natl Cancer lnst 46:539-559, 1971 (22) - - - : Tumeurs experimentales d'etiologie virale chez le chien . Rev Neurol (Paris) 126:347-370, 1972 (23) SHEIN HM: Propagation of human foetal spongioblastoma ancl astrocytes in clispersecl cell cultures. Exp Cell Res 40:554-569, 1965 (24) KRASNICKA Z, BOROWICZ JW, GAJKOWSKA B: Ultrastructure of glial ceIl cultured in vitro. Pol MeclJ Xl:1687-1698, 1972 (25) SATO G: Tissue culture of the nervous system. In Current Topics in Neurobiology, voll. New York: Plenum, 1974, pp 1-288 (26) BENDA P, LIGHTBODY J, SATO G: Differentiated rat glial cell strain in tissue culture. Science 161:370-371, 1968

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I.-Phase-contrast rnicroscopy of glial cells in cultu re . Not e fin e protoplasm extensions that confer a stretched aspccr 10 the cells . Always separated , the cells are multipolar. mostly bipolar and tripolar. X 170 FI GURE 2.-AI a high magnification , the cell body is voluminous . Nuclei arc wund 0 1' ovoid . regular , and clea r, with n u rne ro us anti welldelimited nuclcoli . x 680 FIGURE

RABOTTI , GOGUSEV, TEUTSCH , MONGIAT-LARDEMER, AND HAGUENAU

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FIGURES

118

3. 4.-Aspect of focus of transformation induced by RSV. Note absence of contact inhibition . Figure 3:

X

iO; figure 4: x 340

RABOTTI, GOGUSEV, TEUTSCH, MONGIAT-LARDEMER,

A~D

HAGUF.!\AU

5.-Typica l aspcct of uu clei from glial cel!. N ot" homogen eo u s and fine ly gra n ular as pe cr of n ucle o pla sm of the glial cc ll, well-d cli mired an d roundr-d contou r o f ih c nuclco lus , a nd absencc of chrom atin ma r gin ati on . X 4,000 F/G UR F. 6 .-Typ ical aspe rt of nuclei fro rn fibro blast.. Nucleoplasrn is less homogeneous in rh e fih robl ast tha n in thc glial ccll ; uurlcol i a rc less weil d elim ited , an d chro matin margination can be seen. x 23JlOO

FIG URE

RABOTTI , GOG U S EV, TEUTSCH, MONGIAT-LARDEMER, AN D HAGUENAU

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FIGDRE

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7.-0ther exam plcs of nucl ci in glial cells .

X

41.400

RABOTTI , GOGUSEV, TEUTS CH , MONGIAT-LARDEMER, AND HAG U ENAU

FIGURE 8.-Glial cell (cf. with fibroblast in fig. 9). Note filiform and truncated aspect of ergastoplasrn in glial cell. x 12,000 FIGURE !l.-Fibroblast. Note dilated protein containing ergastoplasrnic cisternae of fibroblast. x 7,200

RABOTTI , GOGUSEV, TEUTSCH, MONGIAT-LARDEMER , AND HAGUENAU

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FIGUKE FIGURE

122

10.-Elongated mitochondria of glial cell, with cristae parallel to their long axes . x 27,000 l L'-e-Detail of cyroplasm of glial cell and truncated aspect of the ergastoplasm. X 32,400

RABOTTI, GOGUSEV, TEUTSCH, MONGIAT-LARDEMER, AND HAGUENAU

12, 13.-Gliofilamcnts (8-9 nm) distributed witbout particular orientation nature of ribosornes. Figure 12: X 65,000; figure 13: x 85,000

FIGURES

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III

thc cytoplasm of glia1 cells . Note intense osm iophilic

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FIGURE FIG URE

14.-Normal glial cells stain ed with anti-S-IOO rabbit globulin (see text) . Note perinuclear tluorescence . x 2,000 15.-Morphology of typical glial tumor obtained by sc inoculation of weanling hamster with 106 RSV-transformed glial cells. Mallory's

stain , x 170

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Transformation in vitro of glial hamster cells by Rous sarcoma virus.

Transformation In Vitro of Glial Hamster Cells by Rous Sarcoma Virus1. 2 G. F. Habottl," J. Gogusev,3 B. Teutsch," F. Mongiat-Lardemer,3 and F. Haquen...
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