hit. J . Cancer: 17, 396-406 (1976)

CHARACTERISTICS OF THREE STRAINS OF FELINE FIBROSARCOMA VIRUS GROWN IN CAT AND MARMOSET MONKEY CELLS Ruth MCDONALD, Baku1 THAKKAR, Lauren G . WOLFEand Friedrich DEINHARDT Department of Microbiology, Rush-Presbyterian-St. Luke's Medical Center, 17.53 West Congress Parkway, Chicago, Ill. 60612; and Graduate College, University of Illinois at the Medical Center, Chicago, Ill. 60612, USA

Summary Two strains ojfeline fibrosarcoma virus (ST-FeSV and GA-FeSV) were found to induce tumors in cats and marmosets, and to transform feline and marmoset cells in vitro after primary inoculation. A third strain (SM-FeSV) failed to induce tumors or transform marmoset cells after primary inoculation; however, when SM-FeSV-infected marmoset cultures were passed 26 times in vitro, the cell cultures released infectious virus which transformed marmoset fibroblasts but still failed to induce tumors in marmosets. ST-FeS V induced mainly round-cell type transformation ( r foci), GA-FeSV induced predominantly mixed round-fusiform cell type transformation (fr foci), and SM-FeSV induced r and f r type foci with a higher proportion of fusiform cells in the f r foci than seen with GA-FeSV. Transforming virus was obtained from r or mixed rjfr foci of ST-FeSV but not from f r foci; heat treatment changed the virus from producing almost exclusively r type foci to inducing an increased number of f r foci. Passage of FeSV in cat cells yielded viruses with a higher ratio of infectivity for feline vs marmoset cells, while passage of FeSV in marmoset cells yielded virus with a relatively higher infectivity ratio for marmoset cells; the three strains differed in the degree of change in the infectivity ratio. Despite the alteration of host range of SM-FeSV propagated in marmoset fibroblasts, the virus retained feline P-30 antigen by CF and FA assays, Neutralization tests did not indicate but also did not exclude an alteration of the surface antigens of ST-FeSV or SM-FeS V propagated in marmoset fibroblasts. The alteration of the relative infectivity of FeSV during passage in marmoset cells may be due to: ( 1 ) the selection of a variant present in the original heterogeneous uncloned population; ( 2 ) mutation; or ( 3 ) recombination with some marmoset genetic material, possibly an as yet unident$ed endogenous marmoset virus. Several strains of C-type RNA viruses have been isolated from naturally-occurring feline fibrosarcomas and examined for their oncogenicity in vivo and transforming property in vitro (Deinhardt ef al., 1972; Hampar et al., 1970; Lee, 1971; McAllister et al., 1971; McDonough et a[., 1971;Pearson et al., 1973; Rabin, 1971; Sarma et al., 1970, 1971a, b, 1972; Snyder, 1971; Snyder and Theilen, 1969; Theilen, 1971; Theilen et al., 1970; Ubertini et al.,

1971; Wolfe et al., 1971, 1972). I n our laboratory, the Snyder-Theilen (ST-FeSV) (Snyder and Theilen, 1969) and Gardner-Arnstein (GA-FeSV) (Gardner et al., 1970, 1971) strains of feline fibrosarcoma virus consistently induced sarcomas in marmoset monkeys (Saguinus fuscicollis, S. nigricollis, S. oediyus) (Deinhardt et al., 1970, 1972; Rabin, 1971 ; Theilen et al., 1970; Wolfe et al., 1971,1972) and transformed feline and marmoset cells in vitro (McDonald et al., 1971,1972; Sarma et al., 1971a, 6,1972). In contrast, the McDonough (SM-FeSV) strain failed to induce tumors in marmosets (Wolfe, McDonald and Deinhardt, unpublished data) and on primary infection transformed feline but not marmoset cells in vitro. However, marmoset cells infected with SM-FeSV became transformed after several passages in vitro, and released virus that transformed both feline and marmoset cells in vitro, although the virus still failed to induce tumors in marmosets. In this communication, we describe: (1) the tumor-cell lines established from cat and marmoset tumors induced by ST-FeSV and GA-FeSV; (2) the transformation of feline and marmoset fibroblasts in vitro by SM-FeSV, ST-FeSV and GA-FeSV; and (3) some of the characteristics of the virus released by the infected feline and marmoset fibroblasts. MATERIAL AND METHODS

Virus

Stocks of ST-FeSV and GA-FeSV were prepared from medium plus cell extracts of 5-day-old cultures of cell lines established from cat and marmoset tumors (McDonald et al., 1972). SM-FeSV was kindly supplied by Dr. Sarma, National Cancer Institute, Bethesda, Md., USA, as clarified fluids from infected feline embryo cells (5 x lo4FFU/ml). Additional stocks of SM-FeSV were prepared from medium plus cell extracts of feline and marmoset cultures infected with the SM-FeSV originally obtained from Dr. Sarma. Cell cultures

Tumor-cell lines were established from cat and marmoset tumors that had been induced by cell-free Received: October 20, 1975, and in revised form December 8, 1975.

FeSV

IN CELL CULTURES

FeSV of cat origin or viable neoplastic tissue of marmoset origin (Wolfe et a!., 1972). The tumor-cell cultures were grown in medium RPMI 1640 supplemented with 20% fetal calf serum (FCS) and 2 mM glutamine. Feline embryonic fibroblast cultures (FEF) and marmoset fibroblast cultures (MF) were established and grown as described previously (McDonald et al., 1972). Cultures were tested by fluorescent antibody (FA) tests for the species-specific P30 (gs-I) antigens of feline C-type RNA viruses (McAllister et al., 1972). Antiserum to the P30 antigen was provided by Dr. Gilden, Flow Laboratories, Rockville, Md., USA. Marmoset cell cultures were consistently negative for RD114 species-specific P30 antigen but all feline cell cultures, even if negative initially, became positive for this antigen during serial cell culture passages (usually between the 12th and 20th passages). Attempts were made to use only cells which were negative by FA for R D l l 4 antigens but this was not always possible. However, there was no indication in control experiments that the presence of RDI 14 antigen influenced in any way the growth of the sarcoma viruses. Tumor-cell lines and FeSV-infected feline and marmoset fibroblast cultures were trypsinized and subcultured every 4 to 7 days by standard tissue culture techniques as described previously (McDonald et al., 1972). Antisera

Antisera were obtained from cats and marmosets bearing tumors induced by ST-FeSV and GA-FeSV. The antisera were used in focus reduction and in indirect immunofluorescent antibody studies. The antiserum used in the fluorescent antibody studies came from a marmoset bearing a tumor induced by GA-FeSV and it contained antibodies to both the species- and interspecies-specific P30 (gs-1 and gs-3) antigens by double diffusion tests. Antisera obtained from marmosets bearing tumors induced by Simian sarcoma virus, type 1, (SSV-I) (Deinhardt et al., 1972) were also used in indirect fluorescent antibody studies. Antisera were produced in guinea-pigs against tween-ether-disrupted feline leukemia virus (FeLV) obtained from a persistently infected F-422 cell line (Rickard et al., 1969) and against SSV-1, obtained from a cell line established from an SSV-I induced marmoset tumor. The antisera were adsorbed with FCS (75 mg/ml serum) until they no longer reacted with FCS by double diffusion. Antisera against F-422 and SSV-1 virus contained antibodies to both species-specificand interspecies-specificP30 (gs-1 and gs-3) antigens in double diffusion tests. The antibody titers of the individual sera were determined in

397

complement fixation tests (CF) with tween-etherdisrupted F-422 or SSV-1 virus. Focus assay

ST-FeSV and GA-FeSV produced by the tumorcell lines and ST-FeSV and SM-FeSV released from infected feline and marmoset cultures were assayed quantitatively for the amount of transforming virus on feline (FEF-R) and marmoset fibroblasts by their ability to induce foci using the agar overlay technique (McDonald et al., 1972). Cultures which had formed an approximately 60 % confluent monolayer were infected with FeSV, incubated for 2 h at 37"C, re-fed with liquid medium, incubated at 37°C for 12-72 h, and then overlaid with 1 % Difco purified bacto-agar. For all incubations, the flasks were sealed and incubated containing atmospheric air. The assays were done on cells not pre-treated with diethylaminoethyl (DEAE)-dextran except where indicated. The titers were expressed as focus-forming units per ml (FFU/ml), and all FFU/ml reported were based on the examination of 12 or more flasks. Foci were counted 14 to 21 days after infection in either viable or fixed cultures. For fixation, 2 ml of fixative were added to agar-overlaid cultures after two rinses with Hanks' balanced salt solution (BBS-H). The fixative consisted of 9 parts 10% formalin, 6 parts absolute ethanol and 2 parts glacial acetic acid. Transformed cells fixed in this manner retained their morphology and high refractility and could be counted at a later date. Effect of DEAE-dextran

The effect of DEAE-dextran treatment of FEF-R and MF-69CBlM cultures on the number and morphology of FeSV-induced foci (large round cells (r) or mixture of small round and fusiform cells (fr)) was examined. FEF-R and MF-69CBlM cultures were seeded and 3-4 h later, 0.1 ml of DEAE-dextran (0.2 mg/ml BSS-H) was added and allowed to adsorb for 1 h. The cultures were then washed three times with BSS-H and infected with either ST-FeSV from marmoset tumor-cell line No. 2 or SM-FeSV from infected FEF-R or MF-69CB1 M cultures. Stability of the viruses inducing r and f r foci

The viruses produced by the two morphological types of foci (r and fr foci) induced in MF-69CB1 M cultures by ST-FeSV from marmoset tumor-cell line No. 2 were examined to see whether they consistently induced foci of the same morphological types. The r and fr foci were collected with Pasteur pipettes; cells from each focus were then grown in four Leighton tubes, cell-free supernatants of these cultures were inoculated into MF-69CB1 M cultures and the cultures were examined for r, fr and mixed r/fr foci.

398

MCDONALD ET AL.

This procedure was performed serially four times for each original focus. Virus-specific antigens

Test samples were prepared from approximately 6 ~ 1 cells 0 ~ from 5-day-old cultures of cat and marmoset tumor-cell lines (passages 4 to 14) and SM-FeSV-infected feline and marmoset cells. The cells were scraped into a small amount of veronal buffered diluent (VBD), PH 7.4 and centrifuged at 8 0 0 x g for 20 min. Cell pellets were resuspended in 4 x volume of VBD, frozen and thawed three times, and again centrifuged as described above. Two-fold dilutions of supernatants were reacted with 2 units of guinea-pig complement and 2 units of antibody, prepared in guinea-pigs against tween-etherdisrupted, tissue-culture-grown FeLV or SSV-1. The degree of the C F reaction was scored on a scale of 0 to 4 (4 = complete fixation). A sample was considered positive for viral antigen if a 3 - 1 or greater fixation of complement was attained in the absence of anticomplementary reactions and absence of reactivity with normal sera (McDonald et al., 1971). These studies were performed before radioimmunoassays for the antigens of FeLV and SSV-I had been developed. Indirect fluorescent antibody studies were performed on the tumor-cell lines and infected cultures at the same passage levels at which they were examined by CF. Coverslip cultures were rinsed with BSS-H, fixed with chilled acetone for 7 min, air dried and stored at -120°C for 1 to 3 months before staining. Fixed cultures were stained for 90 min at 37" C and then 15-18 h at 4" C with antisera to GA-FeSV or SSV-I obtained from tumor-bearing marmosets; antisera were diluted 1 :4with phosphatebuffered saline (PBS) containing 2.5 % bovine serum albumin (BSA), PH 7.2. The cultures were washed twice with PBS, reacted for 30min at 37" C with anti-human IgG conjugated with fluorescein isothiocyanate (FITC) (diluted 1 :20 in PBS containing 1 % BSA), rinsed four times with PBS and mounted with elvanol, PH 8.2; strong cross-reactivity of anti-human IgG with marmoset IgG had previously been shown in our laboratory. Neutralization tests

Neutralization tests were performed to compare the reactions of sera obtained from cats and marmosets, bearing tumors induced by ST-FeSV or GAFeSV, against: (1) ST-FeSV isolated from cat or marmoset tumor-cell lines; and (2) SM-FeSV propagated in feline or marmoset fibroblasts. Approximately 3 0 0 FFU/ml of virus in RPMI 1640 medium were kept at 25" C for 1 h in a 1 :1 ratio with serial dilutions of heat-inactivated cat or marmoset sera (heat-inactivated 56" C, 3 0 min and

diluted in BSS-H). FEF-R cultures were infected with 0.4 mI of the virus-serum mixture. The neutralizing antibody titer was recorded as the highest serum dilution which inhibited development of 50% of the foci. All values listed in Table VII are the averages of two or three experiments; 2-fold serum dilutions in each experiment were performed in duplicate. Irnmunoelectro-osmophoresis (IEOP)

Counterimmunoelectrophoresis for measuring serum antibody titers against viral proteins was performed as described previously (Hoekstra and Deinhardt, 1971). Electron microscopy

Cell monolayers were scraped from the culture flasks, suspended in maintenance medium and centrifuged for 10 min at 800 x g . Cell pellets were fixed for 3 0 min in 2 % glutaraldehyde buffered with 0.1 M sodium cacodylate, postfixed in 1 % osmium tetroxide, dehydrated in graded alcohols and embedded in Epon 812. Thin sections were stained with lead citrate and uranyl acetate; unless virus was readily found, at least 100 cells from each preparation were examined for virus. RESULTS

Three cat and four marmoset ST-FeSV tumor-cell lines were easily distinguishable from four cat and five marmoset GA-FeSV tumor-cell lines. The ST-FeSV tumor-cell lines contained large round cells, small round cells and fusiform cells, whereas the GA-FeSV tumor-cell lines contained small round cells and fusiform cells but only a few large round cells (McDonald et al., 1972). This morphological distinction was stable in all cell lines during the observation period of 10 to 87 passages. Feline sarcoma-leukemia P30 group-specific (gs) antigen was demonstrated by C F and/or FA in all 18 tumor-cell lines examined (Table I). Antigen was present even when no virions were found by electron microscopy and when no transforming virus was recovered. As shown in Table I, ST-FeSV and GA-FeSV tumor-cell lines derived from cat tumors had higher antigen titers than the marmoset tumorcell lines. Furthermore, no correlation existed between the antigen titer and the amount of infectious virus produced by a tumor-cell line. The antigens detected with the particular sera used were predominantly P30 but the presence of additional viral glycoprotein or protein antigens was not studied further. Extracellular C-type virus and virus budding from plasma membranes were found in 17 of 19 tumor-cell lines. Morphological descriptions of the cells and

399

FeSV IN CELL CULTURES TABLE I ANTlGEN CONTENT A N D VIRUS PRODUCTION OF FeSV-INDUCED TUMOR-CELL LINES Virus production Virus strain

species

ST-FeSV

Cat

Marmoset

Tumor-cell line number

Cat

Marmoset

'

FFU/ml EM

CF

FA

+ + + + + + +

+ + + + t + +

1 2 3

> 1 :32 >1:32 >1:32

1

4

ND 1 :8 I :8 I :8

1 2

1 :32

f

f

> 1 :32

+

t

25 3

GA-FeSV

Gs antieen

3

>1:32

4

> 1 :32

1 2 3

1 :8 1 :I6 1 :16 1 :16 1 :I6 1 :16 1 :8

4

5 6 7

-

+

+ +

-

+ t -

f +

-

t t

ND

+ + +

t-

+ t -

FEF-R (cat)

MF-69CBIM (marmoset)

Infectivity ratio : FEF-R/MF-69CBIM

2,200 20,750 31,000

28 370 375

80 56 83

3,450 5,150 41,500 43,750

160 883 1,800 2,100

22 6 23 21

0 163 875 1,300

0 0 50 483

>163 18 3

0 0 15 83 95 113 700

23 0 750 60 500 10 230

-

Characteristics of three strains of feline fibrosarcoma virus grown in cat and marmoset monkey cells.

hit. J . Cancer: 17, 396-406 (1976) CHARACTERISTICS OF THREE STRAINS OF FELINE FIBROSARCOMA VIRUS GROWN IN CAT AND MARMOSET MONKEY CELLS Ruth MCDONAL...
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