Transformation of Rat Embryo Cells by Adenovirus Type 1

Transcription

1 J. gen. ViroL (1969), 4, With I plate Printed in Great Britain 29 Transformation of Rat Embryo Cells by Adenovirus Type 1 By R. M. McALLISTER,* M. O. NICOLSON,* A. M. LEWIS, Jus.,t I. MACPHERSON:~ AND R. J. HUEBNERt * Children's Hospital of Los Angeles, Los Angeles, Calif. 90o27. ~Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md , ~ Institute of Virology, University of Glasgow, Scotland (Accepted 24 June, 1968) SUMMARY Primary rat embryo cells were transformed by adenovirus I. The transformed cells formed foci of multilayered growth in monolayer cultures; cell lines were derived from such foci. A virus stock containing I.I x lo 9 virus particles and 1.5 lo8 p.f.u./ml, contained 2-3 x lo 2 focus-forming units/ml. when assayed in rat embryo cells. Approximately 4"8 lo 6 total virus particles or 6"5 x lo 5 p.f.u, were required to induce one focus of transformed cells. The transformation rate was o'o0o3 ~o for cells exposed to about 2 p.f.u./cell. The transformed cells contained adenovirus I specific tumour antigens when tested by immunofluorescent and complement-fixation reactions. They also synthesized adenovirus I specific RNA which forms RNase-resistant hybrids with adenovirus I DNA. INTRODUCTION Previous studies by Freeman et al. (I 967a) demonstrated the capacity of adenovirus (Ad) types 2 and 5, hitherto not suspected to be oncogenic types, to transform primary cultures of rat embryo cells in vitro. These investigators found that pulse-labelled RNA isolated from Ad-2 transformed cells hybridized to a significant extent with the DNA of adenovirus types I, 2 and 5. They therefore suggested that in addition to subgroup A viruses (types I2, 18, 30, which are highly oncogenic in hamsters, and the weakly oncogenic subgroup B (types 3, 7, 14, 16, 2I), a third group, subgroup C, composed of types I, 2 and 5, had demonstrated (types 2, 5) or suspected (type 1) transforming potential in vitro. This paper describes the transformation in vitro of rat embryo cells by adenovirus type I. METHODS Virus. Adenovirus type I (MOST strain) was recovered from a human lymphoma and propagated once in HeLa cells (McAllister, Landing & Goodheart, 1964). The virus was typed using antiserum from the Research Reference Reagents Branch (National Institute of Allergy and Infectious Diseases); the typing was confirmed by Dr Wallace P. Rowe (personal communication). The stock contained I.I x lo 9 virus particles/ml. when counted by electron microscopy (Watson, Russell & Wildy, 1963) and 1.5 IO8 p.f.u./ml, when assayed on human embryo kidney cells. No adenovirus-associated virus was detected in the stock by electron microscopy. :~ Present address: The Imperial Cancer Research Fund, Lincoln's Inn Fields, London.

2 30 R.M. MCALLISTER AND OTHERS Cell cultures. Near-term embryos from inbred Hooded Lister rats were trypsinized and cultures seeded with 3 x lo 5 cells/ml, in Eagle's basal medium containing antibiotics, twice the normal concentration of amino acids and vitamins, and supplemented with IO % foetal calf serum. Plastic Petri dishes (5 cm. diameter) were seeded with 5 ml. of the cell suspension, incubated at 37 in 5 % CO2 in air, re-fed on the third day, and used when confluent 6 days after seeding. Liquid and agar media. Transformation assays were made in Eagle's medium without calcium and with twice the normal concentration of amino acids and vitamins, supplemented with 5 % calf serum and 2 % foetal calf serum (F medium). Media for agar suspension cultures (Macpherson & Montagnier, I964) consisted of Eagle's medium supplemented with 2o % foetal calf serum and either o'33 % or o'5 % agar. Transformation assays. The method used was described in detail by McAllister & Macpherson (1968) and was similar to that of Freeman et al (I967a) and Freeman et al. (I967b), except that Petri dish cultures were used instead of tube cultures. Confluent monolayers of rat embryo cells (approx. 1.4 x Io 6 cells]culture) in 5 cm. plastic Petri dishes were rinsed with tris + saline solution (ph 7"4), exposed to o. I ml. of virus suspension for 3 hr at 37 and fed with 4 ml. of F medium. The cultures were incubated at 37 in a humidified atmosphere constantly gassed with 5 % CO2 in air. The medium was replaced every 2 to 3 days for 50 days. Foci of transformed cells were counted after the medium had been poured off and the cultures fixed with methanol and stained with Giemsa stain. Tests for infectious virus. The transformed cell lines were tested for infectious virus by passing lo 5 viable cells on each of IO confluent human embryo kidney cultures. The cultures were observed for 14 days when both o-z ml. of supernatant fluid and lo 5 cells were subcultured into fresh human embryo kidney cell cultures which were observed for a further 21 days. There was no evidence of cytopathic effect (CPE). Cultivation of transformed cells in agar medium. Trypsin-dispersed transformed cells (5 x lo 5) were mixed in 1.5 ml. of o'33 % agar medium and the mixture added to 5 cm. Petri dishes containing a pre-set base of 0"5 % agar medium (Macpherson & Montagnier, 1964; McAllister & Macpherson, 1968). After I day, 2 ml. of Eagle's medium containing 20 % foetal calf serum was pipetted on top of the agar and replaced weekly. Cell colonies were counted after 21 days. Histological studies. Colonies of transformed cells were removed from the agar medium 19 days after plating, fixed in fresh Bouin's solution, stained with haematoxylin and eosin (McAllister, Reed & Huebuer, I967) and examined by Drs George Reed, Hart Isaacs, Benjamin Landing, and Howard Igel. Serological tests. The Ad-I transformed rat embryo cell line used for preparing antigens for immunofluorescent tests was in the I8th to the 21 st tissue culture passage. Cells were grown on cover-glasses, fixed in cold acetone, and stained by the indirect method of Pope & Rowe (1964) using the appropriate goat anti-hamster, sheep antirabbit, or horse anti-human conjugate diluted in tissamine rhodamine-conjugated bovine serum albumin as a counter-stain. For the complement-fixation tests (Huebner et al. I963) frozen and thawed extracts of 20 % cell suspensions (11th tissue culture passage) were used for antigens. Individual sera were obtained from animals bearing transplanted tumours induced initially by Ad-I-SV 40 and Ad-2-SV 40 hybrid virus populations (Lewis et al. 1966; Black et al. I967). Additional sera were obtained from hamsters immunized as

3 Transformation of rat embryo cells by adenovirus type 1 weanfings with extracts of transplanted tumours induced by Ad-I-SV 40 hybrid (Gilden, Beddow & Huebner, 1967) and a child with an Ad-i infection who subsequently developed antibody to both Ad-I T and viral antigen (Lewis, Wiese & Rowe, I967). Serum pools were prepared from animals bearing transplanted tumours induced by Ad-7 (PINCKNEY strain), Ad-12 (HUIE strain), SV 40 (strain 776) or the AHLSTROM strain of Rous sarcoma virus. The antigens used in determining the fluorescent antibody titres of the serum specimens were homologous adenovirus-induced T or V antigen-containing cells fixed on cover-glasses (Lewis et al. 1966; Black et al. 1967). The 8426 line of rat embryo cells transformed by Ad-3 which was used as a positive control was kindly provided by Dr A. E. Freeman (Freeman et al. I967c). Studies of nucleic acid homology. Transformed cells in their 12th tissue culture passage were incubated for 30 min. at 37 in the presence of IOO/zc/ml. of [3~p]_ orthophosphate. The cells were harvested and whole-cell RNA extracted by amodification of the method of Scherrer, Latham & Darnell (I963), employing sodium dodecyl sulphate (SDS) and hot phenol. No carrier RNA was added. The purified RNA was treated with DNase (electrophoretically purified, Worthington Biochemicals) and re-extracted with hot phenol+ SDS. The RNA was precipitated several times with ethanol, dissolved in o'o15 M-NaC1, and o.oo16 M-Naa citrate, and dialysed overnight. Specific activity of the whole-cell RNA was 12oo counts/min.//zg. Purified adenovirus-i DNA was kindly supplied by Dr Maurice Green. Escherichia coli [SH]DNA was prepared according to Marmur (1961) from stocks grown in [ZH]thymidine. DNA and RNA were hybridized on membrane filters by the methods of Gillespie & Spiegelman (I 965) and Fuj inaga & Green (I 966). Virus-specific [3~P]RNA was identified by the formation of RNase-resistant hybrids with Ad-t DNA. Transplantation studies. Sixty-four newborn inbred Hooded Lister rats, 24 to 48 hr old, were injected subcutaneously with I to 5 x lo 6 transformed cells. 3 I Transformation of rat embryo cells RESULTS When confluent monolayers of rat embryo cells were exposed to virus, loci of multilayered growth were visible 25 days after infection (P1. 12 a). The loci were easily recognized against a monolayer background of untransformed cells and were identical with those induced in rat cells by adenovirus types 2, 3 and 12 (Freeman et al. I967a, b, c; McAllister & Macpherson, I968). No areas on morphologically altered cells were seen in uninoculated control cultures (Table 1). The smaller number of loci appearing in cultures exposed to lo p.f.u./cell compared with those exposed to 2 p.f.u./cell may have been due to the slight cytopathic effect noted in cultures exposed to the larger dose of virus. The number of Ad-1 virus particles or infectious units required to induce one focus was similar to that required by type I2 (McAllister & Macpherson, 1968 ) (Table 2). Freeman et al. (1967 a) showed that IO 7.3 ID 50 of Ad-2 induced loci in 17 of 27 tubes. Our studies with Ad-2 also showed that it was less effective than our Ad-I strain in transforming rat cells (Table 2).

4 Journal of General Virology, Vol. 4, No. ~ Plate I a. Focus of transformed rat embryo cells 2 5 days after infection. Unstained. b. Colony of Ad-l transformed rat ceils from agar suspension culture. The cytological characteristics resemble those of cells transformed by other adenoviruses; these include the uniform cell type, nuclear characteristics and occasional giant cells. Haematoxylin and eosin. c. Higher magnification of 2. d. Malignant lymphoma from which the Ad-i was isolated. Haematoxylin and eosin. R. M. McALLISTER AND OTHERS (Facll'lg p. 3I)

5 32 R. M. MCALLISTER AND OTHERS Cell line derivation Two foci of Ad-I transformed cells appearing in one culture 25 days after infection were picked with finely drawn Pasteur pipettes and each was subcultured in a plastic Petri dish in Eagle's medium supplemented with Io% foetal calf serum. Both loci yielded cell lines with an epithelioid morphology typical of adenovirus transformed cells. In another experiment a culture with foci of transformed cells was passaged. After the 5th passage, the culture consisted mainly of epithelioid cells. Table I. Transformation of rat embryo cells by adenovirus I Number of foci per monolayer culture (I-4 x io s cells/ culture exposed to virus) Virus input ~ - - ~, (p.f.u./cell) Expt I Expt 2 Io'7 4, 4 4, 4, 3, o 2.I 9, 7, 3, i 8, 8, 7, 5, 5, 3, 2, I, I i.i ND* 6, 4, 4, 2, 2, 2, i, i, i, o o'5 4, I, i, o, o ND* Control o, o o. o * ND = Not done. Table z. Transformation of rat embryo cells by adenoviruses* Ad-I Ad-2 Focus forming units 2"3 IO z 50 (f.f.u.)/ml. virus stock) Virus particles/f.f.u. 4"8 IOs 2-4 to a p.f.u./f.f.u. 6"5 x Io Ad-12 I I0 3 5 X lo s I'I X IO ~ * Ad-I = I"~ x IO 9 virus particles/rnl, and I'5 x io 8 p.f.u./ml. Ad-z = I-2 x io 1 virus particles/ml, and 2 x io 9 p.f.u./ml. Ad-]z = 5 x io 9 virus particles/ml, and I.I x xo 9 p.f.u./ml. Cultivation in agar medium The transformed cells in 8th passage formed colonies in agar medium with a plating efficiency of 5 %. When the colonies were processed by standard pathological techniques, they were found to have the cytological characters of adenovirus-induced hamster tumours (Berman, x967) or of agar suspension colonies derived from tumour cells (McAllister et al. I967) (PI. i b, c). The cytological characters of the colonies also resembled those of the human lymphoma from which the Ad-I was recovered (PI. I d); however, the significance, if any, of this observation is obscure and may well be a coincidence, especially since adenovirus-induced hamster tumours do not appear to be derived from the reticulo-endothelial system (Dr H. Igel, personal communication). Antigenic analysis of Ad-I transformed cells Immunofluorescence tests. The cells contained at least one antigen reacting specifically with sera from hamsters with high titre Ad-I T antibody. Appropriate controls were not stained by the reacting sera and the Ad-I transformed rat embryo cells

6 SCHMIDT-RUPPIN I:1 t~ Table 3. Immunofluorescent studies of Ad-I transformed rat embryo cells Type of serum From 2rid weanling hamster passage of Adq-SV 40 hybrid-induced tumour Serum from hyperimmune hamsters immunized with extracts of Adq-SV 40 hybrid-induced turnout From 4th weanling hamster passage of Ad-2-SV 40 hybrid induced tumour Serum from patient with Ad-I infection Ad-I hyperimmune-rabbit antiserum From hamster with Ad-i2 induced tumour From hamster with Ad-7 induced tumour From hamster with SV 4o induced turnout From hamster with AHLSTROM RSV induced tumour Antisera... h... FA titre Antigens (positive nuclei, ~) V, r A.~ homologous Screening Normal Adq trans. Ad-3 trans. Serum antigen dilution rat embryo rat embryo rat embryo H 438 x-h I/5 o o H 438I-H 5 40 t/5 o 4o-50 o R Io88-H 3 16o I/5 o 8o-9o I-5 R Io88-H 5 64o 1/5 o 8o-9o o R 726-H 2 I6o* I/5 o 8o-9o o R 726-H 3 16o* I/5 o o Acute Io I/IO 0 < I NT Convalescent x 60 i / i o o 5-I o NT LRAS- i i 640 i/4o o o NT FA pool G /> too i/io o o FA pool H /> Ioo I/IO 0 o 7o-80 FA pool D /> Iooo I/Io o o o FA pool 3 I> 16or ffio o o NT CF pool 23 NT~ ~/5 o o NT * Titrated against Ad-i T antigen containing cells. t Titrated against SV 4o T antigen induced by E CF antigen of I/8o against 4-8 units of AHLSTROM hamster tumour antigen. Table 4. Specificity of adenovirus tumour antigen as determined by complement fixation Antigen Adq transformed rat cells 32-64* I6-32 Ad-2 transformed rat cells Ad-12 transformed rat cells o o * Reciprocal of complement fixing antigen titre (2o ~ cell extracts). Hamster tumour antisera ^ Ad-~-SV 4o Ad-2-SV 4o Ad-5-SV 40 Adq2 SV Ot 0 ND O 0 ND o t 0-<4. ND = not done.

7 34 R.M. MCALLISTER AND OTHERS failed to react with sera containing other specific, high titre viral and T antibodies (Table 3). Morphologically, the nuclear staining pattern was indistinguishable from the staining of Ad-I and -2 T antigens in human embryo kidney cells (Black et al. 1967) and that described by Freeman et al. (I967a) for the Ad-2 transformed rat embryo cells. Most nuclei contained small irregular particles or dots which gave a dust-like appearance quite distinct from the thin needle-like nuclear flecks seen in Ad-7 or Ad-I2 transformed hamster or rat cells. In addition to the dust-like pattern some nuclei contained small dots or ovoid bodies and IO % to 20 % of the cells contained I or 2 highly staining ovoid bodies in the cytoplasm. The nuclear staining with human convalescent serum, while morphologically similar, was less frequent and less intense than that with sera from hamsters bearing tumours induced by hybrid virus. The cytoplasmic bodies were not stained by either of the two convalescent human sera tested. These findings suggest a different range of antigens in the Ad-I transformed rat embryo cells from those in Ad-I infected human and monkey ceils. Complement fixation tests. Antigens prepared from the Ad-I transformed cells reacted with sera from hamsters bearing tumours induced by Ad-I-SV 40, Ad-2-SV 40 or Ad-5-SV 4o hybrid viruses but not with sera from hamsters bearing tumours induced by Ad-I2, SV 40 or SCHMIDT-RUPPIN viruses (Table 4). Table 5. Nucleic acid homology Ad-i transformed whole-cell DNA RNA input (counts/rain.) RNA bound (counts/min.)* Input bound (%) Adenovirus-i, 3 #g. 26,9o0 269,00o 2o " Escherichia coli K 996, 3 #g. 26,9OO 24 O-O8 269,000 3 O'0OI * Blank value: filter incubated in [82P]RNA in absence of DNA was subtracted from each sample. Nucleic acid homology The results are shown in Table 5. It is readily apparent that Ad-I transformed cells synthesized Ad-I specific RNA which formed RNase-resistant complexes with Ad-I DNA. By comparison, hybrids were formed with Escherichia colidna only to alimited extent. In the range of RNA concentration examined, a tenfold increase in RNA resulted in about a fourfold increase in hybrid formed. Although a saturation plateau cannot be determined from two points, the data suggest thatthe viralrna represented a small fraction of the total transformed cell RNA. Transplantation studies Forty-eight of the 64 Hooded Lister rats which were injected with Ad-I transformed cells survived infancy and failed to develop tumours during I2 months' observation. DISCUSSION An unexpected result of our studies as well as those of Freeman et al. (I967a) has been the failure of Ad-I or Ad-2 transformed cells to induce tumours in rats. Why

8 Transformation of rat embryo cells by adenovirus type I 35 Ad-i transformed cells do not induce tumours is unknown and of interest since rat cells transformed by Ad-3 and Ad-I2 do induce tumours (Freeman et al. i967b, c). Ad-I and Ad-2 may produce strong transplantation antigens in the ceils they transform, thus minimizing their chances of initiating a focus of tumour cells. Tests to measure adenovirus-specific surface antigens on these cells are in progress. The authors wish to thank Dr E. A. C. Follett for the electron microscopy and Miss Joan Beveridge, Mrs Linda Golden, Mrs Mary Peer, Miss Mary Ann Gaffey and Mrs Lianne Dixon for technical assistance. This investigation was supported by Public Health Service Research Grant no. CA-o4865 from the National Cancer Institute and Postdoctoral Scholarship Grant no. PS-32 from the American Cancer Society. REFERENCES BERMA~, L. D. (1967). Comparative morphologic study of the virus-induced solid tumors of Syrian hamsters. J. natn. Cancer Inst. 39, 847. BLACK. P. H., LEwd, A. M., JUN., BLAeKLOW, N.R., AUS~N, J.B. & ROWE, W.P. (1967). The presence of adenovirus-specific antigens in hamster cells rendered neoplastic by adenovirus I-SV 40 and adenovirus 2-SV 40 hybrid viruses. Proc. natn. Acad. Sci. U.S.A. 57, FREEMAN, A. E., BLACK, P. H., WALFORD, R. & HUEBNER, R. I. (1967b). The adenovirus type 12-rat embryo transformation system. J. Virol. x, 362. FREEMAN, A.E., VANDERPOOL, E.A., BLACK, P.H., TLmNEa, H.C. & H~BNER, R.J. (1967c). Transformation of primary rat embryo cells by weakly oncogenic adenovirus type 3. Nature, Lond. 2x6, 171. FREEMAN, A. E., BLACK, P. H., VANDERPOOL, E. A., HENRY, P. H., AUSTIN, J. B. & HUEBNER, R. J. (I967a). Transformation of primary rat embryo cells by adenovirus type 2. Proc. hath. Acad. Sci. U.S.A. 58, 12o5. FUJINAGA, K. & GREEN, M. (I966). The mechanism of viral carcinogenesis by DNA mammalian viruses: viral-specific RNA in polyribosomes of adenovirus tumor and transformed cells. Proc. natn. Acad. Sci. U.S.A. 55, GILOEN, R. V., BEDDOW, T. G. & HtmaNER, R. J. (1967). Production of high-titer antibody in serum and ascitic fluid of hamsters for a variety of virus-induced tumour antigens. AppL Microbiol. I5, 657. GILL~Pm, D. & SPImELMAN, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. molec. Biol. 12, 829. HUEBNER, R..L, RowE, W. P., TURNER, H. C. & LANE, W. T. (1963). Specific adenovirus complementfixing antigens in virus-free hamster and rat turnouts. Proc. natn. Acad. Sci. U.S.A. 5o, 379- LEwis, A. M., JUN., WIESE, W. H. & ROWE, W. P. (1967). The presence of antibodies in human serum to early (T) adenovirus antigens. Proc. ham. Acad. Sci. U.S.A. 57, 622. LEWIS, A. M., JUN., BAUM, S. G., PRIGGE, K. O. & ROWE, W. P. (1966). Occurrence of adenovirus- SV 4o hybrids among monkey kidney cell adapted strains of adenovirus. Proc. Soc. exp. Biol. Med. 122, 214. MCALLISTER, R. i. & MACPHERSON, I. (I968). Transformation of a hamster cell line by adenovirus type 12. J. gen. ViroL 2, 99- MCALLISTER, R. i., LANDING, B. H. & GOODHEART, C. R. (1964). Isolation of adenoviruses from neoplastic and non-neoplastic tissues of children. Lab. Invest. x3, 894. MCALLLSTER, R. M., REED, G. & HUEBNER, R. J. (1967). Colonial growth in agar of cell derived from adenovirus-induced hamster tumours. J. hath. Cancer Inst. 39, 43. MACI'HERSON, I. & MONTAGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. mole& BioL 3, 2o8. 3-z

9 3 6 R.M. MCALLISTER AND OTHERS POPE, J. H. & ROWE, W. P. (1964). Immunofluorescent studies of adenovirus i2 tumours and of cells transformed or infected by adenoviruses. J. exp. Med. x2o, 577. SCI-IERRER, K., LATHAM, H. & DARN'ELL, J. E. (1963). Demonstration of an unstable RNA and of a precursor to ribosomal RNA in HeLa cells. Proc. hath. Acad. Sci. U.S.A. 49, 24o. WATSON, D. H., RUSSELL, W. C. WILOY, P. (1963). Electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique. Virology xg, 25o. (Received 9 April I968)