NEOPLASMA 19, 5, 1972 405

Characterization of Chicken Sarcoma Viruses Induced with Chemical Carcinogens and Mutagens in Transformed Hamster Cells

V. A L T A N E R O Y Á , Č. A L T A N E R

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Czechoslovakia

Received May 4, 1972

Some properties of the viruses induced by chemical carcinogens and mutagens from hamster cell line transformed with Schmidt-Ruppin strain of avian sarcoma virus were investigated. Avian sarcoma viruses induced with 3-m.ethylcholanthrene, 4-nitroquinoline-l-oxide and 5-azacytidine were oncogenic for young hamsters. The most potent virus in the ability to induce tumours in hamsters was the virus induced by 5-azacytidine. This virus was different from control virus in its high level of RNA-dependent DXA-polymerase activity and has also high relative plating efficiency for duck cells. It was concluded that induction of infectious virus from virogenic hamster cell line by chemical carcinogens is leading to mutation of virus especially in the case of virus induction with mutagen 5-azacytidine.

Chemical carcinogens and mutagens are able to induce RNA tumour viruses from several mouse cell lines [2, 13, 17, 21] or from nonproducer cell lines transfor­med with murine sarcoma virus [1, 3]. Viral particles of C-type were also induced in embryo chicken cells by W E I S S et al. [29]. Recently was shown by A L T A N E R O ­VÁ [6] t h a t some chemical carcinogens and mutagens are able to induce virus pro­duction from hamster virogenic cell line transformed with Schmidt-Ruppin strain of avian sarcoma virus. For a successful induction of virus production also 5-aza­cytidine was used, agent which is known as a potent mutagen of viruses [11, 19, 28]. Therefore it was of interest to follow the properties of induced viruses as far as their tumorigenicity, content of DNA-polymerases and their plating in different avian cells.

In this paper we describe a mutation of Schmidt-Ruppin virus after induction from virogenic hamster cell by 5-azacytidine and properties of viruses induced with 3-methyl-cholanthrene, 4-nitroquinoline-l-oxide and 5-azacytidine.

M a t e r i a l a n d M e t h o d s

Tissue Culture and Media

Hamster cell line Ha (SR) transformed by Schmidt-Ruppin strain of Rous sar­coma virus and general procedures were the same as used previously [6]. Chicken embryo cells were prepared from RTF-free eggs supplied by Dr. I. H L O Ž A N E K , Inst i tute of Experimental Biology and Genetics, Prague.

Polymerase Assays

The standard polymerase assay of T E M I N and M I Z T J T A N I [27] was used. Re­action were started by adding 0.1 ml of s tandard reaction mixture containing 2.5 /лС\

406 ALTANEROVA, ALTANER

of 3 H-thymidme-5'-triphosphate ( 3 H-TTP; 5Ci/mM; Amersham, England) with 10 nmoles of the other three uiilabelled deoxynucleoside triphosphates to a 0.05 ml of virus sample disrupted by t reatment with Tris-EDTA buffer containing 0.25 per cent Nonidet P-40 (Schell Chemical Co, New York, N. Y.) and 1 per cent dithio-threitol (DTT, Sigma Chemical Co. St Louis. Mo). Reaction mixture was incubated at 40 CC and at the times indicated samples of 0.025 ml were spotted on paper discs (Whatman No. 541) and dried. Filters were fixed in 10 per cent trichloracetic acid (TCA), twice in 5 per cent TCA, washed in ethanol and dried. Paper filters were placed in toluene-based scintillation fluid and counted in a Packard Tri-Car b liquid scintillation counter. Exogenous DNA-polymerase was assayed using high-mole­cular weight calf thymus DNA as a template.

Virus Concentration

Viruses were concentrated from tissue culture fluid harvested from transformed chicken cells. 100-fold concentration was achieved by ultracentr if ligation using the same procedure described previously [5].

Virus Tiunorigenicity on Hamsters

One week old Syrian hamsters were injected into the back with different dose of viruses. Animals were checked for tumours by palpation every 3 days starting one week after injection. E D 5 0 was calculated according to R E E D and M T J E N C H [20].

Induced Viruses

Viruses induced with chemical agents arc designed according to cells from where they were obtained. SR-azaCR-virus induced with 5-azacytidine, SR-MCA-virus induced with 3-methycholanthrene (1 ^g/ml), SR-BeP-virus induced with a medium containing 1 /ťg/ml benzo(a)pyrene, SR-NQO-virus induced with treatment with 4-nitroquinoline-l-oxide, SR-virus obtained for Ha(SR) cells by fusion with chicken embryo cells.

R e s u l t s

Induction of Viruses

Treatment of hamster Schmidt-Ruppin transformed cells (Ha(SR)) with diffe­rent chemical carcinogens led to a production of small amount of virus. Infectious virus was detected from treated cell lines in subsequent cell passages where no more chemical agent was present [6]. A chicken tumour which was obtained by injection of filtered concentrated medium from treated Ha(SR) cells was as a source of indu­ced virus. The extract from this tumour was used for infection of chicken embryo secondary cells. Three days after infection cells were transferee! and six days after transfer the virus from converted cells was harvested. A pool of medium from con­verted cells from different induced viruses was concentrated by ultra centrifugation. Such virus preparation were used for all studies done in this work. Control virus was obtained by fusion of irradiated Ha(SR) cells with the same chicken embryo cells used for preparations of stock viruses.

VIRUS INDUCTION WITH CHEMICAL CARCINOGENS 4 0 7

20

10 -

RNA-DNA POLYMERASE DNA-DNA POLYMERASE

20П

- 150

fOO

- 50

• О SR-MCA

MINUTES OF REACTION

-V SR-a.za.CR A A SR-NqO • - • SR-BeP - • SR

Fig. 1. DNA polymerase activities in disrupted virions of induced viruses. Concentrated virus preparations were assayed for DNA polymerase action with no additions (endoge­nous DNA polymerase) or with the addition of 5 /ig of calf thymus DNA per reaction (exogenous DNA polymerase). Trichloracetic acid-insoluble radioactivity was determined

at the indicated times.

Presence of DNA-Polymerase Activities

RNA-tumour viruses contain endogenous RNA-dependent DNA polymerase activity and exogenous DNA-dependent DNA polymerase activity [27, 7]. These enzymes probably play an important role in the process of cell infection (see T E M I N and B A L T I M O R E [26] for a review). Viruses induced by chemical carcinogens and mutagens were studied for the presence of both DNA-polymerase activities. Kinetics of DNA-polymerase assays with these viruses is presented in Fig. 1. I t was proved t h a t all virus preparations contain both DNA polymerase activities. Kinetics of polymerase reaction was the same in all virus preparations studied.

Tumorigenicity of Induced Viruses

To determine whether chemical carcinogen and mutagen induced viruses of Schmidt-Ruppin strain avian sarcoma virus are tumorigenic in hamsters the follo­wing experiments were done. One week old Syrian hamsters were injected with different doses of viral preparation and the number of focus-forming unit (FFU)

408 ALTANEROVÄ, ALTANhR

10

CD

Š

SR-a.za.CR SR-MCA SR-BtP

VIRUSES

SR- NQO SR

TUMORIGENICITY RNA-DNA POLYMERASE 'ĹA

DNA - DNA POLYMERASE

Fig. 2. Tumorigenicity and content of DNA-polymerase of induced viruses. Viruses were assayed on chicken embryo cells to determine number of FKU/ml. Tumorigenicity of viruses was estimated by injection to one week old hamsters. The same virus prepa­

ration were assayed for DXA-polymcrase activities.

needed for induction of tumours in 50 per cent of animals was determined (Fig. 2). All tumours induced in hamsters were progressively growing sarcomas. Several tu­mours we have tested for presence of infectious virus. All hamster tumours were virogenic, but no virus production we were able to detect.

Fig. '2. represents an a t t e m p t to correlate tumorigenicity of these viruses with level of DNA-polymerase activities determined in viral preparations used for in­duction of tumours. Virus induced by 5-azacytidine from Ha(SR) cells shown a high ability to induce tumour in hamsters in comparison with control virus and virus induced with 3-methylcholanthrene, benzo(a)pyrene and 4-nitroquinoltne-l-oxide. A dose of 3 F F U (on chicken cells) was sufficient to induce tumours in 50 per cent of inoculated hamsters. All other induced viruses and control one were highly on­cogenic too. but E D 5 0 dose was around 1000 F F U . When content of endogenous RNA-dependent DXA polymerase activity to one F F U on chicken cell is compared it was shown that the level of this enzyme activity was higher in all viruses induced by chemical carcinogens in comparison with control virus. The most pronounced difference was observed in the case of virus induced b v mutagen 5-azaevtidine.

VIRUS INDUCTION WITH CHEMICAL CARCINOGENS 409

о г

4 10 -k.

u. Lu

О

г

10

CELLS

Fig 3. Relative plating efficiency of induced viruses on duck cells. All viruses were assayed on the same chicken secondary embryo cells and on duck embryo cells and relative

plating efficiency was calculated.

The level of exogenous DNA-polymerase was 1000-fold higher as was detected in control virus.

Differences on exogenous DNA-polymerase in investigated viruses were not so expressive. Virus induced by 5-azacytidine again had high level of DNA-DNA-polymerase activity. Therefore the induction of virus from transformed hamster cells by 5-azacytidine had caused some change of virus which is accompanied with increase of RNA-dependent DNA-polymerase activity in virions and an increase of virus tumorigenicity for hamsters.

Plating Efficiency of Induced Viruses

To see whether the induced viruses were mutated as far as their ability to transform other avian cells concerns, we have assayed these viruses on duck cells. I t was found (Fig. 3.) t h a t they differ in relative plating efficiency on duck cells from control virus. Virus induced with 5-azacytidine was 100-fold more efficient in transformation of duck cells as virus rescued from untreated Ha(SR) cells.

D i s c u s s i o n

Treatment of hamster virogenic cell line containing whole genome of avian sarcoma virus with chemical carcinogens and mutagens is able to induce production

410 ALTANEROVA, ALTANER

of infectious virus [6]. For induction experiments two highly mutagenic compounds were used. 4-nitroquinoline-l-oxide is known as mutagen for bacteria [18] and an inductor of phage from lysogenic bacteria [9] and 5-azacytidine has been shown to be highly mutagenic for arboviruses [19] and also for avian sarcoma virus Б77 [28]. Mutation of B77 virus with 5-azacytidine yielded temperature sensitive mutants of this avian sarcoma virus. I t was therefore of interest to see whether induced viru­ses are m u t a n t s in some of their properties.

One from the unusual property of RNA tumour viruses is presence of RNA-dependeut DNA polymerase activity and DNA-DNA-polymerase activity. These enzymes play probably an important role in the process of cell infection by RNA tumour viruses [26]. All viruses induced with chemical carcinogens and mutagens contain both DNA-polymerase activities. The level of RNA-dependent DNA poly­merase in the virus induced with 5-azacytidine is much higher in comparison with control virus and viruses induced with chemical carcinogens. I t was shown t h a t this virus is also highly oncogenic for hamsters comparing number of focus forming units necessary for induction of tumours in 50 per cent of animals. These data support the conclusion t h a t with 5-azacytidino induced virus from hamster SR-transformed cells is a m u t a n t of avian sarcoma virus which is highly oncogenic for young hamsters.

I t was shown by A L T Á N E K and T E M I N [5] tha t viral genome plays an impor­t an t role in infection of heterologous mammalian cell and tha t duck plating is also connected with the character of viral genome, while viral envelope determines virus plating on homologous chicken cells. From this point of view it was interesting to find tha t with 5-azacytidine induced virus has very different plating efficiency for duck cells in comparison with control virus. All these data have shown tha t pro­perties of induced viruses from transformed cells with chemical carcinogens and mutagens are different from virus rescued from hamster cells by fusion with chicken cells. Virus induced with 5-azacytidine is a mutant highly oncogenic for hamsters with high content of DNA-polymerase activities and high relative plating efficiency for duck cells.

I t was interesting tha t morphology of transformed chicken embryo cells with induced viruses with carcinogens were different. Chicken cells infected with viruses induced by 4-nitroquinoline-l-oxide, 5-azacytidine and benzo(a)pyrene give rise to epitheloid-like transformed cells, while viruses induced by 3-methylcholanthrene and control virus give typical foci of round chicken transformed cells. I t has been proved by Т Е М IN [24] t h a t morphology of chicken transformed cells is partially controlled by the genetic character of the infecting virus. Our observations of diffe­rent cell morphology after infection with induced viruses support our other evidences t h a t induced viruses are m u t a n t s .

There is nothing known about the level on which the viral genome was mutated during the induction with 5-azacytidine. 5-azacytidine appears to be incorporated into RNA [14] and through the ribonucleotide reductase system [15] also to DNA [16]. FuČÍK et al. [10] reported that 5-azacytidine caused chromosomal mutat ion in the root meristen of Vicia faba.

Mammalian cells transformed with avian sarcoma viruses contain whole viral genome, but usually do not produce detectable infectious virus, except a case of rat tumour induced with B77 virus [4]. Viral genome is probably integrated into host genome as a DNA provirus (see T E M I N [25] for a review). I t was shown by C O F F I N and T E M I N [8] t h a t in such cells virus-specific ribonucleic acid is synthe-

VIRUS INDUCTION WITH CHEMICAL CARCINOGENS 411

s ized, b u t t h e c o n t e n t of t h i s R N A is m u c h less t h a n in v i r u s infected, ch i cken cells. R e c e n t l y i t w a s r e p o r t e d b y H I L L a n d H I L L O V Á [12] a n d b y S V O B O D A e t a l . [22] t h a t f rom m a m m a l i a n v i rogen ic cells d e o x y r i b o n u c l e i c ac id c a n bo e x t r a c t e d w h i c h c o n t a i n s i n f o r m a t i o n for a v i a n s a r c o m a v i r u s , w h i c h s t r o n g l y s u p p o r t p r o v i r u s h y p o t h e s i s of TEMIN [23].

M u t a t i o n s of i n d u c e d v i r u s f rom h a m s t e r cells b y 5 - a z a c y t i d i n e cou ld be o n t h e level of v i r u s specific R N A or o n t h e level of D N A p r o v i r u s . T h e fac t t h a t a m u ­t a n t was i so l a t ed f rom h a m s t e r cells i n l a t e r cell p a s s a g e s , w h e r e no m o r e 5-azacy­t i d i n e w a s p r e s e n t in t i s sue c u l t u r e m e d i u m cou ld prefer t h e h y p o t h e s i s t h a t m u t a ­t i o n occu r r ed on t h e level of i n t e g r a t e d D N A p r o v i r u s .

W e wish to t h a n k Dr . P . K O S S B Y for histological examinat ion of t u m o u r s and Mrs. V. P R I S T A Š O V Á for technical ass is tance.

Ле/егенсеа

[1] A A R O N S O N , S. A. : Chemical i n d u c t i o n of focus-forming virus from n o n p r o d u e e r cells t r a n s f o r m e d by m u r i n e sarcoma virus . P r o c . N a t . Acad. Sei. U. S., ÔS, 1972, 30G9.

[2] A A B O N S O N , S. A., T O D A R O , G. J . , SCOLNICK, E . M.: Induc t ion of m u r i n e C-type viruses from clonal lines of virus-free B A L B / 3 T 3 cells. Science, 174, 1971, 157.

[3] A L T Á N E K , C , H L A V A Y O V Á , E . : Transformat ion of ra t liver cells wi th chicken sar­coma virus 1377 and mur ine sarcoma v i rus . ( In press.)

[4] A L T Á N E K , С , Š V E C , F . : Virus p roduc t ion in ra t t umors induced by chicken sarcoma v i rus . J . N a t . Cancer Ins t . , 37, 1966, 745.

[5] A L T Á N E K , С , T E M I N , H . M.: Carcinogenesis b y R N A sarcoma viruses. N I L A quan­t i t a t i v e s t u d y of infection of r a t cells in v i t ro b y av ian sarcoma viruses. Virology, 40, 1970, 118.

[6] A L T A N E B O V Á , V. : I nduc t i on of virus p roduc t ion b y var ious chemical carcinogens a n d m u t a g e n s in virogenic h a m s t e r cell line t ransformed b y R o u s sarcoma v i rus . J . N a t . Cancer Ins t . ( In press.)

[7] B A L T I M O R E , D . : R N A - d e p e n d e n t D N A polymerase in virions of R N A t u m o r viruses . N a t u r e (London), 226, 1970, 1209.

[8] C O F F I N , J . M., T E M I N , H . M. : Ribonuclease-sensi t ive deoxyribonucleic acid poly­merase ac t iv i ty in uninfected r a t cells a n d r a t cells infected wi th R o u s sarcoma v i rus . J . Virol. , 8, 1971, 630.

[9] E N D O , H . , I S H I Z A W A , M., K A M I Y A , Т . : I n d u c t i o n of b a c t e r i o p h a g e format ion in lysogenic bac ter ia by a p o t e n t carcinogen 4-nitroquinoline-l-oxide a n d its derivat i­ves. N a t u r e , 198, 1963, 195.

[10] F U Č Í K , V., ŠORMOVÁ, Z., Š O K M , F . : T h e effect of 5-azacytidine on t h e root m e r i s t e m of Vicia f aba . Biol. P lant . Acad. Sei. bohemoslov. , 7, 1965. 58.

[11] H A L L E , S.: 5-azacytidine as a m u t a g e n for arboviruses . J . Virol., ,'.;, 1968, 1226. [12] H I L L , M., H I L L O V Á , L : P r o d u c t i o n virale d a n s les fibroblastcs de poule t r a i t é e

p a r ľ a c i d e déoxyr ibonuclé ique de cellules N C de r a t t rans formers p a r le v i rus do R o u s . C. R. Acád. Sei., ; % 197], 3094.

[13] IciEL, H . J . , H U E H N E R , R. .L, T U R N E R , H . C , K O T I N , P . , F A L K , H . L. : Mouse leuke­m i a v irus ac t iva t ion b y chemical carcinogens. Science, 166, 1969, 1624.

[14] J U R O V Č Í K , M., R A Š K A , K., Š O R M , F . , ŠORMOVÁ, Z . : Anabolic t r a n s f o r m a t i o n s of t h e new a n t i m e t a b o l i t e 5-azacytidine a n d its i n c o r p o r a t i o n into r ibonucleic acid. Collection Czech. Chem. C o m m o n . , 30, 1965, 3370.

[15] L A R S O N , A., R E I C H A R D , P . : E n z y m a t i c r e d u c t i o n of r ibonucleot ides . I n : Progress in nucleic acid research a n d molecular biologv. E d . J . N . D a v i d s o n , W . E . Cohn. N e w York, Academic Press , I n c . 1967, 303—347.

[16] Li , L. H , O L I N , E. J . , B U S K I R K H . H . , R E I N C K E , L. M.: Cytotoxic i ty a n d m o d e

of act ion of 5-azacytidine on L1210 leukemia . Cancer Res . , 30, 1970, 2760. [17] L O W Y . D . R., R OWE, W . P. , T E I C H , N. , H A R T L E Y , J . W . : Murine l e u k e m i a v i r u s :

High-frecjuency ac t iva t ion in v i t ro b y 5-iodo-dcoxyuridine a n d 5-bromodeoxyuri-dine. Science, 174, 1971, 155.

412 ALTANEROVA, ALTANER

[18] O K A B A Y A S H I , Т . : Mutagenic ac t iv i ty of 4-nitroquinoline-N-oxide. Chem. p h a r m . Bull . , 10, 1962, 1127.

[19] P R I N G L E , C. R . : Genetic character i s t ics of condit ional l e t h a l m u t a n t s of vesicular s t o m a t i t i s v i rus induced b y 5-fluorouracil, 5-azacytidine a n d m e t h y l sul fonate. J . Virol., 5, 1970, 559.

[20] R E E D , L. J . , MTTENCH, H . : A s imple m e t h o d of e s t i m a t i n g fifty p e r cent e n d p o i n t s . Am. J . H y g . , 27, 1938. 493.

[21] R OWE, W . P. , H A R T L E Y , J . W., L A N D E R . M. R., P U G H , W . E. , T E I C H , N . : Nonin­

fectious A K R embryo cell l ines in which each cell h a s t h e c a p a c i t y to be a c t i v a t e d t o p r o d u c e infectious m u r i n e leukemia v i rus . Virology, 46, 1971, 866.

[22] S V O B O D A , J . , H L O Ž Á N E K , I . , M A C H , О.: D e t e c t i o n of chicken sarcoma v i rus after t rans fect ion of chicken fibroblasts w i t h D N A isolated from m a m m a l i a n cells t rans­formed w i t h R o u s v i rus . Fol ia biol., IS. 1972. 150.

[23] T E M I N , H . M.: N a t u r e of t h e p r o v i rus of R o u s sarcoma. N a t . Cancer I n s t . Monogr. . 17, 1964, 557.

[24] T E M I N , H . M.: T h e control of cellular m o r p h o l o g y in embryonic cells infected wi th R o u s s a r c o m a v i rus in v i t ro . Virology, 10, 1960. 182.

[25] T E M I N , H . M.: Mechani sm of cell t rans format ion b y R N A t u m o r viruses. Ann. R e v . Microbiol. . 25, 1971, 609.

[26] T E M I N . H . M., B A L T I M O R E , D . : R N A - d i r e c t e d D N A synthes i s a n d R N A t u m o r viruses . Adv. Virus Res . , 17, 1972, N N N .

[27] T E M I N , H . M.. M I Z U T A N I , S.: R N A - d e p e n d e n t D N A polymerase in vir ions of R o u s s a r c o m a v i rus . N a t u r e , 226, 1970. 1211.

[28] T O Y O S H I M A . K.. V O G T . P . K . : T e m p e r a t u r e sensitive m u t a n t s of an av ian sarcoma v i rus . Virology. 39, 1969, 930.

[29] W E I S S , R . A., F R I T S , R. R., K A T Z . E. . V O G T , P . K . : I n d u c t i o n of av ian t u m o r v iruses in n o r m a l cells b y phys ical a n d chemical carcinogens. Virology, 49, 1972, 920.