Archives of Virology 49, 127--139 (t975) © by Springer-Verlag 1975
Simian Virus 40=Chinese Hamster Kidney Cell Interaction I. Relationship of Chromosome Chanfes to Transformation By C/t. LAVIALLE, J. STEVENET, A. G. MORI~IS, H. G. SUAlCEZ, S. ESTI~ADE, J.-C. SALOMON, and ~. CASSINGENA Institu~ de I¢echerches Scienfifiques sur le Cancer, Villejuif, France With 3 Figures Accepted June 23, 1975
Summary After approximately 20 in vitro passages, Chinese hamster kidney (CHK) cell cultures transformed upon exposure to different strains of SV40 can show a diploid modal chromosome number of 22 with chromosome counts exclusively or essentially in the diploid range (20--25). In primary culture and at the 5 t h - - 7 t h subculture, tumors produced in nude mice b y such cells can display a diploid modal value of 22 chromosomes. Numerical chromosome variations and karyotype abnormalities observed in C H K cells transformed upon infection with SV40, are apparently indistinguishable from those that can be observed in uninfected C H K cells undergoing "spontaneous" -transformation. These results indicate t h a t polyploidization is not a necessary step either in the process of transformation or in that of tumorigenic conversion with SV40.
Introduction I t has been reported that Simian virus 40 (SVdO) infection of diploid Chinese hamster embryo cells causes an alteration of the normal pattern of cellular DNA synthesis, as a result of which viable tetraploid-polyploid cells are formed (LEHMAN and DE~EXDI, 1970; LEHMAN, 1974; I{ORAX et al., 1974). Morphologically transformed ceil samples isolated within 2 - - 3 weeks after infection contain a high percentage of tetraploid-pol)Tloid cells (LEHMAN and DEFENDI, 1970; HIRAI et al., 1974). Stably SV40-transformed Chinese or Syrian hamster cells or primary tumors induced by SV40 in Syrian hamsters are heteroploid or pol)Tloid ( C o o £ ~ and :BLACK, t964; NAO~tTIt~ALet at., t968; LEI~i,a~and DEF~W])I, 1970; LEI~?aA~ and BLOt'STmN, 1974; HIRAI et al., 1974). 9*
128
C m LAVIALLE et al. :
T h e s e r e s u l t s h a v e b e e n c o n s i d e r e d as b e i n g g o o d b u t n o t a s y e t c o n c l u s i v e e v i d e n c e f o r a d i r e c t l i n e a g e f r o m t e t r a p l o i d t o t r a n s f o r m e d cells (LEtIMAN a n d D E F E ~ m , 1 9 7 0 ; LEHMA~ a n d BLOVSTWIN, 1 9 7 4 ; HIRAI et al., 1974). In an attempt to verify this hypothesis, we have undertaken to extend these o b s e r v a t i o n s b y t h e a n a l y s i s of t h e c h r o m o s o m e s of C h i n e s e h a m s t e r k i d n e y cells "spontaneously"-transformed or t r a n s f o r m e d b y d i f f e r e n t s t r a i n s of S V 4 0 , as w e l l as of t u m o r s p r o d u c e d b y i n j e c t i n g t h e s e cells i n t o n u d e m i c e .
Materials and Methods Animals Chinese h a m s t e r s a n d t h y m u s - l e s s n u d e mice n u / n u ( r a n d o m - b r e d ) were s u p p l i e d f r o m stocks b r e d in our I n s t i t u t e . Viruses T h r e e s t r a i n s of p l a q u e p u r i f i e d S V 4 0 were u s e d : a) S V L P , a large p l a q u e v a r i a n t (SLT~Ez et al., 1974); b) S V S P , a s m a l l p l a q u e v a r i a n t (SUAREZet al., 1974); c) t s A 30, a t e m p e r a t u r e - s e n s i t i v e m u t a n t (TEGTMEYEI¢, 1972). All v i r u s stocks were p r e p a r e d a n d a s s a y e d i n A f r i c a n g r e e n m o n k e y k i d n e y CV-1 cells (JENSEN et al., 1964} as p r e v i o u s l y d e s c r i b e d (SuA~EZ et al., 1974). T h e S V L P a n d S V S P v a r i a n t s were g r o w n a n d a s s a y e d a t 37 ° C, t h e t s A 3 0 m u t a n t a t 33 ° C. T r y p s i n and Media D i s a g g r e g a t i o n of m i n c e d tissues a n d d e t a c h m e n t of cells f r o m c u l t u r e vessels were p e r f o r m e d u s i n g twice eristallized t r y p s i n (Choay, P a r i s ) a t t h e c o n c e n t r a t i o n of 0.15 g/1 p h o s p h a t e b u f f e r e d saline (PBS) w i t h o u t Ca ++ a n d w i t h o u t Mg ++. P r i m a r y a n d s e c o n d a r y cell c u l t u r e s were g r o w n in m o d i f i e d E a g l e m e d i u m (MEM ; E u r o b i o , Paris) c o n t a i n i n g 10 p e r c e n t t r y p t o s e p h o s p h a t e b r o t h (Difco), 20 p e r c e n t calf s e r u m , 100 u n i t s penicillin a n d 100 ~zg s t r e p t o m y c i n / m l . Serial s u b c u l t u r e s were m a d e i n M E M c o n t a i n i n g 10 p e r c e n t t r y p t o s e p h o s p h a t e b r o t h , 10 p e r c e n t calf s e r u m a n d n o a n t i b i o t i c s . T h e s a m e m e d i u m , s u p p l e m e n t e d w i t h 10 p e r c e n t fetal calf s e r u m , w a s u s e d for cell cloning. Isolation and Establishment o/ "Spontaneously"- Transformed and S V40- Trans/ormed Chinese Hamster K i d n e y Cell Lines and Clones A k i d n e y was r e m o v e d f r o m a w e a n l i n g f e m a l e Chinese h a m s t e r , w a s h e d in P B S , d e c a p s u l a t e d , a n d t h e m e d u l l a was discarded. A f t e r fine m i n c i n g of t h e tissues a n d d i s a g g r e g a t i o n w i t h t r y p s i n , t h e cells were collected b y c e n t r i f u g a t i o n , r e s u s p e n d e d in m e d i u m , c o u n t e d , s e e d e d in 4-ounce glass p r e s c r i p t i o n b o t t l e s (3 × 106 cells/bottle) a n d i n c u b a t e d a t 37 ° C. T h e m e d i u m was c h a n g e d e v e r y 3 d a y s and, a t d a y 10 a f t e r seeding, t h e c o n f l u e n t p r i m a r y m o n o l a y e r c u l t u r e s were t r y p s i n i z e d a n d pooled. S e c o n d a r y c u l t u r e s were m a d e i n 30 m m F a l c o n p l a s t i c dishes (5 × 104 cells/dish) a n d i n c u b a t e d a t 37 ° C in a i r w i t h 5 p e r c e n t CO2. T w o d a y s later, t h e m e d i u m was r e m o v e d , t h e cells were w a s h e d w i t h P B S a n d e q u a l n u m b e r s of c u l t u r e s were e i t h e r m o c k - i n f e c t e d o r i n f e c t e d w i t h S V L P , S V S P or t s A 30 v i r u s (0.2 m l / d i s h ) a t a n i n p u t m u l t i p l i c i t y of 200 p l a q u e f o r m i n g u n i t s ( P F U ) / c e l l . A f t e r 2 h o u r s a d s o r p t i o n a t 37 ° C, t h e ceils were w a s h e d 3 t i m e s w i t h P B S , c o v e r e d w i t h m e d i u m (2 m l / d i s h ) a n d r e i n c u b a t e d a t 37 ° C i n air w i t h 5 p e r c e n t COs. T h e m e d i u m w a s c h a n g e d twice a week. F o u r t e e n d a y s l a t e r , m o r p h o l o g i c a l l y t r a n s f o r m e d cell colonies were o b s e r v e d in S V L P , S V S P a n d t s A 3 0 i n f e c t e d c u l t u r e s , while n o s u c h colonies were n o t i c e d i n c o n t r o l cultures. All c u l t u r e s were p u t o n a rigid s u b c u l t u r i n g schedule, b e i n g p a s s a g e d e v e r y 4 d a y s a n d a l w a y s a t t h e s a m e cell d e n s i t y (10 ~ cells/ml), w i t h a m e d i u m c h a n g e o n t h e s e c o n d day. B y t h e s i x t h s u b c u l t u r e (the earliest tested), t h e cells e x p o s e d to S V L P , S V S P or t s A 3 0 v i r u s were 100 p e r c e n t p o s i t i v e for S V 4 0 t u m o r (T) a n t i g e n ; t h e u n i n f e c t e d c o n t r o l cells were all n e g a t i v e . F o u r e s t a b l i s h e d cell lines were o b t a i n e d : one " s p o n t a n e -
Chromosomes and SV40-Transformation
129
ously"-transformed (CHK) and three SV40-transformed (CHK/SVLP, CHI~/SVSP, CHK/tsA30). Each of them is currently over the 50th in vitro passage. F r o m these lines, clones were derived at the following passage levels: C H K (p. 16): C1. 1, 3, 4, 6, 8, 9, 10, 11. C H K / S V L P (p. 5): C1. 2, 4, 5. CI-IK/SVLP C1. 5 (p. 4): C1. 5.1. C H K / S V S P (p. 5): C1. 1, 3, 5. C H K / S V S P C1. 3 (p. 3): C1. 3.1. CHK/tsA30 (p. 5): C1. 4, 5. CHK/tsA30 C1. 4 (p. 3): C1. 4.1. Clones were isolated with glass cylinders (PUCK et al., 1956) from 60 m m Falcon plastic dishes containing 1 to 4 well separated colonies/dish.
SVgO Tumor ( T ) Antigen The presence of SV40 specific T antigen in cultures examined at different in vitro passages was demonstrated by the indirect fluorescein isothioeyanate staining procedure (WICKER and AVRAMEAS, 1969).
Growth in Semisolid Suspension 2yledium ( S S M ) The agar technique described by MAcP~ExSOZ~ and MO~TAONIE~ (1964) was used. One hundred to one hundred-thousand cells were seeded per 60 m m Falcon plastic dish (4 dishes/cell concentration). The plates were incubated at 37°C in air wi~h 5 per cent COg. They were observed weekly during 4 weeks for colony formation and colony counts were made at day 30 of incubation.
Tumorigenicity in Nude Mice For each cell line tested, monolayer cells were harvested by trypsinization and 5× 106 cells suspended in 0.2 ml MEM were injected subcutaneously into the left and right, side of each of 3 nude mice at three weeks of age. The animals were regularly examined for t u m o r development for at least 3 months. In vitro Culture o/ Tumors Tumors were removed, washed in PBS, decapsulated, minced and trypsinized. Primary monolayer cultures were made on 12 × 32 m m eoverslips in Leighton tubes (for chromosome analysis) and in 4-ounce prescription bottles (for serial subculture) using a cell density of 3 × 105 cells/ml medium, Subcultures were performed following the schedule described before.
Chromosomes Cultures in exponential growth on coverslips in Leighton tubes were treated for 4 hours aV 37°C with 1 l~g eolchicine (Houd6, Paris)/mI medium. Thereafter, the eoverMips were withdrawn, immersed for 20 minutes in a hypotonie solution of PBS diluted 1 : 4 in distilled water, fixed for 30 minutes in a 1 : 4 solution of acetic acid in methanol, air-dried, stained with aeetic-orcein (Gurr, London) and mounted on slides without squashing. Well-spread, apparently intact metaphases were photographed. F o r each sample studied, 50 metaphase photographs were used for chromosome counting and 25 of them for detailed analysis of karyotype morphology. The chromosomes were arranged in karyotypes according to Yerganian (Hsu, 1963).
Results Cell Trans/ormation "Spontaneous"-Trans/ormation o / C H K Cells. A f t e r a b o u t 10 passages in vitro, C H K cells s t a r t e d to grow in a r a n d o m l y oriented a n d m u l t i l a y e r e d m a n n e r , w i t h a higher r a t e of cell division. T h o u g h i n c a p a b l e (p. 2 8 - - 5 2 ) of division in semisolid
130
CH. LAVIALLE et al. :
suspension medium (SSM), they were able (p. 27) to produce tumors upon injection into nude mice (tumor incidence = 100 per cent). This is evidence that these cells, now over their 50th subculture and growing as permanently established cultures, are "spontaneously".transformed. The ability of C H K C1. 4, 6, 10 and 11 (p. 23--31) to divide in SSM has also been repeatedly examined. Colonies were formed only b y C H K C1. 10 cells, when tested at passage 31 but not at earlier passages. SV40-Trans/or~ation o/ CHK Cells. Transformation of CHK/SVLP, C H K / SVSP and CHK/tsA 30 cells was sho~n b y their altered shape and growth pattern (see Materials and Methods) and by their high rate of division. The persistance of the SV40 specific intranuclear T antigen in 100 per cent of the cells through serial subculturing indicated that these cells were SV40-transformed. When these different SV40-transformed cells were tested for their capacity to produce tumors in nude mice and to grow in SSM, the following results were obtained : C H K / S V L P (p. 45), CHK/SVSP (p. 26) and CHK/tsA30 (p. 26, 40) were all tumorigenic (tumor incidence -- i00 per cent), all the tumor cells b e i n g - - i n each c a s e - - S V 4 0 - T antigen positive; C H K / S V L P (p. 46) and CHK/SVSP (p. 45) formed colonies in SSM, while CHK/tsA30 (p. 40) were unable to grow in SSM; C H K / S V L P C1.5.1 (p. 26), CHK/SVSP C1. 3.1 (p. 27) and CHK/tsA30 C1. 4.1 (p. 27) cells were all capable of division in SSM.
Chromosome Analysis Numerical Chromosome Variations in CHK Cells. The histograms are presented in Figures 1 and 3. C H K cells (p. 14) exhibited a 211 (diploid) modal value of 22 chromosomes and a distribution of chromosome numbers between 20--25. Four C H K clones showed different chromosome counts. C1. 4 (p. 5) had chromosome numbers mainly (88 per cent) between 3 ~ n , few of them (12 per cent) between 2 - - 3 n and a modal value of 41 chromosomes. C1.6 (p. 5) was comparable to the parental C H K cells. C1. t0 (p. 6) had most (96 per cent) of the chromosome numbers between 20--24, some (4 per cent) at 44 and a modal value of 24 chromosomes. C1. 11 (p. 5) showed two cell populations, one (46 per cent) with chromosome numbers in the diploid range and a stem line with 22 chromosomes; the other (54 per cent) with chromosome counts in the tetraploid range and a stem line with 44 chromosomes. A tumor obtained in a nude mouse b y the injection of C H K cells (p. 27), analyzed at the 9th in vitro passage, displayed a modal value of 35 chromosomes, most (90 per cent) of the chromosome numbers between 26--38 and few of them (10 per cent) between 49--77. Numerical Chromosome Variations in S V 40-Trans/ormed CHK Cells. The histograms are presented in Figures 2 and 3. C H K / S V L P cells (p. 22) had a modal chromosome number of 22 with a range of 20--24. C H K / S V L P C1. 5.1 (p. 8), showed a stem line with 21 chromosomes, the majority (88 per cent) of the chromosome counts in the 2n region, a small proportion of them (12 per cent) in the hypotetraploid region. CHK/SVSP cells (p. 23) also exhibited a diploid modal value of 22 chromosomes and a distribution of chromosome numbers between 20--23. Essentially similar findings were obtained with CHK/SVSP C1. 3.1 cells (p. ll).
Chromosomes
and SV40-Transformation
131
i~ CHK
2C
(p.t4)
1C
~p.5)
CHK CL11 (p.5)
' 4'4
2'2 CHK CI. 4
l]
olN ~Z'2
m 30 z
l[
CHK Ct.6 (p.5)
' 4'4 CHK CL10 (p.6)
20
10 '2'2
'4'4 CROMOSOME
' 4'n4
NUMBER
Fig. 1. Distribution of the chromosome numbers in C H K ceils
40 CHK/tsA30 (p.18)
~1 CHK/SVSP (p. 23)
CHK/SVLP (p.22)
30 .a 2o £3 10
m 30
2'2
'
'2'2
'4'4
'
*4
' 4'4
44
'2'2 ' 4'4 '2'2 CHROMOSOME NUMBER
Fig. 2. Distribution of the chromosome numbers in SV40-transformed CI-IK cells
20f
CHK Tum.(p,9)
10
~q
'Y2
, , nn 44
~
n.
n
8'8
CHK / t s A30 Tum. A ( prim.cult J
20
20
CHK/SVSP Turn (prim,cult,)
10 44 CHK / t s A 3 0 (p.5)
"8'8 2C
Tum. 8
'2'2 ' '4'4 CH K/SVSP Turn. (p.7)
lC
22
n rn
' 4'4 ' CHROMOSOME
8'8
r~ '2'2
~ ~
irl ' 4'4 '
NUMBER
Fig. 3. Distribution of the chromosome numbers in tumors produced by '"Spontaneously"- or 8V404ransformed CI~K cells
C~. LAVIALLE et al. :
132
W h e n a t u m o r p r o d u c e d i n a n u d e m o u s e b y C H K / S V S P cells (p. 26) w a s c y t o g e n e t i c a l l y a n a l y z e d i n p r i m a r y c u l t u r e , t w o cell p o p u l a t i o n s w e r e o b s e r v e d : o n e (36 p e r c e n t ) w i t h a m o d a l c h r o m o s o m e n u m b e r of 22 a n d a r a n g e of 2 1 - - 2 8 ; t h e o t h e r (64 p e r c e n t ) w i t h t h r e e m o d a l c h r o m o s o m e n u m b e r s of 38, 42, 4 4 a n d a r a n g e of 3 4 ~ 6 . A t a n a d v a n c e d (Tth) in vitro p a s s a g e of C t I K / S V S P t u m o r cells o n l y t h e s t e m l i n e w i t h 2 2 c h r o m o s o m e s w a s f o u n d , m o s t (92 p e r c e n t ) of t h e cells s h o w e d c h r o m o s o m e n u m b e r s b e t w e e n 2 0 - - 2 7 , a s m a l l p r o p o r t i o n of t h e m (8 p e r c e n t ) b e t w e e n 34 44. C H K / t s A 3 0 cells (p. 19), as n o t i c e d f o r C H K / S V L P a n d C H K / S V S P cells, s h o w e d a m o d a l v M u e of 22 c h r o m o s o m e s . H o w e v e r , s l i g h t l y m o r e t h a n h a l f (60 p e r c e n t ) of t h e c h r o m o s o m e c o u n t s w e r e i n t h e d i p l o i d r a n g e , t h e r e m a i n i n g (40 p e r c e n t ) b e i n g i n t h e 3 - - 4 n r e g i o n . A m o d a l v a l u e of 35 c h r o m o s o m e s a n d a d i s t r i b u t i o n of c h r o m o s o m e n u m b e r s b e t w e e n 3 0 - 4 1 was observed in CHK/ t s A 3 0 C1. 4.1 cells (p. 6). T w o t u m o r s (A a n d B ) d e v e l o p e d i n n u d e m i c e a f t e r i n j e c t i o n of C H K / t s A 3 0 cells (p. 26 f o r A ; p. 41 f o r B ) w e r e e x a m i n e d c y t o g e n e t i c a l l y . T u m o r A w a s
T a b l e 1. Karyotype Analysis o / C H K Cells (1). 14) Mitoses n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
30 4
2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 1 1 2 1 2 2 2 2 2 2 2 2 1 2 2 1 3 2 2 2 1 2 2 2 2 2 2 t 2 2 2 2 2 2 2 2 2 2 2 t 3 1 1 3 t 2 2 2 2 2 2 2 2 1 3 2 2 1 2 3 2 0 2 2 2 2 2 3 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2
5
6
7
8
9
101t
2 2 2 2 2 2 0 2 2 1 2 i 2 2 2 2 2 2 2 2 1 1 2 2 2
1 2 2 2 2 2 t 3 2 2 2 2 3 2 1 3 3 2 2 2 2 2 2 2 2
3 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 3 2 2 2 3 2 1 2 4 3 2 2 2 2 2 2 2 3 2 2 2 3 2 2 3 2 2 2 2 2 2 3 2 2 l 2 3 2 2 2 2 2 2 2 2 3 2 2 2 3 3 1 2 2 2 2 3 2 2 2 2 2 2 t 2 1 2 2 2 2 2 2 2 3 2 2 3 3 3 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2
Abnormal ehromosomes~
Total number of c h r o m o s o m e s
1A 1A 1MSM
22b 22 b 22 b 22b 22b 21 22 b 22 b 23 21 23 22 b 22 b 23 23 23 24 23 22 b 20 21 22 b 24 25 21
1MSM 1ST
1A
2 1
1ST, 1A, l m i m 1T
2 2 t 2 2 2
1T IMSM tMSM 1MSM 1T
1
1T
a A a c r o c e n t r i c c h r o m o s o m e ; MSM s u b m e d i a n m e t a c e n t r i e c h r o m o s o m e ; ST s u b t e l o centric chromosome; mini minute chromosome; T telocentric chromosome. b P s e u d o d i p l o i d mitosis. c 3=×.
Chromosomes
and SV40-Transformation
133
a n a l y z e d in p r i m a r y culture. I t h a d a m o d a l chromosome n u m b e r of 22, a prop o r t i o n (68 per cent) of t h e chromosome counts b e t w e e n 2 L - - 2 4 a n d a p r o p o r t i o n (32 p e r cent) s p r e a d b e t w e e n 3 6 - - 8 5 w i t h almost half of the counts (14 per cent) a t 44. T u m o r B was a n a l y z e d a t the 5th in vitro passage. A m o d a l value of 22 to 24 chromosomes a n d a d i s t r i b u t i o n of chromosome n u m b e r s b e t w e e n 2 1 - - 3 4 were found. Karyotype Analysis el CHK and SV40-Trans/ormed CItK Cells. T h e results of the k a r y o t y p e analysis of C H K (p. 14), C H K / S V L P (p. 22), C I t K / S V S P (p. 23) a n d C H K / t s A 3 0 (p. 19) cells are shown in Tables 1, 2, 3 a n d 4. I n t h e four cell populations, loss a n d a d d i t i o n of chromosomes a n d a b n o r m a l chromosomes were observed, b u t no specific p a t t e r n was found. A m o n g the diploid cells e x a m i n e d , i 0 0 p e r cent C H K , 53 per cent C H K / S V L P , 84 p e r cent C H K / S V S P a n d 69 p e r cent C H K / t s A 3 0 cells were p s e u d o d i p l o i d . Chromosome b r e a k s a n d dicentric chromosomes were occasionally n o t i c e d in diploid, p s e u d o d i p l o i d a n d / o r aneuploid m e t a p h a s e s of the three SV40-transf o r m e d C t t K lines a n d of C H K cells as well. Table 2. Karyotype Analysis o/CHK/SVLP Cells (p. 22) Mitoses n°
Chromosomes
1 2 3c 4 5 6 7 8 9
10 11
1 2 3 4 5 6 7 8 9 i0 11 12 13 14 15 16 17 18 19 20 21 22
2 2 2 2 2 1 2 2 2 2 2 2 2 l 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
23 24 25
2 2 1 2 2 2 3 2 2 2 2 2 2 2 t 2 2 2 2 2 1 2 1 2 2 2 2 1 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2
1 2 1 2 2 2 2 2 2 2 2 2 2 1 2 2 2 1 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1
3 2 2 2 2 2 2 2 2 2 2 2 3 3 2 2 2 2 1 2 2 2
1 2 1 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2
2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 1 2 2
2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 1 2 2
2 1 2 2 2 2 1 2 2 1 2 2 2 2 2 1 2 3 2 2 0 2
Abnormal chromosomes a
Total nmnber of chromosomes
IMSM
22b 22b 22b 22 22 22b 21 22 22
IA
IST
21
IMS~
22 22 22b 22b 21 21 22 24 23 20 20 22b
1MSM tMSM
22 b 2t 21
IMSM, I T IMSM
a MSM sublr, edian metaeentrie chromosome; A acroeentric chromosome; eentric chromosome; T telocentrie chromosome. b Pseudodiploid mitosis.
c 3=X.
ST
subtelo-
C]~. LAWALLE et al. :
134
T a b l e 3. Karyotype Analysis o / C H K / S V S P Cells (p. 23) Mitoses n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
3c 4
5
6
7
8
9
10 11
2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 2 1 1 2 2 1 1 1 1 2 2 1 2 1 2 2 2 1 2 0 1 1 2 1
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2
2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 1 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 1 3 3 2 i 2
2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 1 1 2 2 2 1 2 2 2 2 1 2 1 2 2 2 2 1 2 2
2 2 2 2 2 2 3 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2
Abnormal chromosomes •
Total number of c h r o m o s o m e s
1MSM 1MSM 1MSM 1ST
22 b 22 b 22 b 21 22 22 b 23 22 b 22b 22 b 22 b 22 22 b 20 22 b 22 b 22 22 b 22 b 22 b 21 22 b 21 22 b 23
1MSM, 1T 1MSM, 1T 1MSM 1MSM 1MSM 1MSM 1MSM
1MSM 1MSM 1MSM 1MSM, 1A
1MSM 1M 2T
a MSM s u b m e d i a n m e t a c e n t r i e c h r o m o s o m e ; ST s u b t e l o e e n t r i c c h r o m o s o m e ; T telocentrie chromosome; A acrocentric chromosome; M metacentric chromosome. P s e u d o d i p l o i d mitosis. c 3=X.
Diseussion The indirect evidence that SV4O-transformed tumorigenic Chinese hamster e m b r y o cell lines, S V 4 0 - t r a n s f o r m c d S y r i a n h a m s t e r k i d n e y cell l i n e s a n d p r i m a r y t u m o r s i n d u c e d i n t h e S y r i a n h a m s t e r w i t h S V 4 0 a r e h e t e r o p l o i d or p o l y p l o i d h a s b e e n c o n s i d e r e d a s s t r o n g l y s u g g e s t i n g t h e r e l e v a n c e of p o l y p l o i d i z a t i o n t o transformation with SV40. Furthermore, these observations have been considered t o s u g g e s t a s i m i l a r p a t t e r n i n t h e e v o l u t i o n of m a l i g n a n t p r o p e r t i e s of cells t r a n s f o r m e d in vitro a n d in vivo w i t h S V 4 0 (LEHMAX a n d DEFENDI, 1970 ; LEHMAN a n d BLOVSTEIN, 1 9 7 4 ; H m A I et al., 1974). A s s h o w n i n t h e p r e s e n t w o r k , S V 4 0 - t r a n s f o r m e d C h i n e s e h a m s t e r k i d n e y cell l i n e s a n d c l o n e s c a n b e i s o l a t e d w h i c h a r e d i p l o i d or n e a r - d i p l o i d . S i m i l a r f i n d i n g s h a v e b e e n r e p o r t e d f o r a C h i n e s e h a m s t e r e m b r y o cell l i n e t r a n s f o r m e d w i t h h u m a n a d e n o v i r u s 12 (BtcAILOVSK¥ et al., 1967), f o r S y r i a n h a m s t e r e m b r y o cell l i n e s t r a n s f o r m e d w i t h d i f f e r e n t h u m a n a d e n o v i r u s - S V 4 0 h y b r i d v i r u s e s (BLACK a n d WHITE, 1967), w i t h p o l y o m a v i r u s (DEFENDI a n d LEgM~N, 1965 ; YAMA~OTO et al., 1973) w i t h h e r p e s s i m p l e x v i r u s a n d h u m a n c y t o m e g a l o v i r u s (NAcgTIGAL et al., 1974), or a f t e r e x p o s u r e t o c h e m i c a l c a r c i n o g e n s (DIPAOLO et al., 1971 ; 1973).
Chromosomes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
2 2 2 4 2 2 3 2 4 2 2 2 3 2 2 2 2 2 3 2 2 2 2 2 2
2 2 2 2 3 3 3 3 4 2 2 2 1 2 2 2 2 2 2 2 2 3 2 2 2
3c 4 3 2 1 4 2 2 2 3 4 2 2 2 2 1 3 2 2 1 2 2 1 2 2 1 2
2 1 2 4 3 4 2 3 2 2 2 2 2 2 2 2
2
6
7
8
9
4 1 1 4 5 4 4 5 4 2 2 1 1 2 1
4 3 2 3 5 2 4 4 4 2 2 2 2 2 3 2 1 1 3 2 2 2 2 2 2
3 2 2 2 4 6 3 4 4 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2
4 2 3 5 3 5 4 4 2 2 2 2 1 2 1 2 2 2 2 2 2 2 3 2 2
3 2 2 4 4 3 4 3 4 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2
2 2 2
1 3 2 1
2
3 1 2 2
2 2
2
5
2 2 2
2 3
2
135
Karyotype Analysis o/CHK/tsA 30 Cells (p. 19)
T a b l e 4. Mitoses n°
and SV40-Transformation
2
10 11 3 3 2 2 4 4 5 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2
2 3 2 5 2 4 2 4 3 2 2 2 2 2 2
Abnormal chromosomesa
Total n u m b e r
1ST, 1MSM
34 23 21 40 38 40 37 39 38 22 22 22 b 22 b 22 b 22 b 22 22 b 22 b 24 22 b 22 b 22 b 24 21 22
1MSM
1MSM 1MSM 1MSM 1MSM
1MSM 1MSM 1T
2 2
1MSM 1T, 1M
2 2 2
1ST
2
of c h r o m o s o m e s
S T s u b t e l o e e n t r i c c h r o m o s o m e ; MSIVi s u b m e d i a n m e t a c e n t r i c c h r o m o s o m e ; T telocentric chromosome; M metacentric chromosome. b Pseudodiploid mitosis. ° 3~X.
In the present investigation we have further shown that SV40-transformed Chinese hamster kidney cell lines characterized by stemlines with 22 chromosomes and either exhibiting (CHK/tsA30) or not exhibiting (CHK/SVSP) detectable chromosome counts above the diploid range, can be tumorigenic. This suggests that the presence of cells with chromosome complements over the 2n range is not a prerequisite in order for a SV40-transformed cell population to be tumorigenic. If, at the passage levels tested, SV40-transformed CIIK cells with chromosome complements above the diploid range had a strong selective advantage in rive or were the only tumorigenic cells, few if any diploid or near-diploid metaphases should have been detected in CHK/tsA 30 tumor A and in CHK/SVSP tumor analyzed in primary culture. This was clearly not the case. The chromosome counts above the 2n range found in these tumors in primary culture, might thus be considered--particularly in the ease of CHK/SVSP tumor--as being a consequence of changing homeostatic relationships between cell and host, of readaptation of cells to in vitro culture conditions, or both. The fact that after 5--7 subcultures, CI-IK/tsA30 tumor B and CIIK/SVSP tumor showed a diploid or neardiploid modal value and less than i0 per cent of cells with chromosome numbers
t36
CH. LAV~A~LEet al. :
over the diploid range indicates that, in vitro, most of the latter cells either had no selective advantage or were not viable. I t has been suggested t h a t near-diploid cells that are present in a SV40-transformed cell population m a y arise from multipolar mitoses (L]~HSIANand BLOUSTEIH, 1974). Though this possibility can not be totally excluded, it does not seem to be able to account for the considerable number of near-diploid and diploid cells found in the majority of the SV40-transformed cell populations that we have studied. The numerical chromosome variations that we have observed in SV40transformed C H K cells were essentially the same as those noticed in uninfected C H K cells. K a r y o t y p e analysis of different SV40-transformed C t t K lines has revealed, in each line; karyotype abnormalities t h a t were also found in the uninfected C H K cell population. I t follows that there is no evidence to suggest t h a t these changes were specific effects of SV40. In earlier eytogenetic studies made with SV40-transformed human cells, the preferential involvement of a chromosome group(s) has been reported (S~IEIH and EHDERS, 1962; YERGANIAN et al., 1962; MOORHEAD and SAKSELA, 1963). The oncogenieity of SV40 has only been demonstrated in mastomys and in Syrian hamster (BUTELet al., 1972) and only few karyologie studies have been so far described on cells transformed in vivo with this virus. The available results show that primary tumors induced b y SV40 in Syrian hamsters, examined either in primary culture or after several in vitro passages, all had a hypertetraploid and/or a hypotetraploid chromosome number distribution (CooPER and BLACK, 1964; NAC~TIGAL et al., 1968; LEHMAN and BLOUSTEIN,1974; I-IIRAI et al., 1974). Until more information is available on primary SV40 tumors developed in Syrian hamsters as well as in other animal species no reasonable conclusion about their ploidy can be drawn, especially since it has been reported that primary and transplanted mouse polyoma tumors (HELLSTR6M et al., 1963) can be diploid, as can also be Syrian hamster adenovirus tumors (BARSKI and CORNEFERT, t964; CASSINGENA and TOURHn~R, 1965; UTZUMI et al., 1965), mouse m a m m a r y carcinomas associated with mouse m a m m a r y tumor virus (TJIo and 0STnRGnES, 1958), Friend leukemias of mice (LEvAH and BII~S:SLs, 1958), Rous sarcomas and erythroleukemias of chickens (PoHT£H, 1963). Cytogenetical analysis of direct preparations of Burkitt tumors associated with Epstein-Barr virus, also showed t h a t the majority of chromosome numbers per cell occurred around the diploid mode in all the tumors analyzed in detail (JAcoss et al., 1963). I t is well known that diploid or near-diploid cell cultures generally shift toward a doubling or tetraploid levels as cultivation continues and t h a t a heteroploid cell population finally evolves. This will quite probably occur also for our diploid or near-diploid SV40-transformed C[-IK cell populations, thus indicating t h a t in the SV40-Chinese hamster system, as observed in the SV40-Syrian hamster system (NACHTIGALet al., 1971) and in cell lines transformed with human eytomegalovirns (NAoHTIOAL et al., 1974), hetcroploidy m a y also be secondary to t r a n s f o r m a t i o n - - i n our case, secondary even to tumorigenic transformation. That heteroploidy m a y also follow the neoplastic conversion has already been shown in the polyoma virus-mouse system (H~LLST~SM et al., 1963). Though the role that the tetraploid-polyploid cells induced following infection with SV 40 might play in transformation with this virus has not yet been defined, the
Chromosomes
and SV40-Transformation
137
results p r e s e n t e d in this w o r k M1 t o g e t h e r s t r o n g l y suggest t h a t p o l y p l o i d i z a t i o n is n o t a necessary s t e p either in t h e process of t r a n s f o r m a t i o n or in t h a t of t u m o r i genic conversion w i t h SV40. T h a t p o l y p l o i d i z a t i o n is not. a prerequisite for transf o r m a t i o n w i t h SV40 is also suggested b y previous results of WEI~STEI~~ a n d MOORHEAD (1967). F~EED~ANN a n d StuN, e x a m i n i n g a large n u m b e r of p e r m a n e n t ceil lines a n d p r i m a r y diploid cell cultures from various species, have r e c e n t l y shown t h a t one i n vitro p r o p e r t y consistently correlated w i t h t u m o r i g e n i c i t y in n u d e mice is t h e c a p a c i t y of t h e cell to form colonies in a semisolid suspension m e d i u m (SSM), in this case methyleellulose m e d i u m (FREEDMAN a n d SHrN, 1974). I n our experience this has n o t been t h e case since, while all p a r e n t a l lines w h e t h e r t r a n s f o r m e d "spont a n e o u s l y " or w i t h SV40 were tumorigenic, n o t all were capable of g r o w t h in SSM. This a p p a r e n t d i s c r e p a n c y b e t w e e n our results a n d those of FREEDM2~N a n d SHI~, m a y p e r h a p s be e x p l a i n e d b y our use of a g a r m e d i u m . The p a r a d o x i e a I o b s e r v a t i o n t h a t some clones d e r i v e d from p a r e n t a l lines unable to grow in agar SSM d i d grow in a g a r SSM is difficult to u n d e r s t a n d , b u t it m a y be due to t h e stress of cloning. Acknowledgments
We are grateful to Marc Girard for providing us with the SV40 strain tsA30 of Peter Tegtmeyer. We are particularly indebted to Prof. l~oger Weft for critieal review of the manuscript. A. G. M. is supported by a lgoyM Society (London) European Exchange Programme fellowship.
References
BARSKI, G., CORNEFEI~T, F. : Charaet6ristiques earyologiques des tumeurs pulmonaires de hamsters produites par l'ad6novirus 12, Aim. Inst. Pasteur 107, 114--d20 (1964). BT.ACK, P. I-I., WHI~E, B. J. : I n vitro transformation by the adenovirus-SV40 hybrid viruses. II. Characteristics of the transformation of hamster cells by the adeno 2-, adeno 3-, and adeno 12-SV4:0 viruses. J. exp. Med. 125, 629--646 (1967). BI~AILOVStg:Y, C., WICKER, R., SUAREZ, H. G., CASSINGEN2k, R . : Transformation i n vitro de eellules de hamster ehinois par l'ad6novirus 12: E t u d e cytog6n6tique. Int. J. Cancer 2, 133--142 (1967). BUTEL, J. S., TEVETIIIA, S. S., MELNICK, J. L. : Oneogenicity and cell transformation by papovavirus SV40: the role of the viral genome. Adv. Cancer Res, 15, 1 55 (1972). CASmNCENA, l~., TOURNIER, P.: Interaction ad6novirus 12-eellules de Mesocricetus auratus. I. Etude ehromosomique. Ann. Inst. Pasteur I08, 277--297 (1965). CooPER, H. L., BLACK, P. H.: Cytogenetic studies of three clones derived from a permanent line of hamster cells transformed by SV40. J. cell. cornp. Physiol. 64, 201--220 (1964). I)EFENDI, V., LEHMAN, J. M.: Transformation of hamster embryo cells in vitro by polyoma virus: morphological, karyologieal, immunological and t.ransplantation characteristics. J. cell. eomp. Physiol. 66, 351--409 (1965).
DIPAOLO, J. A., NELSON, R. L., DOI'~-OVAN,P. J. : Morphological, oncogenie, and karyological characteristics of Syrian hamster embryo cells transformed in. vitro b y carcinogenic polyeyclie hydrocarbons. Cancer ges. 31, 1118-- 1127 ( t 97 t ). DIPAOLO, J. A., POPESCU, N. C., NELSON, t~. L. : Chromosomal banding patterns and i n vitro transformation of Syrian hamster cells. Cancer l~es. 33, 3250--3258 (1973). FREEDMAN, V. H., SHIN, S. : Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell 3, 355--359 (1974).
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HEImS~'I~61Vl, K. E., HELLSTR6M, I., SJ~)GIaEN, H. O. : Further studies on karyotypes of a variety of primary and transplanted mouse polyoma tumors. J. Nat. Cancer Inst. 31, 1239 1253 (1963). HI:aAI, K., CAMPBELL, G., DEFENDI, V. : Changes of regulation of host DNA synthesis and viral DNA integration in SV40 infected cells. In: Cold Spring Harbor Symposium on Control of Proliferation of Animal Cells, 151--166 (1974). HOlCAN, P. K., JETT, J. H., 1%OMERO, A., LEHMAN, J. M.: Flow microfluorometry analysis of DNA content in Chinese hamster cells following infection with Simian virus 40. Int. J. Cancer 14, 514--521 (1974). HsI7, T. C. : Mammalian chromosomes in vitro, XVI-Analysis of chromosomes breakages in cell population of Chinese hamster. Can. Cancer Conf. 5, 117--127 (1963). JACOBS, 1%. A., TOD'GH, I. M., ~¥EIGH% D. A. : Cytogenetic studies in Burkitt's lymphoma. Lancet ii, 1144--1146 (1963). JENSEN, F. C., GIEA~aDI, A. J., GII~DEN, 1%. V., KOPEOWSKI, H.: Infection of human and Simian tissue cultures with 1%ous sarcoma virus. Prec. Nat. Acad. Sei. (Wash.) 52, 53--59 (1964). LEHMAN, J. M. : Early chromosome changes in diploid Chinese hamster cells after infection with Simian virus 40. Int. hr . Cancer 13, 164--172 (1974). LEHMAN, J. M., DEFEI~DI, ~7. : Changes in deoxyribonucleic acid synthesis regulation in Chinese hamster cells infected with Simian virus 40. J. Virol. 6, 738--749 (t970). LEHMAN, J. M., BLOUSTEIN,P. : Chromosome analysis and agglutination by concanavalin A of primary Simian virus 40-induced tumors. Int. J. Cancer 14, 771--778 (1974). LEVAN, A., BIESELE, J. J. : The role of chromosomes in carcinogenesis, as studied in serial tissue culture of mammalian cells. Ann. N.Y. Acad. Sei. 6, 1022--1053 (1958). MACPI-IEIgSON, I., MONTAG~IER, L. : Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291--294 (1964). MOOI~HEAD, P. S., SAKSELA, E.: Non-random chromosomal aberrations in SV40transformed human cells. J. cell. comp. Physiol. 62, 57--84 (1963). NACHTIGAL, M., LUIgGEANN, A., NACHTIOAI,, S., ADERCA, A. : Cytogenetie study of two tumor lines induced in the golden hamster by SV40 virus. Rev. l~oum, d'Inframierobioi. 5, I19--127 (1968). NACII~rIGAL, M., MELNICK, J. L., BU~:EL, J. S. : Chromosomal changes in Syrian hamster cells transformed by Simian virus 40 (SV40) and variants of defective SV40 (PAl%A). J. Nat. Cancer Inst. 47, 35--45 (1971). NACtlTIGAL, M., ALBRECtIT, T., 1%APP, F. : Analysis of chromosomes of Syrian hamster cells transformed with h u m a n cytomegalovirus. Intervirology 4, 77--90 (1974). PONT]gN, J. : Chromosome analysis of three virus-associated chicken tumors: Rous sarcoma, erythroleukemia, and R P L 12 lymphoid tumor. J. Nat. Cancer Inst. 30, 8 9 7 - 9 2 1 (1963). PucK, T., MA~cTzs, P., CIEcIumt, S. : Clonal growth of mammalian cells i n vitro. J . exp. Med. 103, 273--284 (1956). SHEIN, H. M., ENDEICS, J. F. : Transformation induced by Simian virus 40 in human renal cell cultures. I. Morphology and growth characteristics. Prec. Nat. Acad. Sei. (Wash.) 48, 1164--1172 (1962). SUAREZ, H. G., C&SSINGENA, R., ESTRADE, S., %VICKER, g., LAX~LLE, Cm, LAZAr, P. : Properties of SV40 rescued from actinomycin D-sensitive and actinomyein Dresistant transformed hamster cells. I. Lyric infection. Arch. ges. Virusforseh. 46, 93--104 (1974). TEn,MEYEr, P. : Simian virus 40 deoxyribonucleic acid synthesis : The virus replieon. J. Virol. 16, 591--598 (1972). T~Io, J. H., ()STE~GREN, G.: The chromosomes of primary m a m m a r y carcinomas in milk virus strains of the mouse. Hereditas 44, 451--465 (1958). UTZUMI, K. tR., KITaMVRA, I., T~E~TI~-, J. J. : Karyologieal studies of normal cells and of adenovirus-type-12-indueed tumor ceils of the Syrian hamster. J. Nat. Cancer Inst. 35, 759--769 (1965).
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%VEIIgSTEIN, I)., MOOR, IIEAD, P. S. : The relation of karyotipic change to loss of contact inhibition of division in human diploid cells after SV 40 infection. J. cell. Physiol. 69, 367--376 (1967). WICKEH, ll.., AVHA?~EAS, S.: Localization of virus antigens by enzyme-labeled antibodies. J. gem Virol. 4, 465--471 (i969). YA.~OTO, T., B.AB£NOWITZ, Z., SACHS, L. : Identification of the chromosomes that control malignancy. Nature (New Biol.) 243, 247--250 (1973). YEI~GA~IAN, G., SHEIJq, H. M., EN/)EICS, J. F.: Chromosomal disturbances observed in h u m a n fetal renal cells transformed in vitro by Simian virus 40 and carried in culture. Cytogenetics 1, 314--324 (1962).
Authors' address: Dr. I~. CASSINGENA,I n s t i t u t de Recherches Scientifiques sur le Cancer, B.P. Nr. 8, F-94800 Villejuif, France. Received May 30, 1975
Simian Virus 40=Chinese Hamster Kidney Cell Interaction I. Relationship of Chromosome Chanfes to Transformation By C/t. LAVIALLE, J. STEVENET, A. G. MORI~IS, H. G. SUAlCEZ, S. ESTI~ADE, J.-C. SALOMON, and ~. CASSINGENA Institu~ de I¢echerches Scienfifiques sur le Cancer, Villejuif, France With 3 Figures Accepted June 23, 1975
Summary After approximately 20 in vitro passages, Chinese hamster kidney (CHK) cell cultures transformed upon exposure to different strains of SV40 can show a diploid modal chromosome number of 22 with chromosome counts exclusively or essentially in the diploid range (20--25). In primary culture and at the 5 t h - - 7 t h subculture, tumors produced in nude mice b y such cells can display a diploid modal value of 22 chromosomes. Numerical chromosome variations and karyotype abnormalities observed in C H K cells transformed upon infection with SV40, are apparently indistinguishable from those that can be observed in uninfected C H K cells undergoing "spontaneous" -transformation. These results indicate t h a t polyploidization is not a necessary step either in the process of transformation or in that of tumorigenic conversion with SV40.
Introduction I t has been reported that Simian virus 40 (SVdO) infection of diploid Chinese hamster embryo cells causes an alteration of the normal pattern of cellular DNA synthesis, as a result of which viable tetraploid-polyploid cells are formed (LEHMAN and DE~EXDI, 1970; LEHMAN, 1974; I{ORAX et al., 1974). Morphologically transformed ceil samples isolated within 2 - - 3 weeks after infection contain a high percentage of tetraploid-pol)Tloid cells (LEHMAN and DEFENDI, 1970; HIRAI et al., 1974). Stably SV40-transformed Chinese or Syrian hamster cells or primary tumors induced by SV40 in Syrian hamsters are heteroploid or pol)Tloid ( C o o £ ~ and :BLACK, t964; NAO~tTIt~ALet at., t968; LEI~i,a~and DEF~W])I, 1970; LEI~?aA~ and BLOt'STmN, 1974; HIRAI et al., 1974). 9*
128
C m LAVIALLE et al. :
T h e s e r e s u l t s h a v e b e e n c o n s i d e r e d as b e i n g g o o d b u t n o t a s y e t c o n c l u s i v e e v i d e n c e f o r a d i r e c t l i n e a g e f r o m t e t r a p l o i d t o t r a n s f o r m e d cells (LEtIMAN a n d D E F E ~ m , 1 9 7 0 ; LEHMA~ a n d BLOVSTWIN, 1 9 7 4 ; HIRAI et al., 1974). In an attempt to verify this hypothesis, we have undertaken to extend these o b s e r v a t i o n s b y t h e a n a l y s i s of t h e c h r o m o s o m e s of C h i n e s e h a m s t e r k i d n e y cells "spontaneously"-transformed or t r a n s f o r m e d b y d i f f e r e n t s t r a i n s of S V 4 0 , as w e l l as of t u m o r s p r o d u c e d b y i n j e c t i n g t h e s e cells i n t o n u d e m i c e .
Materials and Methods Animals Chinese h a m s t e r s a n d t h y m u s - l e s s n u d e mice n u / n u ( r a n d o m - b r e d ) were s u p p l i e d f r o m stocks b r e d in our I n s t i t u t e . Viruses T h r e e s t r a i n s of p l a q u e p u r i f i e d S V 4 0 were u s e d : a) S V L P , a large p l a q u e v a r i a n t (SLT~Ez et al., 1974); b) S V S P , a s m a l l p l a q u e v a r i a n t (SUAREZet al., 1974); c) t s A 30, a t e m p e r a t u r e - s e n s i t i v e m u t a n t (TEGTMEYEI¢, 1972). All v i r u s stocks were p r e p a r e d a n d a s s a y e d i n A f r i c a n g r e e n m o n k e y k i d n e y CV-1 cells (JENSEN et al., 1964} as p r e v i o u s l y d e s c r i b e d (SuA~EZ et al., 1974). T h e S V L P a n d S V S P v a r i a n t s were g r o w n a n d a s s a y e d a t 37 ° C, t h e t s A 3 0 m u t a n t a t 33 ° C. T r y p s i n and Media D i s a g g r e g a t i o n of m i n c e d tissues a n d d e t a c h m e n t of cells f r o m c u l t u r e vessels were p e r f o r m e d u s i n g twice eristallized t r y p s i n (Choay, P a r i s ) a t t h e c o n c e n t r a t i o n of 0.15 g/1 p h o s p h a t e b u f f e r e d saline (PBS) w i t h o u t Ca ++ a n d w i t h o u t Mg ++. P r i m a r y a n d s e c o n d a r y cell c u l t u r e s were g r o w n in m o d i f i e d E a g l e m e d i u m (MEM ; E u r o b i o , Paris) c o n t a i n i n g 10 p e r c e n t t r y p t o s e p h o s p h a t e b r o t h (Difco), 20 p e r c e n t calf s e r u m , 100 u n i t s penicillin a n d 100 ~zg s t r e p t o m y c i n / m l . Serial s u b c u l t u r e s were m a d e i n M E M c o n t a i n i n g 10 p e r c e n t t r y p t o s e p h o s p h a t e b r o t h , 10 p e r c e n t calf s e r u m a n d n o a n t i b i o t i c s . T h e s a m e m e d i u m , s u p p l e m e n t e d w i t h 10 p e r c e n t fetal calf s e r u m , w a s u s e d for cell cloning. Isolation and Establishment o/ "Spontaneously"- Transformed and S V40- Trans/ormed Chinese Hamster K i d n e y Cell Lines and Clones A k i d n e y was r e m o v e d f r o m a w e a n l i n g f e m a l e Chinese h a m s t e r , w a s h e d in P B S , d e c a p s u l a t e d , a n d t h e m e d u l l a was discarded. A f t e r fine m i n c i n g of t h e tissues a n d d i s a g g r e g a t i o n w i t h t r y p s i n , t h e cells were collected b y c e n t r i f u g a t i o n , r e s u s p e n d e d in m e d i u m , c o u n t e d , s e e d e d in 4-ounce glass p r e s c r i p t i o n b o t t l e s (3 × 106 cells/bottle) a n d i n c u b a t e d a t 37 ° C. T h e m e d i u m was c h a n g e d e v e r y 3 d a y s and, a t d a y 10 a f t e r seeding, t h e c o n f l u e n t p r i m a r y m o n o l a y e r c u l t u r e s were t r y p s i n i z e d a n d pooled. S e c o n d a r y c u l t u r e s were m a d e i n 30 m m F a l c o n p l a s t i c dishes (5 × 104 cells/dish) a n d i n c u b a t e d a t 37 ° C in a i r w i t h 5 p e r c e n t CO2. T w o d a y s later, t h e m e d i u m was r e m o v e d , t h e cells were w a s h e d w i t h P B S a n d e q u a l n u m b e r s of c u l t u r e s were e i t h e r m o c k - i n f e c t e d o r i n f e c t e d w i t h S V L P , S V S P or t s A 30 v i r u s (0.2 m l / d i s h ) a t a n i n p u t m u l t i p l i c i t y of 200 p l a q u e f o r m i n g u n i t s ( P F U ) / c e l l . A f t e r 2 h o u r s a d s o r p t i o n a t 37 ° C, t h e ceils were w a s h e d 3 t i m e s w i t h P B S , c o v e r e d w i t h m e d i u m (2 m l / d i s h ) a n d r e i n c u b a t e d a t 37 ° C i n air w i t h 5 p e r c e n t COs. T h e m e d i u m w a s c h a n g e d twice a week. F o u r t e e n d a y s l a t e r , m o r p h o l o g i c a l l y t r a n s f o r m e d cell colonies were o b s e r v e d in S V L P , S V S P a n d t s A 3 0 i n f e c t e d c u l t u r e s , while n o s u c h colonies were n o t i c e d i n c o n t r o l cultures. All c u l t u r e s were p u t o n a rigid s u b c u l t u r i n g schedule, b e i n g p a s s a g e d e v e r y 4 d a y s a n d a l w a y s a t t h e s a m e cell d e n s i t y (10 ~ cells/ml), w i t h a m e d i u m c h a n g e o n t h e s e c o n d day. B y t h e s i x t h s u b c u l t u r e (the earliest tested), t h e cells e x p o s e d to S V L P , S V S P or t s A 3 0 v i r u s were 100 p e r c e n t p o s i t i v e for S V 4 0 t u m o r (T) a n t i g e n ; t h e u n i n f e c t e d c o n t r o l cells were all n e g a t i v e . F o u r e s t a b l i s h e d cell lines were o b t a i n e d : one " s p o n t a n e -
Chromosomes and SV40-Transformation
129
ously"-transformed (CHK) and three SV40-transformed (CHK/SVLP, CHI~/SVSP, CHK/tsA30). Each of them is currently over the 50th in vitro passage. F r o m these lines, clones were derived at the following passage levels: C H K (p. 16): C1. 1, 3, 4, 6, 8, 9, 10, 11. C H K / S V L P (p. 5): C1. 2, 4, 5. CI-IK/SVLP C1. 5 (p. 4): C1. 5.1. C H K / S V S P (p. 5): C1. 1, 3, 5. C H K / S V S P C1. 3 (p. 3): C1. 3.1. CHK/tsA30 (p. 5): C1. 4, 5. CHK/tsA30 C1. 4 (p. 3): C1. 4.1. Clones were isolated with glass cylinders (PUCK et al., 1956) from 60 m m Falcon plastic dishes containing 1 to 4 well separated colonies/dish.
SVgO Tumor ( T ) Antigen The presence of SV40 specific T antigen in cultures examined at different in vitro passages was demonstrated by the indirect fluorescein isothioeyanate staining procedure (WICKER and AVRAMEAS, 1969).
Growth in Semisolid Suspension 2yledium ( S S M ) The agar technique described by MAcP~ExSOZ~ and MO~TAONIE~ (1964) was used. One hundred to one hundred-thousand cells were seeded per 60 m m Falcon plastic dish (4 dishes/cell concentration). The plates were incubated at 37°C in air wi~h 5 per cent COg. They were observed weekly during 4 weeks for colony formation and colony counts were made at day 30 of incubation.
Tumorigenicity in Nude Mice For each cell line tested, monolayer cells were harvested by trypsinization and 5× 106 cells suspended in 0.2 ml MEM were injected subcutaneously into the left and right, side of each of 3 nude mice at three weeks of age. The animals were regularly examined for t u m o r development for at least 3 months. In vitro Culture o/ Tumors Tumors were removed, washed in PBS, decapsulated, minced and trypsinized. Primary monolayer cultures were made on 12 × 32 m m eoverslips in Leighton tubes (for chromosome analysis) and in 4-ounce prescription bottles (for serial subculture) using a cell density of 3 × 105 cells/ml medium, Subcultures were performed following the schedule described before.
Chromosomes Cultures in exponential growth on coverslips in Leighton tubes were treated for 4 hours aV 37°C with 1 l~g eolchicine (Houd6, Paris)/mI medium. Thereafter, the eoverMips were withdrawn, immersed for 20 minutes in a hypotonie solution of PBS diluted 1 : 4 in distilled water, fixed for 30 minutes in a 1 : 4 solution of acetic acid in methanol, air-dried, stained with aeetic-orcein (Gurr, London) and mounted on slides without squashing. Well-spread, apparently intact metaphases were photographed. F o r each sample studied, 50 metaphase photographs were used for chromosome counting and 25 of them for detailed analysis of karyotype morphology. The chromosomes were arranged in karyotypes according to Yerganian (Hsu, 1963).
Results Cell Trans/ormation "Spontaneous"-Trans/ormation o / C H K Cells. A f t e r a b o u t 10 passages in vitro, C H K cells s t a r t e d to grow in a r a n d o m l y oriented a n d m u l t i l a y e r e d m a n n e r , w i t h a higher r a t e of cell division. T h o u g h i n c a p a b l e (p. 2 8 - - 5 2 ) of division in semisolid
130
CH. LAVIALLE et al. :
suspension medium (SSM), they were able (p. 27) to produce tumors upon injection into nude mice (tumor incidence = 100 per cent). This is evidence that these cells, now over their 50th subculture and growing as permanently established cultures, are "spontaneously".transformed. The ability of C H K C1. 4, 6, 10 and 11 (p. 23--31) to divide in SSM has also been repeatedly examined. Colonies were formed only b y C H K C1. 10 cells, when tested at passage 31 but not at earlier passages. SV40-Trans/or~ation o/ CHK Cells. Transformation of CHK/SVLP, C H K / SVSP and CHK/tsA 30 cells was sho~n b y their altered shape and growth pattern (see Materials and Methods) and by their high rate of division. The persistance of the SV40 specific intranuclear T antigen in 100 per cent of the cells through serial subculturing indicated that these cells were SV40-transformed. When these different SV40-transformed cells were tested for their capacity to produce tumors in nude mice and to grow in SSM, the following results were obtained : C H K / S V L P (p. 45), CHK/SVSP (p. 26) and CHK/tsA30 (p. 26, 40) were all tumorigenic (tumor incidence -- i00 per cent), all the tumor cells b e i n g - - i n each c a s e - - S V 4 0 - T antigen positive; C H K / S V L P (p. 46) and CHK/SVSP (p. 45) formed colonies in SSM, while CHK/tsA30 (p. 40) were unable to grow in SSM; C H K / S V L P C1.5.1 (p. 26), CHK/SVSP C1. 3.1 (p. 27) and CHK/tsA30 C1. 4.1 (p. 27) cells were all capable of division in SSM.
Chromosome Analysis Numerical Chromosome Variations in CHK Cells. The histograms are presented in Figures 1 and 3. C H K cells (p. 14) exhibited a 211 (diploid) modal value of 22 chromosomes and a distribution of chromosome numbers between 20--25. Four C H K clones showed different chromosome counts. C1. 4 (p. 5) had chromosome numbers mainly (88 per cent) between 3 ~ n , few of them (12 per cent) between 2 - - 3 n and a modal value of 41 chromosomes. C1.6 (p. 5) was comparable to the parental C H K cells. C1. t0 (p. 6) had most (96 per cent) of the chromosome numbers between 20--24, some (4 per cent) at 44 and a modal value of 24 chromosomes. C1. 11 (p. 5) showed two cell populations, one (46 per cent) with chromosome numbers in the diploid range and a stem line with 22 chromosomes; the other (54 per cent) with chromosome counts in the tetraploid range and a stem line with 44 chromosomes. A tumor obtained in a nude mouse b y the injection of C H K cells (p. 27), analyzed at the 9th in vitro passage, displayed a modal value of 35 chromosomes, most (90 per cent) of the chromosome numbers between 26--38 and few of them (10 per cent) between 49--77. Numerical Chromosome Variations in S V 40-Trans/ormed CHK Cells. The histograms are presented in Figures 2 and 3. C H K / S V L P cells (p. 22) had a modal chromosome number of 22 with a range of 20--24. C H K / S V L P C1. 5.1 (p. 8), showed a stem line with 21 chromosomes, the majority (88 per cent) of the chromosome counts in the 2n region, a small proportion of them (12 per cent) in the hypotetraploid region. CHK/SVSP cells (p. 23) also exhibited a diploid modal value of 22 chromosomes and a distribution of chromosome numbers between 20--23. Essentially similar findings were obtained with CHK/SVSP C1. 3.1 cells (p. ll).
Chromosomes
and SV40-Transformation
131
i~ CHK
2C
(p.t4)
1C
~p.5)
CHK CL11 (p.5)
' 4'4
2'2 CHK CI. 4
l]
olN ~Z'2
m 30 z
l[
CHK Ct.6 (p.5)
' 4'4 CHK CL10 (p.6)
20
10 '2'2
'4'4 CROMOSOME
' 4'n4
NUMBER
Fig. 1. Distribution of the chromosome numbers in C H K ceils
40 CHK/tsA30 (p.18)
~1 CHK/SVSP (p. 23)
CHK/SVLP (p.22)
30 .a 2o £3 10
m 30
2'2
'
'2'2
'4'4
'
*4
' 4'4
44
'2'2 ' 4'4 '2'2 CHROMOSOME NUMBER
Fig. 2. Distribution of the chromosome numbers in SV40-transformed CI-IK cells
20f
CHK Tum.(p,9)
10
~q
'Y2
, , nn 44
~
n.
n
8'8
CHK / t s A30 Tum. A ( prim.cult J
20
20
CHK/SVSP Turn (prim,cult,)
10 44 CHK / t s A 3 0 (p.5)
"8'8 2C
Tum. 8
'2'2 ' '4'4 CH K/SVSP Turn. (p.7)
lC
22
n rn
' 4'4 ' CHROMOSOME
8'8
r~ '2'2
~ ~
irl ' 4'4 '
NUMBER
Fig. 3. Distribution of the chromosome numbers in tumors produced by '"Spontaneously"- or 8V404ransformed CI~K cells
C~. LAVIALLE et al. :
132
W h e n a t u m o r p r o d u c e d i n a n u d e m o u s e b y C H K / S V S P cells (p. 26) w a s c y t o g e n e t i c a l l y a n a l y z e d i n p r i m a r y c u l t u r e , t w o cell p o p u l a t i o n s w e r e o b s e r v e d : o n e (36 p e r c e n t ) w i t h a m o d a l c h r o m o s o m e n u m b e r of 22 a n d a r a n g e of 2 1 - - 2 8 ; t h e o t h e r (64 p e r c e n t ) w i t h t h r e e m o d a l c h r o m o s o m e n u m b e r s of 38, 42, 4 4 a n d a r a n g e of 3 4 ~ 6 . A t a n a d v a n c e d (Tth) in vitro p a s s a g e of C t I K / S V S P t u m o r cells o n l y t h e s t e m l i n e w i t h 2 2 c h r o m o s o m e s w a s f o u n d , m o s t (92 p e r c e n t ) of t h e cells s h o w e d c h r o m o s o m e n u m b e r s b e t w e e n 2 0 - - 2 7 , a s m a l l p r o p o r t i o n of t h e m (8 p e r c e n t ) b e t w e e n 34 44. C H K / t s A 3 0 cells (p. 19), as n o t i c e d f o r C H K / S V L P a n d C H K / S V S P cells, s h o w e d a m o d a l v M u e of 22 c h r o m o s o m e s . H o w e v e r , s l i g h t l y m o r e t h a n h a l f (60 p e r c e n t ) of t h e c h r o m o s o m e c o u n t s w e r e i n t h e d i p l o i d r a n g e , t h e r e m a i n i n g (40 p e r c e n t ) b e i n g i n t h e 3 - - 4 n r e g i o n . A m o d a l v a l u e of 35 c h r o m o s o m e s a n d a d i s t r i b u t i o n of c h r o m o s o m e n u m b e r s b e t w e e n 3 0 - 4 1 was observed in CHK/ t s A 3 0 C1. 4.1 cells (p. 6). T w o t u m o r s (A a n d B ) d e v e l o p e d i n n u d e m i c e a f t e r i n j e c t i o n of C H K / t s A 3 0 cells (p. 26 f o r A ; p. 41 f o r B ) w e r e e x a m i n e d c y t o g e n e t i c a l l y . T u m o r A w a s
T a b l e 1. Karyotype Analysis o / C H K Cells (1). 14) Mitoses n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
30 4
2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 2 2 2 1 1 1 2 1 2 2 2 2 2 2 2 2 1 2 2 1 3 2 2 2 1 2 2 2 2 2 2 t 2 2 2 2 2 2 2 2 2 2 2 t 3 1 1 3 t 2 2 2 2 2 2 2 2 1 3 2 2 1 2 3 2 0 2 2 2 2 2 3 2 1 2 2 2 2 2 2 2 2 2 2 2 2 1 2
5
6
7
8
9
101t
2 2 2 2 2 2 0 2 2 1 2 i 2 2 2 2 2 2 2 2 1 1 2 2 2
1 2 2 2 2 2 t 3 2 2 2 2 3 2 1 3 3 2 2 2 2 2 2 2 2
3 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 3 2 2 2 3 2 1 2 4 3 2 2 2 2 2 2 2 3 2 2 2 3 2 2 3 2 2 2 2 2 2 3 2 2 l 2 3 2 2 2 2 2 2 2 2 3 2 2 2 3 3 1 2 2 2 2 3 2 2 2 2 2 2 t 2 1 2 2 2 2 2 2 2 3 2 2 3 3 3 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2
Abnormal ehromosomes~
Total number of c h r o m o s o m e s
1A 1A 1MSM
22b 22 b 22 b 22b 22b 21 22 b 22 b 23 21 23 22 b 22 b 23 23 23 24 23 22 b 20 21 22 b 24 25 21
1MSM 1ST
1A
2 1
1ST, 1A, l m i m 1T
2 2 t 2 2 2
1T IMSM tMSM 1MSM 1T
1
1T
a A a c r o c e n t r i c c h r o m o s o m e ; MSM s u b m e d i a n m e t a c e n t r i e c h r o m o s o m e ; ST s u b t e l o centric chromosome; mini minute chromosome; T telocentric chromosome. b P s e u d o d i p l o i d mitosis. c 3=×.
Chromosomes
and SV40-Transformation
133
a n a l y z e d in p r i m a r y culture. I t h a d a m o d a l chromosome n u m b e r of 22, a prop o r t i o n (68 per cent) of t h e chromosome counts b e t w e e n 2 L - - 2 4 a n d a p r o p o r t i o n (32 p e r cent) s p r e a d b e t w e e n 3 6 - - 8 5 w i t h almost half of the counts (14 per cent) a t 44. T u m o r B was a n a l y z e d a t the 5th in vitro passage. A m o d a l value of 22 to 24 chromosomes a n d a d i s t r i b u t i o n of chromosome n u m b e r s b e t w e e n 2 1 - - 3 4 were found. Karyotype Analysis el CHK and SV40-Trans/ormed CItK Cells. T h e results of the k a r y o t y p e analysis of C H K (p. 14), C H K / S V L P (p. 22), C I t K / S V S P (p. 23) a n d C H K / t s A 3 0 (p. 19) cells are shown in Tables 1, 2, 3 a n d 4. I n t h e four cell populations, loss a n d a d d i t i o n of chromosomes a n d a b n o r m a l chromosomes were observed, b u t no specific p a t t e r n was found. A m o n g the diploid cells e x a m i n e d , i 0 0 p e r cent C H K , 53 per cent C H K / S V L P , 84 p e r cent C H K / S V S P a n d 69 p e r cent C H K / t s A 3 0 cells were p s e u d o d i p l o i d . Chromosome b r e a k s a n d dicentric chromosomes were occasionally n o t i c e d in diploid, p s e u d o d i p l o i d a n d / o r aneuploid m e t a p h a s e s of the three SV40-transf o r m e d C t t K lines a n d of C H K cells as well. Table 2. Karyotype Analysis o/CHK/SVLP Cells (p. 22) Mitoses n°
Chromosomes
1 2 3c 4 5 6 7 8 9
10 11
1 2 3 4 5 6 7 8 9 i0 11 12 13 14 15 16 17 18 19 20 21 22
2 2 2 2 2 1 2 2 2 2 2 2 2 l 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
23 24 25
2 2 1 2 2 2 3 2 2 2 2 2 2 2 t 2 2 2 2 2 1 2 1 2 2 2 2 1 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 2 2 2
1 2 1 2 2 2 2 2 2 2 2 2 2 1 2 2 2 1 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 1
3 2 2 2 2 2 2 2 2 2 2 2 3 3 2 2 2 2 1 2 2 2
1 2 1 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2
2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 1 2 2
2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 1 2 2
2 1 2 2 2 2 1 2 2 1 2 2 2 2 2 1 2 3 2 2 0 2
Abnormal chromosomes a
Total nmnber of chromosomes
IMSM
22b 22b 22b 22 22 22b 21 22 22
IA
IST
21
IMS~
22 22 22b 22b 21 21 22 24 23 20 20 22b
1MSM tMSM
22 b 2t 21
IMSM, I T IMSM
a MSM sublr, edian metaeentrie chromosome; A acroeentric chromosome; eentric chromosome; T telocentrie chromosome. b Pseudodiploid mitosis.
c 3=X.
ST
subtelo-
C]~. LAWALLE et al. :
134
T a b l e 3. Karyotype Analysis o / C H K / S V S P Cells (p. 23) Mitoses n° 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
3c 4
5
6
7
8
9
10 11
2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
1 2 1 1 2 2 1 1 1 1 2 2 1 2 1 2 2 2 1 2 0 1 1 2 1
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2
2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 1 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 1 3 3 2 i 2
2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
2 2 2 2 2 1 1 2 2 2 1 2 2 2 2 1 2 1 2 2 2 2 1 2 2
2 2 2 2 2 2 3 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2
Abnormal chromosomes •
Total number of c h r o m o s o m e s
1MSM 1MSM 1MSM 1ST
22 b 22 b 22 b 21 22 22 b 23 22 b 22b 22 b 22 b 22 22 b 20 22 b 22 b 22 22 b 22 b 22 b 21 22 b 21 22 b 23
1MSM, 1T 1MSM, 1T 1MSM 1MSM 1MSM 1MSM 1MSM
1MSM 1MSM 1MSM 1MSM, 1A
1MSM 1M 2T
a MSM s u b m e d i a n m e t a c e n t r i e c h r o m o s o m e ; ST s u b t e l o e e n t r i c c h r o m o s o m e ; T telocentrie chromosome; A acrocentric chromosome; M metacentric chromosome. P s e u d o d i p l o i d mitosis. c 3=X.
Diseussion The indirect evidence that SV4O-transformed tumorigenic Chinese hamster e m b r y o cell lines, S V 4 0 - t r a n s f o r m c d S y r i a n h a m s t e r k i d n e y cell l i n e s a n d p r i m a r y t u m o r s i n d u c e d i n t h e S y r i a n h a m s t e r w i t h S V 4 0 a r e h e t e r o p l o i d or p o l y p l o i d h a s b e e n c o n s i d e r e d a s s t r o n g l y s u g g e s t i n g t h e r e l e v a n c e of p o l y p l o i d i z a t i o n t o transformation with SV40. Furthermore, these observations have been considered t o s u g g e s t a s i m i l a r p a t t e r n i n t h e e v o l u t i o n of m a l i g n a n t p r o p e r t i e s of cells t r a n s f o r m e d in vitro a n d in vivo w i t h S V 4 0 (LEHMAX a n d DEFENDI, 1970 ; LEHMAN a n d BLOVSTEIN, 1 9 7 4 ; H m A I et al., 1974). A s s h o w n i n t h e p r e s e n t w o r k , S V 4 0 - t r a n s f o r m e d C h i n e s e h a m s t e r k i d n e y cell l i n e s a n d c l o n e s c a n b e i s o l a t e d w h i c h a r e d i p l o i d or n e a r - d i p l o i d . S i m i l a r f i n d i n g s h a v e b e e n r e p o r t e d f o r a C h i n e s e h a m s t e r e m b r y o cell l i n e t r a n s f o r m e d w i t h h u m a n a d e n o v i r u s 12 (BtcAILOVSK¥ et al., 1967), f o r S y r i a n h a m s t e r e m b r y o cell l i n e s t r a n s f o r m e d w i t h d i f f e r e n t h u m a n a d e n o v i r u s - S V 4 0 h y b r i d v i r u s e s (BLACK a n d WHITE, 1967), w i t h p o l y o m a v i r u s (DEFENDI a n d LEgM~N, 1965 ; YAMA~OTO et al., 1973) w i t h h e r p e s s i m p l e x v i r u s a n d h u m a n c y t o m e g a l o v i r u s (NAcgTIGAL et al., 1974), or a f t e r e x p o s u r e t o c h e m i c a l c a r c i n o g e n s (DIPAOLO et al., 1971 ; 1973).
Chromosomes
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Chromosomes 1
2
2 2 2 4 2 2 3 2 4 2 2 2 3 2 2 2 2 2 3 2 2 2 2 2 2
2 2 2 2 3 3 3 3 4 2 2 2 1 2 2 2 2 2 2 2 2 3 2 2 2
3c 4 3 2 1 4 2 2 2 3 4 2 2 2 2 1 3 2 2 1 2 2 1 2 2 1 2
2 1 2 4 3 4 2 3 2 2 2 2 2 2 2 2
2
6
7
8
9
4 1 1 4 5 4 4 5 4 2 2 1 1 2 1
4 3 2 3 5 2 4 4 4 2 2 2 2 2 3 2 1 1 3 2 2 2 2 2 2
3 2 2 2 4 6 3 4 4 2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2
4 2 3 5 3 5 4 4 2 2 2 2 1 2 1 2 2 2 2 2 2 2 3 2 2
3 2 2 4 4 3 4 3 4 2 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2
2 2 2
1 3 2 1
2
3 1 2 2
2 2
2
5
2 2 2
2 3
2
135
Karyotype Analysis o/CHK/tsA 30 Cells (p. 19)
T a b l e 4. Mitoses n°
and SV40-Transformation
2
10 11 3 3 2 2 4 4 5 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2
2 3 2 5 2 4 2 4 3 2 2 2 2 2 2
Abnormal chromosomesa
Total n u m b e r
1ST, 1MSM
34 23 21 40 38 40 37 39 38 22 22 22 b 22 b 22 b 22 b 22 22 b 22 b 24 22 b 22 b 22 b 24 21 22
1MSM
1MSM 1MSM 1MSM 1MSM
1MSM 1MSM 1T
2 2
1MSM 1T, 1M
2 2 2
1ST
2
of c h r o m o s o m e s
S T s u b t e l o e e n t r i c c h r o m o s o m e ; MSIVi s u b m e d i a n m e t a c e n t r i c c h r o m o s o m e ; T telocentric chromosome; M metacentric chromosome. b Pseudodiploid mitosis. ° 3~X.
In the present investigation we have further shown that SV40-transformed Chinese hamster kidney cell lines characterized by stemlines with 22 chromosomes and either exhibiting (CHK/tsA30) or not exhibiting (CHK/SVSP) detectable chromosome counts above the diploid range, can be tumorigenic. This suggests that the presence of cells with chromosome complements over the 2n range is not a prerequisite in order for a SV40-transformed cell population to be tumorigenic. If, at the passage levels tested, SV40-transformed CIIK cells with chromosome complements above the diploid range had a strong selective advantage in rive or were the only tumorigenic cells, few if any diploid or near-diploid metaphases should have been detected in CHK/tsA 30 tumor A and in CHK/SVSP tumor analyzed in primary culture. This was clearly not the case. The chromosome counts above the 2n range found in these tumors in primary culture, might thus be considered--particularly in the ease of CHK/SVSP tumor--as being a consequence of changing homeostatic relationships between cell and host, of readaptation of cells to in vitro culture conditions, or both. The fact that after 5--7 subcultures, CI-IK/tsA30 tumor B and CIIK/SVSP tumor showed a diploid or neardiploid modal value and less than i0 per cent of cells with chromosome numbers
t36
CH. LAV~A~LEet al. :
over the diploid range indicates that, in vitro, most of the latter cells either had no selective advantage or were not viable. I t has been suggested t h a t near-diploid cells that are present in a SV40-transformed cell population m a y arise from multipolar mitoses (L]~HSIANand BLOUSTEIH, 1974). Though this possibility can not be totally excluded, it does not seem to be able to account for the considerable number of near-diploid and diploid cells found in the majority of the SV40-transformed cell populations that we have studied. The numerical chromosome variations that we have observed in SV40transformed C H K cells were essentially the same as those noticed in uninfected C H K cells. K a r y o t y p e analysis of different SV40-transformed C t t K lines has revealed, in each line; karyotype abnormalities t h a t were also found in the uninfected C H K cell population. I t follows that there is no evidence to suggest t h a t these changes were specific effects of SV40. In earlier eytogenetic studies made with SV40-transformed human cells, the preferential involvement of a chromosome group(s) has been reported (S~IEIH and EHDERS, 1962; YERGANIAN et al., 1962; MOORHEAD and SAKSELA, 1963). The oncogenieity of SV40 has only been demonstrated in mastomys and in Syrian hamster (BUTELet al., 1972) and only few karyologie studies have been so far described on cells transformed in vivo with this virus. The available results show that primary tumors induced b y SV40 in Syrian hamsters, examined either in primary culture or after several in vitro passages, all had a hypertetraploid and/or a hypotetraploid chromosome number distribution (CooPER and BLACK, 1964; NAC~TIGAL et al., 1968; LEHMAN and BLOUSTEIN,1974; I-IIRAI et al., 1974). Until more information is available on primary SV40 tumors developed in Syrian hamsters as well as in other animal species no reasonable conclusion about their ploidy can be drawn, especially since it has been reported that primary and transplanted mouse polyoma tumors (HELLSTR6M et al., 1963) can be diploid, as can also be Syrian hamster adenovirus tumors (BARSKI and CORNEFERT, t964; CASSINGENA and TOURHn~R, 1965; UTZUMI et al., 1965), mouse m a m m a r y carcinomas associated with mouse m a m m a r y tumor virus (TJIo and 0STnRGnES, 1958), Friend leukemias of mice (LEvAH and BII~S:SLs, 1958), Rous sarcomas and erythroleukemias of chickens (PoHT£H, 1963). Cytogenetical analysis of direct preparations of Burkitt tumors associated with Epstein-Barr virus, also showed t h a t the majority of chromosome numbers per cell occurred around the diploid mode in all the tumors analyzed in detail (JAcoss et al., 1963). I t is well known that diploid or near-diploid cell cultures generally shift toward a doubling or tetraploid levels as cultivation continues and t h a t a heteroploid cell population finally evolves. This will quite probably occur also for our diploid or near-diploid SV40-transformed C[-IK cell populations, thus indicating t h a t in the SV40-Chinese hamster system, as observed in the SV40-Syrian hamster system (NACHTIGALet al., 1971) and in cell lines transformed with human eytomegalovirns (NAoHTIOAL et al., 1974), hetcroploidy m a y also be secondary to t r a n s f o r m a t i o n - - i n our case, secondary even to tumorigenic transformation. That heteroploidy m a y also follow the neoplastic conversion has already been shown in the polyoma virus-mouse system (H~LLST~SM et al., 1963). Though the role that the tetraploid-polyploid cells induced following infection with SV 40 might play in transformation with this virus has not yet been defined, the
Chromosomes
and SV40-Transformation
137
results p r e s e n t e d in this w o r k M1 t o g e t h e r s t r o n g l y suggest t h a t p o l y p l o i d i z a t i o n is n o t a necessary s t e p either in t h e process of t r a n s f o r m a t i o n or in t h a t of t u m o r i genic conversion w i t h SV40. T h a t p o l y p l o i d i z a t i o n is not. a prerequisite for transf o r m a t i o n w i t h SV40 is also suggested b y previous results of WEI~STEI~~ a n d MOORHEAD (1967). F~EED~ANN a n d StuN, e x a m i n i n g a large n u m b e r of p e r m a n e n t ceil lines a n d p r i m a r y diploid cell cultures from various species, have r e c e n t l y shown t h a t one i n vitro p r o p e r t y consistently correlated w i t h t u m o r i g e n i c i t y in n u d e mice is t h e c a p a c i t y of t h e cell to form colonies in a semisolid suspension m e d i u m (SSM), in this case methyleellulose m e d i u m (FREEDMAN a n d SHrN, 1974). I n our experience this has n o t been t h e case since, while all p a r e n t a l lines w h e t h e r t r a n s f o r m e d "spont a n e o u s l y " or w i t h SV40 were tumorigenic, n o t all were capable of g r o w t h in SSM. This a p p a r e n t d i s c r e p a n c y b e t w e e n our results a n d those of FREEDM2~N a n d SHI~, m a y p e r h a p s be e x p l a i n e d b y our use of a g a r m e d i u m . The p a r a d o x i e a I o b s e r v a t i o n t h a t some clones d e r i v e d from p a r e n t a l lines unable to grow in agar SSM d i d grow in a g a r SSM is difficult to u n d e r s t a n d , b u t it m a y be due to t h e stress of cloning. Acknowledgments
We are grateful to Marc Girard for providing us with the SV40 strain tsA30 of Peter Tegtmeyer. We are particularly indebted to Prof. l~oger Weft for critieal review of the manuscript. A. G. M. is supported by a lgoyM Society (London) European Exchange Programme fellowship.
References
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Authors' address: Dr. I~. CASSINGENA,I n s t i t u t de Recherches Scientifiques sur le Cancer, B.P. Nr. 8, F-94800 Villejuif, France. Received May 30, 1975
