Biol Cell (1990) 70, 9-18

9

© Elsevier, Paris

Original article

Low tumorigenicity of canine cells transformed by the human cytomegalovirus Jocelyn Yelle, Gilles Lussier, Albena Pramatarova, Claude Hamelin* Centre de recherche en virologie, Institut Armand-Frappier, CP 100, Laval-des-Rapides, Qudbec, H 7 N 423 Canada (Received 11 December 1989; accepted 6 November 1990)

Summary - Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non-permissive in-

fection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies ( < 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus. canine cells / human cytomegalovirus / immortalization / oncogenic transformation / simian virus 40

Introduction

N e o p l a s t i c t r a n s f o r m a t i o n o f d i p l o i d cells is c o n s i d e r e d as a g r a d u a l process involving an early i m m o r t a l i z a t i o n step a n d further p h e n o t y p i c changes t o w a r d tumorigenicity. A n u m b e r o f e x p e r i m e n t s have s h o w n that the h u m a n c y t o m e g a l o v i r u s ( H C M V ) , a m e m b e r o f the herpes virus f a m i l y , c o u l d t r a n s f o r m cells in vitro a n d give t h e m the c a p a c i t y to induce t u m o r s in vivo [ 1 , 2 , 6]. The viral D N A sequence r e s p o n s i b l e for n e o p l a s t i c or t u m o r i g e n i c transf o r m a t i o n o f the cells has been identified [15, 16]. T h e i m m o r t a l i z a t i o n process for H C M V has, however, only been studied by the group o f Rosenthal. A D N A fragm e n t (Xba I-E, fig l) c a r r y i n g the i m m o r t a l i z i n g activity was l o c a t e d on the viral g e n o m e [3, 5, 13, 18]. Despite all their e f f o r t s , the role a n d m e c h a n i s m o f a c t i o n o f this sequence have not yet been elucidated. O n e o f the p r o b l e m s faced in this type o f study lies in the choice o f a cell system. R o d e n t cells c u r r e n t l y used in these w o r k s show a high rate o f s p o n t a n e o u s t r a n s f o r m a tion. I m m o r t a l i z e d cells f r e q u e n t l y a c q u i r e t u m o r i g e n i c p r o p e r t i e s d u r i n g p a s s a g e in vitro. A system based on the utilization o f canine cells was recently d e v e l o p e d in o u r l a b o r a t o r y to s t u d y the i m m o r talizing p r o p e r t i e s o f d i f f e r e n t D N A viruses [27]. Preliminary experiments showed the t r a n s f o r m a t i o n o f dog cells by H C M V [26, 27]. O u r p u r p o s e here was to e x a m i n e the i m m o r t a l i z a t i o n status o f these cell lines.

M a t e r i a l and m e t h o d s

Viral infection Subconfluent JLR cell cultures in 25-cm: flasks (5 × 105 cells) were infected either with HCMV (strain AD 169) or simian virus 40 (SV40) at a multiplicity of infection of l0 plaque-forming units (PFU) per cell. The cells were incubated with the virus for 90 min at 37°C. Growth medium was then added to the culture and the incubation continued. After 5 d, infected cells were trypsinized, diluted 1/2 in culture medium, and incubated at 37°C until transformation foci were detected.

DNA transfection Approximately 5 x 105 cells per 60-mm dish were transfected with 20/zg of HCMV DNA (cleaved by restriction endonuclease EcoR I) using the glycerol-calcium phosphate method described earlier [25]. Transfected cells were maintained at 37°C in culture medium until the appearance of transformation foci.

Transformed cell lines Colonies of densely packed cells, slowly overgrowing the infected or transfected cultures, were isolated by spot-trypsinization, grown to confluence in 6-well tissue culture plates, then serially passaged in vitro. Proliferating cells were routinely seeded at a density of 3 × l05 cells/cmL The growth medium was changed twice a week. After l0 d of incubation at 37°C, cells were counted and viability determined by trypan blue dye exclusion.

Immunofluorescence

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Cultures of primary dog cells (JLR) were obtained by trypsinizing kidneys from 60-d-old embryos as described in [8]. Cells used for infection or transfection were passaged no more than twice

*Correspondence and reprints

(split 1/2) and grown in 199 H a n k ' s / M E M Earle (1/1) medium with 10% fetal calf serum (FCS) and 50 tzg/ml gentamicin in plastic tissue culture vessels (flasks or plates).

The presence of HCMV antigens in transformed dog cells fixed at - 70°C with acetone was detected by indirect immunofluorescence, using a human convalescent serum, followed by fluorescein-conjugated goat antihuman gammaglobulin. The human serum reacts specifically with HCMV nuclear antigens and has been used for years in this institution for the clinical diagnosis of HCMV infections. Antiserum purchased from Flow Laboratories was used, on the other hand, to detect the large T antigen in SV40-transformed cells.

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Fig 1. Localization of transforming sequences on the genome of herpes simplex types 1 and 2, and on that of the human cytomegaiovirus. Restriction maps of HSV-1 and HSV-2 (a) as well as those of HCMV (b) were constructed according to [4, 7, 10, 16, 19]. imm, immortalization; tum, complete oncogenic transformation (tumorigenic).

Agarose plating Cells were prepared and plated in 0.3% agarose as described in [26]. Colonies formed in semi-solid medium by the transformed dog ceils were counted and measured after 8 wks of incubation at 37°C.

sages 3 and 31) cells in 0.1 ml of phosphate-buffered saline. JLR and JLR-SV40 cells were used at the same concentration as negative and positive controls, respectively. Mice kept under sterile conditions were examined at regular intervals for 11 wks in order to detect the formation of tumors.

Tumor induction

Histologic examination

Four-wk-old male Balb-C (Nu/Nu) mice obtained from Jackson Laboratory Inc were subcutaneously inoculated with 5 x 106 JLR-HCMV (passages 3 and 52) or JLR-HCMV/Eco (pas-

Tissues were removed from animals bearing a tumor, fixed in Perfix (Fisher), sectioned (5 tzm), and stained with hemat0xilineosin [11].

Canine cells transformed by HCMV

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Table I. Properties of immortalized canine cell lines.

Cell line

Saturation density °

Expression of viral particles ~

JLR JLR-SV40 JLR-HCMV

0.90 x 105 1.05 x 106 0.80 x 106

-

JLR-HCMV/Eco

0.95 x l06

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Expresion of viral antigens (%y 0 90 75 70 70 1

Growth in semi-solid medium (%)a 0 20 2 2 2 40

(p3) (p32) (p3) (p36)

(p3) (p32) (p3) (923)

~Number of cells per cm ~ of surface. Values were obtained with medium containing 10% FCS and represent the average of three independent experiments; bDetection of virus particles by induction of a cytopathic effect on indicator cells or by examination of biologic material from cultures with the electron microscope; :Cells reacting positively with an antiserum against HCMV or SV40 antigens. The cell passage number is indicated between parentheses; aCells forming colonies in suspension in medium containing 0.3°70 agarose. The cell passage number is indicated between parentheses.

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Fig 2. Morphology of canine cells immortalized by simian virus 40 or the human cytomegalovirus. A. Normal JLR cells. B. JLRSV40 cells. C. JLR-HCMV cells. D. JLR-HCMV/Eco cells (Giemsa; magnification x 130).

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J Yelle

et al

Results

Immortalization o f canine cells Approximately 5 × 105 J L R cells were infected at a multiplicity of infection of 10. Cells were trypsinized at confluency and seeded again. After 5 weeks at 37°C, 11 spots of cells in expansion appeared in the flasks. One of the cell spots was trypsinized separately and seeded again to give rise to the JLR-SV40 cell line. These ceils were subcultured for more than 15 months on a weekly basis and are still maintained as a culture in our laboratory. J L R cells were immortalized in the same way with H C M V . After 7 weeks of incubation at 37°C, 8 spots of refringent, densely packed, fast growing cells were detected in the culture flasks. These clones were similar in shape to those obtained using SV40 as a transforming agent. One of them was isolated to give rise to the immortalized JLRH C M V cell line. It has been subcultured more than 55 times in our laboratory and is still showing the same growth properties. This cell line has always reached confluency within 5-'7 days after seeding 1 x 105 cells per cm ~ of surface. J L R cells transfected with H C M V D N A cleaved with the EcoR I restriction endonuclease also yielded immortalization centers. In these experiments, about 10 spots of immortalized cells were obtained for each/zg of viral D N A digested by EcoRI. One of the clones was isolated and cul..

b

Fig 3. Expression of viral antigens in canine cells immortalized by the human cytomegalovirus or fragments of its genome. a. Human IAFP cells infected for 96 h by HCMV. b. JLRHCMV cells after 3 passages, c. JLR-HCMV/Eco after 3 passages, d. JLR-HCMV/Eco cells after 36 passages, e. normal JLR cells, f.. JLR-SV40 cells after 5 passages (antiserum against HCMV antigens; magnification × 250).

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Fig 4. Growth in semi-solid medium oi~ canine cells immortalized by SV40 or the human cytomegalovirus. A. Normal JLR cells. B. JLR-SV40 cell line. C. JLR-HCMV cell line. D. JLR-HCMV/Eco cell line. Cells were incubated at 37°C for 8 weeks in medium containing 0,3% agarose (Magnification × 60).

Canine cells transformed by HCMV tured to give rise to the J L R - H C M V / E c o cell line. These cells were maintained without any difficulty during many passages, like the other immortalized cell cultures obtained in this study.

Cell morphology The morphology of canine cells immortalized by SV40 or HCMV was compared. Normal JLR cells were fibroblastic in appearance and were rather spread on the plastic surface. Spaces found between the cells remained empty, even after several days in culture. The JLR-SV40, JLR-HCMV and J L R - H C M V / E c o cell lines, in contrast, are made of relatively smaller fibroblasts (fig 2b, c, d). Immortalized cells grow in a parallel fashion, and this is particularly evident when the cells are reaching confluency. Orientation of the cells is more easily seen with JLR-SV40 and JLRH C M V / E c o cells (fig 2b, d). At confluency, the immortalized cells are pressed one against the other and are difficult to distinguish.

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Density of saturation The density of saturation reached by the different cell liries was evaluated in growth medium containing 10°70 FCS. While the JLR cell culture never produced more than 0.9 × 105 cells/cm 2 of surface, the JLR-SV40, JLRblCMV and J L R - H C M V / E c o cells reached saturation densities varying between 0.8 and 1.05 x 106 cells/cm 2 (table I). These values remained constant during passage of the cells. Similar results were obtained with medium containing only 2°7o FCS, except for the JLR cell cultures which showed no sign of growth under these conditions.

Expression of virus particles in immortalized cells The presence of virus particles in culture supernatants of immortalized cell lines was verified. Vero and IAFP indicator cells were incubated for several days at 37°C with samples of medium from the JLR-SV40, JLR-HCMV and J L R - H C M V / E c o cell lines. Cytopathic effects reflecting

B Fig 5. In vivo induction of tumors. A. Tumor induced by JLR-SV40 cells. B. Tumor induced by JLR-HCMV/Eco ceils.

14

J Yelle ef a/

Fig 6. Microscopic section of tumor induced with JLR-SV40 cellsin athymic nude mouse showing epitheloid appearance.‘Hematoxylin

eosine; magnifications (A) x 25 ; (J3) x 75 ; (C) x 200.

Canine cells transformed by HCMV

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Fig 7. Microscopic section of tumor induced with JLR-HCMV/Eco cells in athymic nude mouse showing fibroblastoi~ appearance. Hematoxylin-eosine; magnifications (A) x 25; (B) x 75; (C) x 200.

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J Yelle et al

the production of infectious virus were never observed. Tests performed with culture fluids from the cells at different passage levels were also negative. Virus particles were also never found in the culture fluids by electron microscopy. Little if any infectious virus was thus produced by the immortalized dog cells in culture. Ultrathin sections prepared from cell cultures were now carefully examined to detect virus inside or outside the cells. Particles morphologically similar to SV40 or HCMV were not observed in the immortalized cell lines, even after the examination of hundreds of sections (table I). Not a single retrovirus was found in the dog cells by electron microscopy. Expression o f viral antigens in immortalized cells

Expression of viral proteins in immortalized cells was detected by immunofluorescence. More than 90% of the JLR-SV40 cells reacted positively with the hamster antiserum (table I). A higher proportion of human IAFP control cells infected with HCMV also expressed virus proteins, detected by the human antiserum (fig 3a). The JLRHCMV and J L R - H C M V / E c o cell lines were also examined at differgnt passages. Both cultures expressed nuclear antigens in more than 70O-/oof the cells at passage 3 (table I; fig 3b,c). Stable with JLR-HCMV cells, this percentage dropped rapidly with JLR-HCMV/Eco cells in culture (table I ; fig 3d). Control JLR cells and JLR-SV40 cells never reacted with the antiserum recognizing HCMV antigens (fig 3e,f). Growth in semi-solid medium

The capacity of JLR-SV40, JLR-HCMV and JLRHCMV/Eco ceils to grow in suspension in semi-solid medium was evaluated. The JLR cells always remained isolated in this type of medium and never formed colonies (fig 4a). The JLR-SV40 cell line, on the other hand, gave rise to several colonies 0.1 mm in diameter or larger (fig 4b). About20oT0 of these cells were able to grow in a medium containing agarose (table I). JLR-HCMV cells yielded rare colonies approximately 0.2 mm in size (fig 4c). Only 2°7o o f the cells showed growth in this type of medium. The J L R - H C M V / E c o cell line, however, gave rise to several colonies between 0.1 and 0.15 mm in diameter (fig 4d). After several passages in culture, about 40°7o of these cells forme.d colonies in semi-solid medium (table I).

Table II. Tumorigenicity of immortalized canine cells in athym-

ic mice. Cell lines

Vero Vero-SV40 JLR JLR-HCMV (p3) JLR-HCMV (p52) JLR-HCMV/Eco (p3) JLR-HCMV/Eco (p31) JLR-SV40 (p3) JLR-SV40 (1940)

No of mice inoculated a

Production of tumors b

5 5 5 10 10 10 10 5 5

0 5 0 0 0 1 0 5 5

aSubcutaneous injection of 5 x 106 cells in 0.1 ml of saline. bMice kept under observation for 11 weeks.

Induction o f tumors

The tumorigenicity of JLR-HCMV (passages 3 and 52) and J L R - H C M V / E c o (passages 3 and 3 I) cells was evaluated in the athymic nude mouse using JLR and JLR-SV40 (passages 3 and 31) cells as negative and positive controls, respectively. As shown in table II, mice injected with JLR cells remained healthy for the duration of the experiment while all those exposed to canine cells transformed by SV40 developed a tumor in less than 2 months (fig 5a). Only one of the 40 mice inoculated with JLR-HCMV or JLRH C M V / E c o cells, however, developed a tumor during the 11-week period of observation (fig 5b). It is interesting to note that the size of the tumors induced by HCMV- or SV40-transformed cells was relatively small as compared to what is seen, for example, when mammary carcinoma cells are injected into a mouse. Under the microscope, the tumor induced by JLRH C M V / E c o cells showed less dense tissues and less mitotic figures than those produced by JLR-SV40 cells (figs 6 and 7). J L R - H C M V / E c o tumor cells were fibroblastic in appearance while the others were rather epitheloid in shape. In both cases, however, we noted that the surrounding tissues were either little or not at all invaded by the tumors.

Discussion

Properties o f immortalized cells

The division potential of dog embryo kidney cells is extremely low [8]. Under our experimental conditions, the JLR cells usually stopped dividing after passage 1. The normal kidney fibroblasts further split 1/2, occasionally reached confluency but only after constant refeeding with fresh medium containing 10°70 serum, and long periods of incubation. At this point, " a g e d " cells could remain alive for many months at 37°C without showing any sign of spontaneous transformation. Normal JLR cells were efficiently transformed by SV40 and HCMV as described in [26, 27]. Colonies of densely packed cells, slowly overgrowing the culture, were observed after 6-8 weeks of incubation at 37°C (1-2 foci/ 105 infected cells). Continuous dog cell lines were also obtained after transfection with EcoR I-cleaved HCMV DNA (" 10 foci/tzg). Small, rapidly dividing cells were isolated on several occasions and subcultured more than 50 times without interruption. The JLR-SV40, JLR-HCMV and J L R - H C M V / E c o cell lines represent well the different clones obtained from isolated spots of dog cells with unlimited division potential. Immortalized dog cells, in contrast to their aging counterparts, could reach a saturation of 106 cells/cm 2 in less than 10 days, even with medium containing only 2°7o serum. They also reacted positively with a specific SV40- or HCMV-antiserum (fig 3) and were able to grow in agarose (fig 4). Cellular immortality, reduced serum requirement, presence of viral antigen(s) in the cells, and anchorageindependent growth were all pointing to a transformation event induced in JLR cells by SV40 or HCMV. As far as the origin of the immortalized dog cells is concerned, we noted that virus-like particles were not found in the numerous ultrathin cell sections examined todate by electron microscopy. This technique, which is the only one that can detect certain types of retroviruses [23, 24] gave negative results with the JLR cells. The presence

Canine cells transformed by HCMV of endogenous retroviruses in dog embryo kidney cells has, in fact, never been reported in the literature. Mammalian cells transformed by SV40 of HCMV are known, on the other hand, not to produce infectious virus [22]. Even if certain parts of the herpes simplex type 1 (HSV-I) genome stimulate the expression of retroviruses in murine cells, these DNA sequences differ from those required for cellular transformation [14]. Expression of the large T antigen in JLR-SV40 cells, as detected by immunofluorescence, confirms the importance of this protein in the transformation of dog cells. With HCMV, however, viral antigens are detected in an extremely variable proportion of the transformed cells [6, 22]. The observed variations could be due, at least in part, to the different antisera used for the detection of HCMV antigens [6]. It remains that the percentage of HCMVtransformed cells reacting positively with a specific antiserum generally decreases with time in culture. Results in table I indicate that viral antigens disappeared more rapidly from J L R - H C M V / E c o cells than from JLR-HCMV cells during in vitro passage. Distinct mechanisms are apparently used by SV40 and HCMV to transform the canine cells. JLR-SV40, JLR-HCMV and J L R - H C M V / E c o cells, in contrast to JLR cells, were able to grow in agarose. Colonies formed in semi-solid medium rarely exceeded 0.2 mm in diameter. Relatively small colonies w~re also obtained by Jariwalla et al [12] with immortalized hamster cells. Despite this important similitude, canine cells remain more appropriate than any rodent cells to study the first step towards carcinogenesis because they are devoid of endogenous retroviruses and rarely escape senescence in a spontaneous manner. Now unlimited growth in vitro, restricted growth in agarose, very low tumorigenic capacity in vivo, low invasive character of the unique tumor obtained with J L R - H C M V / E c o cells at passage 3, all these observations support the idea of an immortalization event rather than an oncogenic transformation event.

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HCMV strains show very similar physical maps, there are variations in the position of many restriction sites. It would be interesting to try to immortalize canine cells with the XbaI-E fragment of the Towne strain, tested by these authors. In the same way the HSV-2 BglII-C immortalizing fragment could be used as an important control tool t'o compare the sensitivity of the two cellular systems. Our results with canine cells confirm data in the literature which indicate the possible existence of two disctinct steps in the in vitro transformation process [17, 21]. The canine cell system could be most useful to complete already existing information, obtained with rodent cell lines, on the phenomenon of viro-induced immortalization of cells. The detection of immortalizing sequences of an EcoRI fragment of HCMV DNA [26] represents, on the other hand, an observation which could lead to the discovery of new transforming regions in the genome of herpetic viruses. Our data fit well into the prolongation of the work carried out by other groups suggesting that distinct DNA sequences of certain herpes viruses are associated with the two stages of transformation [3, 12, 13]. It will be interesting, to see if in the next few years, this characteristic is also found in other viruses of this family. A library of pAT 153 plasmids carrying EcoRI fragments of the HCMV genome is now under construction. Each of the 36 possible recombinant plasmids will be cleaved by EcoRI, co-precipitated with calcium phosphate, and applied to subconfluent monolayers of JLR cells. The HCMV DNA fragment(s) inducing immortality will be closely mapped and sequenced. More information concering the oncogenic potential of this herpes virus should be obtained after comparison of the different transforming regions of its genome. A wide variety of mammalian cells should be used as a target for tumor DNA viruses in order to fully understand the multistep process leading to neoplastic disease.

Acknowledgments Importance o f the cellular system & the study o f the transforming properties o f herpes viruses Results obtained with different cell systems indicate that distinct regions of the HSV-2 genome are required for immortalization and tumorigenic transformation of cells in culture [22]. As far as HCMV is concerned, the same situation apparently prevails (fig 1). Work carried out by Nelson et al [15, 16] with immortalized cell lines shows that the HindIII-E fragment of the viral genome (strain AD-169) carries DNA sequences related to tumorigenic transformation. Data obtained by Jariwalla et al [3, 13] suggest, on the other hand, that the same viral DNA fragment carries a distinct region associated with immortalization. These results underline the capital importance of the type of cells and the method of selection for transformed clones, in experiments carried out with this group of viruses. Because our results were obtained with a cellular sys'tem allowing the study of immortalization, it is not surprising that they differ from those of Nelson et al [15, 16]. Data obtained with the canine cells indicate that, in contrast with the indications of Clanton et al [3], the HindIII-E fragment of the virus strain AD-169 does not provoke the immortalization of cell cultures. Because these authors have performed their experiments with a virus strain and cells different to ours, it is difficult to establish the exact meaning of these differences. Even if the two

We thank L Cousineau and G Godin for excellent technical assistance. This work was supported by grant A3373 from the Natural Sciences and Engineering Research Council of Canada and by funds from the Institut Armand-Frappier.

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