Vol. 15, No. 2 Printed in U.SA.

JOURNAL OF VIROLOGY, Feb. 1975, p. 420-422 Copyright 0) 1975 American Society for Microbiology

Malignant Transformation of Hamster Kidney Cells by BK Virus MARINELLA PORTOLANI, GIUSEPPE BARBANTI-BRODANO,* AND MICHELE LA PLACA Institute of Microbiology, University of Bologna, Bologna, Italy Received for publication 8 August 1974

Primary hamster kidney cells were transformed by BK virus, a new human Transformed (HKBK) cells produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.

papovavirus.

BK virus is a new human papovavirus isolated by Gardner et al. (6) from a patient receiving a kidney graft. Several studies have been performed recently on the biological, antigenic, and structural properties of this virus (10, 11, 18). In particular, serological investigations (6, 13, 18) have established that an antigenic relationship exists between the structural antigens as well as the T antigen of BK virus and the corresponding antigens of simian virus 40 (SV40). The identification of BK virus as a papovavirus, the wide diffusion of BK virus infection in human populations (5, 14, 16), and the presence of antibodies to BK virus T antigen in some human sera (15) raised the question of its possible role in human oncogenesis. Major and Di Mayorca (9) reported neoplastic transformation of BHK-21 cells by BK virus, but no adequate proof was given in their experiments of the specificity of transformation, since neither the virus-specific T antigen nor virus rescue was demonstrated in transformed cells. We describe here the neoplastic transformation of hamster kidney cells by BK virus. BK virus was grown in Vero cells and titrated in human embryonic fibroblasts by the fluorescent antibody (FA) focus assay according to Aaronson and Todaro (1) and by hemagglutination of human type 0 erythrocytes according to Portolani et al. (14). The virus pool used in these experiments had a titer of 3.7 x 105 FA focus-forming units per ml and 105 hemagglutinating units (HAU) per ml. Primary hamster kidney cells, obtained by trypsinization of kidneys from 72-h-old baby hamsters, were infected with 2 FA focus-forming units per cell. After infection, cells were fed with Eagle minimal essential medium supplemented with 5% calf serum and were split twice a week. At the

eighth passage control cells started to die out, whereas colonies of morphologically modified cells appeared in the infected cultures. These cells showed a fast growth rate, loss of contact inhibition, and a very high saturation density; at the 12th passage a cell line was established (HKBK) that is now in the 45th passage. HKBK cells were tested at different passage levels for virus production by assaying the hemagglutinating activity of the supernatant medium and by fluorescent staining of capsid antigens, using a specific guinea pig serum to BK virus. These tests were consistently negative. HKBK cells (2 x 106) at the 16th passage produced palpable tumors 20 days after injection in 100% of suckling and in 66% of adult hamsters (Table 1). These tumors grew fast and eventually killed the animals 2 to 3 months after the inoculation of transformed cells. Sera from all tumor-bearing hamsters were tested for the presence of antibodies to BK virus T antigen. Seventy-eight percent of sera from animals injected when suckling and 50% of sera from animals injected when adults produced a typical T antigen fluorescent staining on HKBK cells (Fig. 1). The same sera stained the T antigen in MKS-BulOO cells transformed by SV40 (17) and in SV40-infected Vero cells. Conversely, hamster serum to the SV40 T antigen gave a typical T antigen staining on HKBK cells. Sera from tumor-bearing hamsters did not contain antibodies to the structural virus antigens, since they gave only a T antigen staining when tested on Vero cells 3 days after infection with BK virus or 2 days after infection with SV40. These sera did not contain hemagglutination-inhibiting antibodies to BK virus. HKBK cells were fused with permissive cells,

420

TABLE 1. Characteristics of HKBK cells and of the six cell lines derived from HKBK cell-induced tumors HKBK

cells T antigen VPC Oncogenicity For suckling hamsters For adult hamsters

421

NOTES

VOL. 15, 1975

+98.2a Negative

Tumor

cell lines +6/6 (100)° Negative

37/37 (100)d 4/6(66)d

with

sera a serum

from tumor-bearing hamsters

or

with

to SV40 T antigen. Four of the six tumor-derived cell lines, when fused with per-

missive cells, exhibited fluorescent nuclei in syncytia (Fig. 2b and Table 2) after staining with a specific serum to BK virus. Attempts to detect BK virus by hemagglutination in the supernatant medium and cell homogenate from fused cultures of HKBK and

Percentage of positive cells. ,'Number of cell lines showing T antigen/number tested and percentage (in parentheses) of positive cells. c VP, Viral coat protein antigen. dNo. of animals with tumors/no. of animals injected with HKBK cells and percentage (in parentheses) of animals with tumors. a

FIG. 2. Fluorescent nuclei in syncytia 3 days after fusion of (a) HKBK cells and (b) tumor cell line no. 2 with human embryonic fibroblasts. Specific guinea pig serum to BK virus was diluted 1:4 and fluoresceinconjugated rabbit antiserum to guinea pig immunoglobulin G (Hyland) was diluted 1:5. TABLE 2. Rescue of infectious BK virus from HKBK cells and from cell lines derived from HKBK cell-induced tumors Cells

HKBK Tumor cell lines FIG. 1. T antigen staining of HKBK cells. Serum from a tumor-bearing hamster was used at the dilution of 1:2. Fluorescein-conjugated rabbit antiserum to hamster immunoglobulin G (Hyland) was diluted 1:5.

human embryonic fibroblasts, and Vero cells by of beta-propiolactone-inactivated Sendai virus (2). In both cases, 72 h after fusion fluorescent nuclei were detected in syncytia by using a specific guinea pig serum to BK virus (Fig. 2a). The percentage of fluorescent-positive syncytia was 2.7 (Table 2). Cells from six HKBK cell-induced tumors were grown in culture and tested for T antigen and virus rescue. All were positive for T antigen when reacted means

1 2 3 4 5 6

Percentage of syncytia

Infectivity

showing

Rescue of BK virus

2.7

256

+

NTd 3.0 2.4 0.5 0 0.3

NT 16 0 NT NT 0

NT + 0 NT NT 0

fluorescent (HAU/ml)b nucleia

of rescued virus for HEFc

a 72 h after fusion of transformed or tumor cells with Vero cells. "At the 10th subculture of fused cells and upon treatment with receptor-destroying enzyme (RDE). Cells were frozen and thawed once in 4 ml of supernatant medium and subjected to sonic oscillation for 3 min. The cell homogenate was incubated overnight at 37 C with RDE at the final dilution of 1:10 and then heated at 56 C for 1 h to inactivate RDE. c Infectivity tests were performed without heat inactivation of the RDE-treated cell homogenate. HEF, Human embryonic fibroblasts. d NT, Not tested.

422

NOTES

J. VIROL.

Vero cells were negative. Fused cultures were cells is limited by the inefficient excision of viral then subcultured, and the cell homogenates DNA from cellular DNA (7, 8). were tested periodically by hemagglutination LITERATURE CITED directly and after treatment with receptordestroying enzyme (Sigma Chemical Co.), which 1. Aaronson, S. A., and G. J. Todaro. 1970. Infectious SV40 and SV40 DNA: rapid fluorescent focus assay. Proc. has been shown to improve the release of Soc. Exp. Biol. Med. 134:103-106. infectious virus from polyoma, JC, and BK 2. Barbanti-Brodano, G., L. Possati, and M. La Placa. 1971. virus-infected cells (3, 4, 12). Although hemagInactivation of polykaryocytogenic and hemolytic activities of Sendai virus by phospholipase B (lysolecithiglutination was always negative when tested nase). J. Virol. 8:796-800. directly, hemagglutinating titers of 32 HAU per 3. Diamond, L. 1964. Cell transformation in vitro and tumor ml at the sixth subculture and 256 HAU per ml induction in vivo by large- and small-plaque polyoma at the tenth subculture were detected upon virus. Virology 23:73-80. treatment of the cell homogenate with receptor- 4. Dougherty, R. M., and H. S. Di Stefano. 1974. Isolation and characterization of a papovavirus from human destroying enzyme. The hemagglutinating acurine. Proc. Soc. Exp. Biol. Med. 146:481-487. tivity of 8 HAU of the cell homogenate was 5. Gardner, S. D. 1973. Prevalence in England of antibody completely inhibited by a 1:100 dilution of a to human polyomavirus (B.K.). Brit. Med. J. 1:77-78. specific guinea pig serum to BK virus. Eight 6. Gardner, S. D., A. M. Field, D. V. Coleman, and B. Hulme. 1971. New human papovavirus (B.K.) isolated days after infection of human embryonic fibrofrom urine after renal transplantation. Lancet blasts with the hemagglutination-positive 1:1253-1257. homogenate, fluorescent nuclei were detected 7. Huebner, K., C. M. Croce, and H. Koprowski. 1974. Isolation of defective viruses from SV40-transformed by using the specific guinea pig serum to BK human and hamster cells. Virology 59:570-573. virus, and infectious virus was recovered from 8. Huebner, K., and H. Koprowski. 1974. Synthesis of SV40 the supernatant medium. One out of three tucapsid antigens in heterokaryocytes formed by fusion of mor cell lines subcultured after fusion with nonpermissive with permissive cells. Virology 58:609-611. Vero cells gave results similar to those obtained 9. Major, E. O., and G. Di Mayorca. 1973. Malignant with HKBK cells (Table 2). of BHK-21 clone 13 cells by BK virus, a transformation In conclusion, hamster kidney cells were human papovavirus. Proc. Nat. Acad. Sci. U.S.A. transformed by BK virus; in fact, they produced 70:3210-3212. the intranuclear BK virus T antigen and in- 10. Mantyijrvi, R. A., P. P. Arstila, and 0. H. Meurman. 1972. Hemagglutination by BK virus, a tentative new duced tumors in hamsters, and the tumor-bearmember of the papovavirus group. Infect. Immun. ing hamsters had antibodies to BK virus T 6:824-828. antigen. Moreover, virus was rescued from the 11. Mullarkey, M. F., J. F. Hruska, and K. K. Takemoto. 1974. Comparison of two human papovaviruses with originally transformed cell line and from one simian virus 40 by structural protein and antigenic line obtained from tumors induced in hamsters. analysis. J. Virol. 13:1014-1019. The low frequency of rescue, in spite of the very 12. Padgett, B. L., and D. L. Walker. 1973. Prevalence of good efficiency of fusion, was probably due to antibodies in human sera against JC virus, an isolate from a case of progressive multifocal leukoencephthe high number of defective particles present alopathy. J. Infect. Dis. 127:467-470. in the virus inoculum. Indeed, even though the J. B., and 0. Narayan. 1973. Studies of the virus pool used for infection showed only 7.2% of 13. Penney, antigenic relationships of the new human papovavicoreless virions when observed by negative ruses by electron microscopy agglutination. Infect. Immun. 8:299-300. staining under an electron microscope, the ratio of physical particles, determined from the he- 14. Portolani, M., A. Marzocchi, G. Barbanti-Brodano, and M. La Placa. Prevalence in Italy of antibodies to magglutinating titer (10), to FA focus-forming a new human papovavirus (BK virus). J. Med. Microb., units was very high (106 to 1). It is likely, then, in press. that very few cells were infected and trans- 15. Shah, K. V., R. W. Daniel, and G. Murphy. 1973. Antibodies reacting to simian virus 40 T antigen in formed by virions containing a fully infectious human sera. J. Nat. Cancer Inst. 51:687-690. genome, whereas most of the cells were proba- 16. Shah, K. V., R. W. Daniel, and R. M. Warszawski. 1973. bly infected by defective virus particles. This High prevalence of antibodies to BK virus, an SV40may explain why essentially all BK virus-transrelated papovavirus, in residents of Maryland. J. Infect. Dis. 128:784-787. formed cells produced T antigen, whereas only P., G. Barbanti-Brodano, B. B. Knowles, and H. few of them were inducible when fused with 17. Swetly, Koprowski. 1969. Response of simian virus 40-transpermissive cells. Alternatively, a large number formed cell lines and cell hybrids to superinfection with of transformed cells may contain a complete simain virus 40 and its deoxyribonucleic acid. J. Virol. 4:348-355. viral genome if the efficiency of the induction is K. K., and M. F. Mullarkey. 1973. Human low. This possibility is supported by the obser- 18. Takemoto, papovavirus, BK strain: biological studies including vation that the activation of the viral genome in antigenic relationship to simian virus 40. J. Virol. SV40-transformed cells fused with permissive 12:625-631.

Malignant transformation of hamster kidney cells by BK virus.

Vol. 15, No. 2 Printed in U.SA. JOURNAL OF VIROLOGY, Feb. 1975, p. 420-422 Copyright 0) 1975 American Society for Microbiology Malignant Transformat...
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