JOURNAL OF VIROLOGY, Apr. 1979, 0022-538X/79/04-0404/06$02.00/0
Vol. 30, No. 1
p. 404-409
Neoplastic Transformation of Cultured Syrian Hamster Embryo Cells by DNA of Herpes Simplex Virus Type 2 RAXIT J. JARIWALLA,1 LAURE AURELIAN,"* AND P. 0. P. TS'O' Division of Biophysics' and Department of Comparative Medicine,2 The Johns Hopkins University Schools of Hygiene and Public Health and Medicine, Baltimore, Maryland 21205
Received for publication 25 October 1978
Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (0.01 jig per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s). Neoplastic transformation by DNA extracted from the DNA-containing animal tumor viruses has led to the identification and mapping of sequences associated with oncogenesis (11). However, neoplastic transformation of cultured cells by purified DNA of herpes simplex virus type 2 (HSV-2), a DNA virus associated with human cancer (24), has not yet been demonstrated. In the absence of nonpermissive in vitro systems, neoplastic transformation by HSV-2 has been observed only with virus whose lytic functions were inactivated (16, 19, 25), thus limiting the significance of these observations with respect to the mechanisms of carcinogenic processes in human disease. In this communication we report the neoplastic transformation of cultured Syrian hamster embryo (SHE) cells by purified HSV-2 DNA at dilutions that lack detectable infectivity, i.e., under conditions most closely reflecting those that may operate in vivo. Viral DNA was isolated as described by Walboomers and Schegget (26) from Vero cells infected with 0.1 to 0.3 PFU of the S-1 strain of HSV-2 per cell. This virus strain was chosen because it originated from intraepithelial human cervical carcinoma (3), and, as such, it was considered that it might possess a high transforming potential. DNA (cellular plus viral) was extracted from the infected cells at the time (24 to 48 h postinfection) of maximal (90 to 100%) cytopathic effect. The cells were lysed with 0.6% (wt/vol) sodium dodecyl sulfate and digested for 3 h at 37°C with Pronase (2 mg/ml) that had been heat treated for 10 min at 80°C. Pronase (2 mg/mi) was again added, and the digestion was continued for 12 to 15 h. After one cycle of centrifugation on NaI bouyant density gradients
containing ethidium bromide (30 ,tg/ml) in a Ti 50 rotor at 44,000 rpm for 48 h at 20°C, two distinct bands were observed. The lower, denser band containing viral DNA was collected and cleared of ethidium bromide by dialysis against Dowex 5OW-2X beads (Bio-Rad Laboratories, Richmond, Calif.), first with two changes of 50 to 100 volumes of a high-salt buffer (1 M NaCl10 mM Tris-hydrochloride [pH 8.0]-1 mM EDTA) and then against three changes of 50 to 100 volumes of a low-salt buffer (10 mM NaCl10 mM Tris-hydrochloride [pH 8.0]-1 mM EDTA). HSV-2 DNA was subjected to sedimentation assay in a model E analytical ultracentrifuge in 1 M NaCl-0.05 M sodium citrate (pH 7.0) at 25,980 rpm (20°C). It sedimented as a rather broad band with an average sedimentation coefficient, calculated according to Studier (23), of 54.5S (Fig. 1). Viral DNA was used to transfect SHE cells grown in subconfluent monolayers (5 x 105 cells per 60-mm dish). Before exposure to viral DNA, one set of dishes was incubated in HEPES (N-2hydroxyethyl piperazine-N'-2-ethanesulfonic acid)-buffered saline (HeBS) (pH 7.05) (12), supplemented with 0.5 mM MgCl2 and 0.9 mM CaCl2, for 135 min at 37°C; another set was subjected to calcium depletion (13) by incubation in HeBS supplemented with 0.5 mM MgC12 for 135 min at 37°C; and the third set was washed with HeBS. Viral DNA, coprecipitated with calcium phosphate in the presence of salmon sperm DNA (5 to 10,ug per dish), was applied to cells at concentrations ranging from 0.01 to 1 ,ug per dish as described by Graham et al. (12). After incubation at 37°C for 8 h, the monolayers were overlaid with growth medium
404
NOTES
VOL. 30, 1979
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12_Mb
0
I-
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20.1..
MIGRATION
FIG. 1. Sedimentation analysis of HSV-2 (S-1) DNA in a model E analytical ultracentrifuge. The band sedimentation was conducted in 1 MNaCI-0.05 Msodium citrate (pH 7.0) at 25,980 rpm (20°C) following the procedure described by Studier (23).
405
containing 0.2% human immunoglobulin G (Merck, Sharp and Dohme, West Point, Pa.) to neutralize progeny virus (1). Plaques developed within 2 to 3 days in cultures exposed to -0.1 pug of DNA. The specific infectivity, defined as number of plaques per microgram of DNA, ranged between 16 and 21 PFU/pg in cells exposed to viral DNA after treatment with HeBS containing Ca2" and Mg2" (Table 1). It was enhanced 7.5-fold in SHE cells pretreated by calcium depletion. On the other hand, cultures exposed to 0.01 ,ug of DNA did not display plaque formation and continued to propagate. They were replenished with growth medium once every 3 to 4 days for 4 to 5 weeks. Monolayers were serially passaged at split ratios of 1:5 or 1:10. Within five to six passages, the growth rate of the cells declined and the cultures entered crisis. Control cultures "mock-infected" by exposure to medium or to salmon sperm DNA senesced 2 to
TABLE 1. Transfection and transformation of SHE cells with HSV-2 (S-i) DNAa Expt. no.
I
Treatment of cells before DNA
HeBS + Ca2+ + Mg2+ Incubation (370C, 135 min)
HeBS + Mg2e only Incubation (370C, 135 min) Washing with HeBS only Growth medium only
Growth medium only
jug of DNA
No. of
No. of transformed
per dish
plaques per
cultures/total no. of
dish
cultures
1.2 0.12 0.012 1.2 0.12 0.012 1.2 0.12 0.012 0¢
25 2 0
0/2a 0/2a 2/2 0/2a 0/2a 2/2 0/2a 0/2a 2/2 0/4
TNTCb 15 0 TNTC 5 0 0
2 0.14 0/2a 0.014 0 2/2 0 0.002 2/2 0.0005 0 0/2 OC 0 0/4 a 3 x 105 SHE cells were seeded in 60-mm dishes containing growth medium consisting of IBR-modified Dulbecco Eagle reinforced medium (Biolabs, Northbrook, Ill) supplemented with 0.22 g of NaHCO3 per 100 ml, 10% Rehatuin F. S. fetal bovine serum (Reheis Chemicals, Kankakee, Ill.), 100 U of penicillin per ml, 100 pg of streptomycin per ml, and 10 U of mycostatin per ml. After 24 h, replicate cultures were treated as shown above and exposed to HSV-2 (S-1) DNA. The viral DNA was isolated from infected Vero cells by a modification of the method described by Walboomers and Scheggett (6). Before its use to infect cells, viral DNA was mixed with salmon sperm DNA to a final concentration of 10 or 20 pg/ml in HeBS consisting of 8.0 g of NaCl, 0.37 g of KCl, 0.125 g of Na2HPO4. 2H20, 1.0 g of dextrose, and 5.0 g of HEPES (pH 7.05) per liter. CaCl2 was added to a final concentration of 125 mM, and the mixture was kept at 220C for 20 to 30 min. Cell monolayers were exposed to 0.5 ml of the DNA/Ca phosphate mixture for 15 to 20 min at 220C, after which 5 ml of Eagle reinforced medium plus 10% Rehatuin fetal bovine serum was added, and the dishes were incubated for 6 to 8 h at 37°C. The medium was then removed and replaced with Eagle reinforced medium plus 10% Rehatuin fetal bovine serum supplemented with 0.2% pooled human gamma globulin. Dishes that did not contain plaques were fed for 4 to 5 weeks, trypsinized, and transferred to 75-cm2 flasks. Resulting cultures were passaged when confluent at split ratios of 1:5 or 1:10. Cell lines established from cultures that escaped senescence were assayed for transformationassociated characteristics. a Dishes containing plaques were fixed with methanol and stained with Giemsa. b TNTC, Too numerous to count. ' Control untreated dishes or dishes treated only with salmon sperm DNA.
II
406
J. VIROL.
NOTES
3 weeks later. In contrast, cultures surviving exposure to HSV-2 DNA developed refractile colonies which, within 1 to 3 weeks, overgrew the cultures. Cell lines established from cultures exposed to 0.01 ,.g of HSV-2 (S-1) DNA (Table 1, experiment I) manifested morphological alteration and loss of contact inhibition (high saturation density and growth in multilayers) characteristic of transformation. Transformation (Table 1, experiment II) was detected only in cells exposed to a narrow range of viral DNA concentrations (0.01 to 0.002 ,ug per dish), suggesting that it was mediated by viral DNA. Assuming that only 1 out of 5 x 105 SHE cells was transformed by 0.002 ,ug of HSV-2 DNA, the frequency of transformation is estimated at approximately 1 transformant per i05 recipient cells per 0.01 ,&g of DNA. Two cell lines, designated SDNA-177 and SDNA-277, established by exposure of Ca2+-depleted and HeBS-washed cultures, respectively, to 0.01 ,ug of HSV-2 DNA, were examined for in vitro phenotypic alterations associated with the neoplastic state (Table 2). One such characteristic is increased cloning efficiency ([number of colonies x 100]/number of seeded cells) (14) in liquid media when seeded at low (100 to 500 cells per 60-mm dish) cell density (20). Normal SHE cells displayed a low (1 to 4%) cloning efficiency when seeded at a density of 5,000 to 10,000 cells per 100-mm dish, but did not clone when seeded at a density of 100 to 500 cells per 60-mm dish. In contrast, both SDNA-177 and SDNA-277 TABLE 2.
cells manifested high cloning efficiency (8 to 18%) when seeded at low cell density (100 to 500 cells per 60 mm dish), indicating a reduced dependence on cross-feeding for growth. An additional criterion of transformation is the ability of cells to grow in medium supplemented with low serum concentrations (8). Indeed, normnal SHE cells do not grow in 2% serum, whereas SDNA-177 and SDNA-277 cells clone under these conditions at an efficiency of 3.5 and 1.5%, respectively, even when seeded at a low cell density (100 to 500 cells per 60-mm dish). The ability of transformed cells to form colonies in soft agar or agarose is associated with their loss of anchorage dependence (17). Colonyforming efficiency in 0.3% agarose was measured as described (5) and expressed as (number of colonies x 100)/number of seeded cells (105 per 60-mm dish) (5). Normal SHE cells do not grow in agarose, but SDNA-177 and SDNA-277 produce colonies with an efficiency of 2.3 and 5%, respectively. Fibrinolytic activity, a biochemical marker associated with neoplasia, was assayed as described (6). The levels of extracellular activity secreted by SDNA-177 and SDNA-277 cells were three- to sixfold higher than those of normal SHE cells. The ability of transformed cells to induce tumors in appropriate hosts is the most critical assay for the study of the mechanisms of transfonnation as they pertain to carcinogenesis. Accordingly, approximately 2 x 106 SDNA-177
Phenotypic characteristics of SHE cells transforned by HSV-2 DNA SDNA-177 (posttreatment pas-
Morphology
Normal SHE (passage3to5) Fibroblast-like
sage4to35) Mixture of epithelial and fibroblast-like
passage4to30) Predominantly spindlelike
Growth in culture
Monolayers
Multilayers
Multilayers
0
17.6 3.5
8 1.5
0
2.3
5.5
Characteristic
SDNA-277 (posttreatment
Cloning efficiency at low cell densitya
in 10% serum (%) in 2% serum (%)
Colony-forming efficiency' in 0.3% agarose (%) Fibrinolytic activityc
0
Low (4.9 + 0.05)
High (12.9
4)
High (26
2)
~~~+ NDd Tumorigenicity (invasive anaplastic fibrosarcomas) a From 100 to 500 cells were seeded per 60-mm dish. Colonies of >20 cells were counted at day 10. Cloning efficiency = (number of colonies x 100)/number of seeded cells. b 105 cells were seeded per 60-mm dish. Colonies of >20 cells were counted at 2 to 3 weeks. Cloning efficiency = (number of colonies x 100)/105. 'Units of fibrinolytic activity per 2 ml of harvest medium ± standard error. d ND, Not done. _
VOL. 30, 1979
NOTES
407
cells at posttreatment passage 21 or 35 were virus specific, as indicated by the observations inoculated subcutaneously on the ventral side of that preimmune rabbit immunoglobulin G's 1- to 3-day-old newborn Golden Syrian hamsters were nonreactive and that Ra-2 and anti-AG-e (Ela: ENG strain, Engle Laboratories, Farmers- 7S immunoglobulins did not fix complement burg, Ind.). Animals were palpated twice weekly with extracts of normal SHE cells. It should be for tumor development at the site of inoculation. pointed out that anti-AG-e serum stains exfolTumor formation was first detected in three of iated cervical tumor but not normal cells in nine inoculated animals after a latent period of indirect immunofluroescence (L. Aurelian, P. K. 6 to 7 weeks. By 13 weeks, all the inoculated Gupta, J. K. Frost, N. B. Rosenshein, C. C. animals were positive for tumors. Histopatholog- Smith, H. W. Tyrer, J. M. Mantione, and C. D. ically, the tumors were invasive anaplastic fibro- Albright, Acta Cytol., in press). sarcomas. Tumors (-1.5 by 1.5 cm) were excised, HSV DNA, the infectivity of which was artitrypsinized, and grown in tissue culture. The ficially inactivated, has been shown to cause tumor cells, designated SDNA-177T1, SDNA- biochemical (4, 18, 27) or morphological (29) 177T2, etc., were morphologically identical to transformation. However, these transformants bear little resemblance to oncogenic processes the SDNA-177 cells. The microquantitative complement fixation occurring in human disease since (i) they do not assay was used to determine the presence of cause tumors in animals and (ii) they were esHSV antigens in extracts of transformed cells tablished with viral DNA, the infectivity of prepared as previously described (2). The results which was artificially inactivated. of these studies (Table 3) may be summarized The salient feature of our studies is the obseras follows. (i) Human sera containing neutralizvation that cells exposed to purified, not intening antibody to HSV-2 (no. 119 and 124) or to tionally inactivated HSV-2 (S-1) DNA display both HSV-2 and HSV-1 (no. 141 and 183) fixed properties characteristic of transformation, concomplement with extracts from transformed tain HSV-specific antigens, and cause invasive cells. The virus specificity of the reaction was tumors when inoculated into appropriate hosts. evidenced by the observations that a human Consistent with the presence of virus-specific serum (no. 317) lacking antibody to either virus antigens in SDNA-177 cells (Table 3), sera from type was nonreactive, and that normal SHE cells tumor-bearing hamsters contain neutralizing andid not fix complement with any of the human tibody to HSV-2 as determined by plaque-resera. (ii) Extracts of SDNA-177 cells also fixed duction neutralization assay (1), and SDNAcomplement with immunoglobulin G from anti- 177T1 cells fix complement with antisera to sera prepared against total viral antigens (Ra-2) HSV-2 antigens (unpublished data). and against a purified type-common antigenic Transformation of SHE cells by high dilutions fraction designated AG-e, and consisting of two of HSV-2 (S-1) DNA is amenable to three interproteins that comigrate with virion structural pretations: (i) transforming molecules are nonproteins VP 5 and 6 (21). The reactions were infectious (aberrant in size and/or complexity) TABLE 3. Detection of HSV antigens in HSV-2 (S-i) DNA-transformed and normal SHE cells by microquantitative complement fixationa Serum or immunoglobulin
Neutralizing antibody to
% Complement fixed by extract (20 itg of protein) of:
HSV-1
HSV-2
SHE
SDNA-177
SDNA-277
+ + -
+ + + + -
9.9 5.4 1.8 0 0
38.7 32.9 44.1 14.5 0
33.8 27.7 28.8 0 0
Human Sera 141 183 119 124 317
Immunoglobulin G of rabbit sera to: Total virion antigens (Ra-2)
+ + 0 16.1 0 0 0 0 AG-eb 0 29.7 10.2 Preimmune 0 0 0 a"Immunoglobulins were fractionated by sucrose gradient centrifugation. Neutralizing potential for HSV-2 and/or HSV-1 was determined by the multiplicity analysis plaque reduction assay (1). The microquantitative complement fixation assay was used with optimal concentrations of antisera or immunoglobulins as previously described (2). Reaction was considered positive if more than 10% of the complement was fixed (2). b AG-e is the 0.4 M phosphate eluate of a calcium phosphate column of total soluble HSV-2 antigens (21).
Preimmune
408 NOTES and do not become encapsidated into virions; (ii) transforming DNA molecules are identical to those 55S molecules that become encapsidated (15) but their expresion is modified in recipient cells by host- and/or virus-determined regulatory mechanisms; or (iii) transforming molecules are fragments of viral DNA generated in vitro by nonspecific shearing forces or produced in recipient cells by the action of cellular nucleases (9). Presently available data appear to argue against the first interpretation but cannot distinguish between the other two interpretations with any degree of certainty. Thus, the majority of viral DNA molecules synthesized in infected nuclei do not become encapsidated and remain associated with the nuclei throughout infection (28). However, these molecules differ from mature virus DNA in their sedimentation characteristics (28), whereas S-1 DNA sediments as a single band with an s20,w of 54.5S (Fig. 1), a value virtually identical to that determined by other investigators for DNA extracted from purified HSV-2 virions (15). Furthermore, the specific infectivity of S-1 viral DNA is relatively low (12), although it does not differ from that reported by Stow and Wilkie (22) for HSV-1 (Glasgow strain 17) DNA. The observation that calcium depletion significantly enhances the specific infectivity of S-1 DNA (Table 1) suggests that a low specific infectivity is not an intrinsic property of this DNA. Indeed, the specific infectivity of DNA extracted from sucrose-gradientpurified S-1 virions is approximately 100-fold higher than that of the DNA extracted from S1-infected cells. Nevertheless, virion-extracted DNA also transforms SHE cells at dilutions (10-4 ,ug of DNA per dish) lacking infectivity, and the transformants express HSV-2 antigens and cause tumors in inoculated hamsters (manuscript in preparation). Finally, it should be pointed out that hamster cells transformed with UV-irradiated HSV-2 contain only a fraction (8 to 32%) of the viral DNA sequences (10), indicating that transformation may be mediated by DNA fragments. Indeed, Camacho and Spear (7) have reported that a fragment of HSV-1 DNA transforms cells in vitro although these transformants do not cause tumors in animals. Farber (9) has shown that 56S viral DNA is degraded in recipient cells to a 20S predominant fragment, suggesting that transforming fragments may also be generated in vivo. Taken as a whole, these observations suggest that the bulk of the S-1 viral DNA molecules (of cellular or virion extraction) are not capable of initiating infection, possibly due to fragmentation occurring in vitro or within the recipient cells. Such molecules retain transforming poten-
J. VIROL.
tial and become the predominant species upon high dilution of the DNA preparation. The possibility cannot be excluded that the neoplastic potential of the SDNA-177 cells is due to the source (cervix cancer) of the S-1 virus. The HSV-DNA-SHE cell system described in this report is a particularly suitable model for the study of the viral genes and gene products involved in neoplastic transfornation. Studies designed to inquire into this question and to examine the relationship between HSV-induced cellular alterations in vitro and tumorigenicity in vivo are now in progress in our laboratory. This work was supported by Public Health Service grant Ca-16043 from the National Cancer Institute. R.J.J. was supported by a National Research Service Award from the National Cancer Institute. We acknowledge helpful discussions with Mark Manak. We also thank M. F. Smith for help with the complement fixation assay, B. Crawford for work on fibrinolytic activity, and L. Trpis for technical assistance.
LITERATURE CITED 1. Aurelian, L., L. Royston, and H. J. Davis. 1970. Antibody to genital herpesvirus: association with cervical atypia and carcinoma in situ. J. Natl. Cancer Inst. 45: 455-464. 2. Aurelian, L, B. Schuman, and R. L Marcus. 1973. Antibody to HSV-2 induced tumor specific antigens in sera from patients with cervical carcinoma. Science 181:161-164. 3. Aurelian, L, J. D. Strandberg, L. V. Melendez, and L A. Johnson. 1971. Herpesvirus type 2 isolated from cervical tumor cells grown in tissue culture. Science 174:485-488. 4. Bacchetti, S., and F. L Graham. 1977. Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA. Proc. Natl. Acad. Sci. U.S.A. 74:1590-1594. 5. Barrett, J. C., B. D. Crawford, D. L Grady, L D. Hester, P.A. Jones, W. F. Benedict, and P. 0. P. Ts'o. 1977. Temporal acquisition of enhanced fibrinolytic activity by Syrian hamster embryo cells following treatment with benzo(a) pyrene. Cancer Res. 37:38153823. 6. Barrett, J. C., B. D. Crawford, and P. 0. P. Ts'o. 1977. Quantitation of fibrolytic activity of syrian hamster fibroblasts using 3H-labeled fibrinogen prepared by reductive alkylation. Cancer Res. 27:1182-1185. 7. Camacho, A., and P. G. Spear. 1978. Transformation of hamster embryo fibroblasts by a specific fragment of the herpes simplex virus genome. Cell 15:993-1002. 8. Dulbecco, R. 1970. Topoinhibition and serum requirement of transformed and untransformed cells. Nature (London) 227:802-806. 9. Farber, F. E. 1976. Comparison of DNA facilitators in the uptake and intracellular fate of infectious herpes simplex virus type 2 DNA. Biochim. Biophys. Acta 454: 410-418. 10. Frenkel, N., H. Locker, B. Cox, B. Roizman, and F. Rapp. 1976. Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor. J. Virol. 18:885-893. 11. Graham, F. L*, P. J. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. J. de Vries, W. Fiers, and A. J. van der Eb. 1974. Studies on in vitro transformation by DNA and DNA fragments of human adenovirues and simian virus 40. Cold Spring Harbor
VOL. 30, 1979 Symp. Quant. Biol. 39:637-650. 12. Graham, F. L, G. Veldhuisen, and N. M. Wilkie. 1973. Infectious herpesvirus DNA. Nature (London) New Biol. 246:265-267. 13. Greene, J. J., C. W. Dieffenbach, and P. 0. P. Ts'o. 1978. Inactivation of interferon mRNA in the shut-off of human interferon synthesis. Nature (London) 271: 81-83. 14. Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc. Natl. Acad. Sci. U.S.A. 63:288-293. 15. Kieff, E. D., S. L. Bachenheimer, and B. Roizman. 1971. Size, composition, and structure of the deoxyribonucleic acid of herpes simplex subtypes 1 and 2. J. Virol. 8:125-132. 16. Kucera, L S., J. P. Gusdon, I. Edwards, and G. Herbst. 1977. Oncogenic transformation of rat embryo fibroblasts with photoinactivated herpes simplex virus: rapid in vitro cloning of transformed cells. J. Gen. Virol. 35:473-485. 17. McPherson, I., and L. Montagnier. 1964. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23:291-294. 18. Maitland, N. J., and J. K. McDougall. 1977. Biochemical transformation of mouse cells by fragments of herpes simplex virus DNA. Cell 11:233-241. 19. Rapp, F., and R. Duff. 1973. Transformation of hamster embryo fibroblasts by herpes simplex viruses type 1 and type 2. Cancer Res. 33:1527-1534. 20. Risser, R., D. Rifkin, and R. Pollack. 1974. The stable classes of transformed cells induced by SV40 infection of established 3T3 cells and primary rat embryonic cells. Cold Spring Harbor Symp. Quant. Biol. 39:317324.
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21. Smith, C. C., R. B. Bell, and L Aurelian. 1978. Immune response to antigen (AG-e) in patients with HSV-2 asociated diseases, p. 528. Proceedings of the 4th International Virology Congress. Centre for Agricultural Publishing and Documentation, Wageningen, The Netherlands. 22. Stow, N. D., and N. M. Wilkie. 1976. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33: 447458. 23. Studier, W. F. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390. 24. Symposium on Immunological Control of Virus-Associated Tumors in Man: Prospects and Problems. 1976. Cancer Res. 36:783-859. 25. Takahashi, M., and K. Yamanishi. 1974. Transformation of hamster embryo and human embryo cells by temperature sensitive mutants of herpes simplex virus type 2. Virology 61:306-311. 26. Walboomers, J. M. M., and J. T. Schegget. 1976. A new method for the isolation of herpes simplex virus type 2 DNA. Virology 74:256-258. 27. Wigler, M., S. Silverstein, L S. Lee, A. Pellicer, Y. C. Cheng, and R. Axel. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223-232. 28. Wilkie, N. M. 1973. The synthesis and substructure of herpesvirus DNA: the distribution of alkali-labile single strand interruptions in HSV-1 DNA. J. Gen. Virol. 21: 453-467. 29. Wilkde, N. M., J. B. Clements, J. C. M. MacNab, and J. H. Subak-Sharpe. 1974. The structure and biological properties of herpes simplex virus DNA. Cold Spring Harbor Symp. Quant. Biol. 39:657-666.
Vol. 30, No. 1
p. 404-409
Neoplastic Transformation of Cultured Syrian Hamster Embryo Cells by DNA of Herpes Simplex Virus Type 2 RAXIT J. JARIWALLA,1 LAURE AURELIAN,"* AND P. 0. P. TS'O' Division of Biophysics' and Department of Comparative Medicine,2 The Johns Hopkins University Schools of Hygiene and Public Health and Medicine, Baltimore, Maryland 21205
Received for publication 25 October 1978
Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (0.01 jig per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s). Neoplastic transformation by DNA extracted from the DNA-containing animal tumor viruses has led to the identification and mapping of sequences associated with oncogenesis (11). However, neoplastic transformation of cultured cells by purified DNA of herpes simplex virus type 2 (HSV-2), a DNA virus associated with human cancer (24), has not yet been demonstrated. In the absence of nonpermissive in vitro systems, neoplastic transformation by HSV-2 has been observed only with virus whose lytic functions were inactivated (16, 19, 25), thus limiting the significance of these observations with respect to the mechanisms of carcinogenic processes in human disease. In this communication we report the neoplastic transformation of cultured Syrian hamster embryo (SHE) cells by purified HSV-2 DNA at dilutions that lack detectable infectivity, i.e., under conditions most closely reflecting those that may operate in vivo. Viral DNA was isolated as described by Walboomers and Schegget (26) from Vero cells infected with 0.1 to 0.3 PFU of the S-1 strain of HSV-2 per cell. This virus strain was chosen because it originated from intraepithelial human cervical carcinoma (3), and, as such, it was considered that it might possess a high transforming potential. DNA (cellular plus viral) was extracted from the infected cells at the time (24 to 48 h postinfection) of maximal (90 to 100%) cytopathic effect. The cells were lysed with 0.6% (wt/vol) sodium dodecyl sulfate and digested for 3 h at 37°C with Pronase (2 mg/ml) that had been heat treated for 10 min at 80°C. Pronase (2 mg/mi) was again added, and the digestion was continued for 12 to 15 h. After one cycle of centrifugation on NaI bouyant density gradients
containing ethidium bromide (30 ,tg/ml) in a Ti 50 rotor at 44,000 rpm for 48 h at 20°C, two distinct bands were observed. The lower, denser band containing viral DNA was collected and cleared of ethidium bromide by dialysis against Dowex 5OW-2X beads (Bio-Rad Laboratories, Richmond, Calif.), first with two changes of 50 to 100 volumes of a high-salt buffer (1 M NaCl10 mM Tris-hydrochloride [pH 8.0]-1 mM EDTA) and then against three changes of 50 to 100 volumes of a low-salt buffer (10 mM NaCl10 mM Tris-hydrochloride [pH 8.0]-1 mM EDTA). HSV-2 DNA was subjected to sedimentation assay in a model E analytical ultracentrifuge in 1 M NaCl-0.05 M sodium citrate (pH 7.0) at 25,980 rpm (20°C). It sedimented as a rather broad band with an average sedimentation coefficient, calculated according to Studier (23), of 54.5S (Fig. 1). Viral DNA was used to transfect SHE cells grown in subconfluent monolayers (5 x 105 cells per 60-mm dish). Before exposure to viral DNA, one set of dishes was incubated in HEPES (N-2hydroxyethyl piperazine-N'-2-ethanesulfonic acid)-buffered saline (HeBS) (pH 7.05) (12), supplemented with 0.5 mM MgCl2 and 0.9 mM CaCl2, for 135 min at 37°C; another set was subjected to calcium depletion (13) by incubation in HeBS supplemented with 0.5 mM MgC12 for 135 min at 37°C; and the third set was washed with HeBS. Viral DNA, coprecipitated with calcium phosphate in the presence of salmon sperm DNA (5 to 10,ug per dish), was applied to cells at concentrations ranging from 0.01 to 1 ,ug per dish as described by Graham et al. (12). After incubation at 37°C for 8 h, the monolayers were overlaid with growth medium
404
NOTES
VOL. 30, 1979
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20.1..
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FIG. 1. Sedimentation analysis of HSV-2 (S-1) DNA in a model E analytical ultracentrifuge. The band sedimentation was conducted in 1 MNaCI-0.05 Msodium citrate (pH 7.0) at 25,980 rpm (20°C) following the procedure described by Studier (23).
405
containing 0.2% human immunoglobulin G (Merck, Sharp and Dohme, West Point, Pa.) to neutralize progeny virus (1). Plaques developed within 2 to 3 days in cultures exposed to -0.1 pug of DNA. The specific infectivity, defined as number of plaques per microgram of DNA, ranged between 16 and 21 PFU/pg in cells exposed to viral DNA after treatment with HeBS containing Ca2" and Mg2" (Table 1). It was enhanced 7.5-fold in SHE cells pretreated by calcium depletion. On the other hand, cultures exposed to 0.01 ,ug of DNA did not display plaque formation and continued to propagate. They were replenished with growth medium once every 3 to 4 days for 4 to 5 weeks. Monolayers were serially passaged at split ratios of 1:5 or 1:10. Within five to six passages, the growth rate of the cells declined and the cultures entered crisis. Control cultures "mock-infected" by exposure to medium or to salmon sperm DNA senesced 2 to
TABLE 1. Transfection and transformation of SHE cells with HSV-2 (S-i) DNAa Expt. no.
I
Treatment of cells before DNA
HeBS + Ca2+ + Mg2+ Incubation (370C, 135 min)
HeBS + Mg2e only Incubation (370C, 135 min) Washing with HeBS only Growth medium only
Growth medium only
jug of DNA
No. of
No. of transformed
per dish
plaques per
cultures/total no. of
dish
cultures
1.2 0.12 0.012 1.2 0.12 0.012 1.2 0.12 0.012 0¢
25 2 0
0/2a 0/2a 2/2 0/2a 0/2a 2/2 0/2a 0/2a 2/2 0/4
TNTCb 15 0 TNTC 5 0 0
2 0.14 0/2a 0.014 0 2/2 0 0.002 2/2 0.0005 0 0/2 OC 0 0/4 a 3 x 105 SHE cells were seeded in 60-mm dishes containing growth medium consisting of IBR-modified Dulbecco Eagle reinforced medium (Biolabs, Northbrook, Ill) supplemented with 0.22 g of NaHCO3 per 100 ml, 10% Rehatuin F. S. fetal bovine serum (Reheis Chemicals, Kankakee, Ill.), 100 U of penicillin per ml, 100 pg of streptomycin per ml, and 10 U of mycostatin per ml. After 24 h, replicate cultures were treated as shown above and exposed to HSV-2 (S-1) DNA. The viral DNA was isolated from infected Vero cells by a modification of the method described by Walboomers and Scheggett (6). Before its use to infect cells, viral DNA was mixed with salmon sperm DNA to a final concentration of 10 or 20 pg/ml in HeBS consisting of 8.0 g of NaCl, 0.37 g of KCl, 0.125 g of Na2HPO4. 2H20, 1.0 g of dextrose, and 5.0 g of HEPES (pH 7.05) per liter. CaCl2 was added to a final concentration of 125 mM, and the mixture was kept at 220C for 20 to 30 min. Cell monolayers were exposed to 0.5 ml of the DNA/Ca phosphate mixture for 15 to 20 min at 220C, after which 5 ml of Eagle reinforced medium plus 10% Rehatuin fetal bovine serum was added, and the dishes were incubated for 6 to 8 h at 37°C. The medium was then removed and replaced with Eagle reinforced medium plus 10% Rehatuin fetal bovine serum supplemented with 0.2% pooled human gamma globulin. Dishes that did not contain plaques were fed for 4 to 5 weeks, trypsinized, and transferred to 75-cm2 flasks. Resulting cultures were passaged when confluent at split ratios of 1:5 or 1:10. Cell lines established from cultures that escaped senescence were assayed for transformationassociated characteristics. a Dishes containing plaques were fixed with methanol and stained with Giemsa. b TNTC, Too numerous to count. ' Control untreated dishes or dishes treated only with salmon sperm DNA.
II
406
J. VIROL.
NOTES
3 weeks later. In contrast, cultures surviving exposure to HSV-2 DNA developed refractile colonies which, within 1 to 3 weeks, overgrew the cultures. Cell lines established from cultures exposed to 0.01 ,.g of HSV-2 (S-1) DNA (Table 1, experiment I) manifested morphological alteration and loss of contact inhibition (high saturation density and growth in multilayers) characteristic of transformation. Transformation (Table 1, experiment II) was detected only in cells exposed to a narrow range of viral DNA concentrations (0.01 to 0.002 ,ug per dish), suggesting that it was mediated by viral DNA. Assuming that only 1 out of 5 x 105 SHE cells was transformed by 0.002 ,ug of HSV-2 DNA, the frequency of transformation is estimated at approximately 1 transformant per i05 recipient cells per 0.01 ,&g of DNA. Two cell lines, designated SDNA-177 and SDNA-277, established by exposure of Ca2+-depleted and HeBS-washed cultures, respectively, to 0.01 ,ug of HSV-2 DNA, were examined for in vitro phenotypic alterations associated with the neoplastic state (Table 2). One such characteristic is increased cloning efficiency ([number of colonies x 100]/number of seeded cells) (14) in liquid media when seeded at low (100 to 500 cells per 60-mm dish) cell density (20). Normal SHE cells displayed a low (1 to 4%) cloning efficiency when seeded at a density of 5,000 to 10,000 cells per 100-mm dish, but did not clone when seeded at a density of 100 to 500 cells per 60-mm dish. In contrast, both SDNA-177 and SDNA-277 TABLE 2.
cells manifested high cloning efficiency (8 to 18%) when seeded at low cell density (100 to 500 cells per 60 mm dish), indicating a reduced dependence on cross-feeding for growth. An additional criterion of transformation is the ability of cells to grow in medium supplemented with low serum concentrations (8). Indeed, normnal SHE cells do not grow in 2% serum, whereas SDNA-177 and SDNA-277 cells clone under these conditions at an efficiency of 3.5 and 1.5%, respectively, even when seeded at a low cell density (100 to 500 cells per 60-mm dish). The ability of transformed cells to form colonies in soft agar or agarose is associated with their loss of anchorage dependence (17). Colonyforming efficiency in 0.3% agarose was measured as described (5) and expressed as (number of colonies x 100)/number of seeded cells (105 per 60-mm dish) (5). Normal SHE cells do not grow in agarose, but SDNA-177 and SDNA-277 produce colonies with an efficiency of 2.3 and 5%, respectively. Fibrinolytic activity, a biochemical marker associated with neoplasia, was assayed as described (6). The levels of extracellular activity secreted by SDNA-177 and SDNA-277 cells were three- to sixfold higher than those of normal SHE cells. The ability of transformed cells to induce tumors in appropriate hosts is the most critical assay for the study of the mechanisms of transfonnation as they pertain to carcinogenesis. Accordingly, approximately 2 x 106 SDNA-177
Phenotypic characteristics of SHE cells transforned by HSV-2 DNA SDNA-177 (posttreatment pas-
Morphology
Normal SHE (passage3to5) Fibroblast-like
sage4to35) Mixture of epithelial and fibroblast-like
passage4to30) Predominantly spindlelike
Growth in culture
Monolayers
Multilayers
Multilayers
0
17.6 3.5
8 1.5
0
2.3
5.5
Characteristic
SDNA-277 (posttreatment
Cloning efficiency at low cell densitya
in 10% serum (%) in 2% serum (%)
Colony-forming efficiency' in 0.3% agarose (%) Fibrinolytic activityc
0
Low (4.9 + 0.05)
High (12.9
4)
High (26
2)
~~~+ NDd Tumorigenicity (invasive anaplastic fibrosarcomas) a From 100 to 500 cells were seeded per 60-mm dish. Colonies of >20 cells were counted at day 10. Cloning efficiency = (number of colonies x 100)/number of seeded cells. b 105 cells were seeded per 60-mm dish. Colonies of >20 cells were counted at 2 to 3 weeks. Cloning efficiency = (number of colonies x 100)/105. 'Units of fibrinolytic activity per 2 ml of harvest medium ± standard error. d ND, Not done. _
VOL. 30, 1979
NOTES
407
cells at posttreatment passage 21 or 35 were virus specific, as indicated by the observations inoculated subcutaneously on the ventral side of that preimmune rabbit immunoglobulin G's 1- to 3-day-old newborn Golden Syrian hamsters were nonreactive and that Ra-2 and anti-AG-e (Ela: ENG strain, Engle Laboratories, Farmers- 7S immunoglobulins did not fix complement burg, Ind.). Animals were palpated twice weekly with extracts of normal SHE cells. It should be for tumor development at the site of inoculation. pointed out that anti-AG-e serum stains exfolTumor formation was first detected in three of iated cervical tumor but not normal cells in nine inoculated animals after a latent period of indirect immunofluroescence (L. Aurelian, P. K. 6 to 7 weeks. By 13 weeks, all the inoculated Gupta, J. K. Frost, N. B. Rosenshein, C. C. animals were positive for tumors. Histopatholog- Smith, H. W. Tyrer, J. M. Mantione, and C. D. ically, the tumors were invasive anaplastic fibro- Albright, Acta Cytol., in press). sarcomas. Tumors (-1.5 by 1.5 cm) were excised, HSV DNA, the infectivity of which was artitrypsinized, and grown in tissue culture. The ficially inactivated, has been shown to cause tumor cells, designated SDNA-177T1, SDNA- biochemical (4, 18, 27) or morphological (29) 177T2, etc., were morphologically identical to transformation. However, these transformants bear little resemblance to oncogenic processes the SDNA-177 cells. The microquantitative complement fixation occurring in human disease since (i) they do not assay was used to determine the presence of cause tumors in animals and (ii) they were esHSV antigens in extracts of transformed cells tablished with viral DNA, the infectivity of prepared as previously described (2). The results which was artificially inactivated. of these studies (Table 3) may be summarized The salient feature of our studies is the obseras follows. (i) Human sera containing neutralizvation that cells exposed to purified, not intening antibody to HSV-2 (no. 119 and 124) or to tionally inactivated HSV-2 (S-1) DNA display both HSV-2 and HSV-1 (no. 141 and 183) fixed properties characteristic of transformation, concomplement with extracts from transformed tain HSV-specific antigens, and cause invasive cells. The virus specificity of the reaction was tumors when inoculated into appropriate hosts. evidenced by the observations that a human Consistent with the presence of virus-specific serum (no. 317) lacking antibody to either virus antigens in SDNA-177 cells (Table 3), sera from type was nonreactive, and that normal SHE cells tumor-bearing hamsters contain neutralizing andid not fix complement with any of the human tibody to HSV-2 as determined by plaque-resera. (ii) Extracts of SDNA-177 cells also fixed duction neutralization assay (1), and SDNAcomplement with immunoglobulin G from anti- 177T1 cells fix complement with antisera to sera prepared against total viral antigens (Ra-2) HSV-2 antigens (unpublished data). and against a purified type-common antigenic Transformation of SHE cells by high dilutions fraction designated AG-e, and consisting of two of HSV-2 (S-1) DNA is amenable to three interproteins that comigrate with virion structural pretations: (i) transforming molecules are nonproteins VP 5 and 6 (21). The reactions were infectious (aberrant in size and/or complexity) TABLE 3. Detection of HSV antigens in HSV-2 (S-i) DNA-transformed and normal SHE cells by microquantitative complement fixationa Serum or immunoglobulin
Neutralizing antibody to
% Complement fixed by extract (20 itg of protein) of:
HSV-1
HSV-2
SHE
SDNA-177
SDNA-277
+ + -
+ + + + -
9.9 5.4 1.8 0 0
38.7 32.9 44.1 14.5 0
33.8 27.7 28.8 0 0
Human Sera 141 183 119 124 317
Immunoglobulin G of rabbit sera to: Total virion antigens (Ra-2)
+ + 0 16.1 0 0 0 0 AG-eb 0 29.7 10.2 Preimmune 0 0 0 a"Immunoglobulins were fractionated by sucrose gradient centrifugation. Neutralizing potential for HSV-2 and/or HSV-1 was determined by the multiplicity analysis plaque reduction assay (1). The microquantitative complement fixation assay was used with optimal concentrations of antisera or immunoglobulins as previously described (2). Reaction was considered positive if more than 10% of the complement was fixed (2). b AG-e is the 0.4 M phosphate eluate of a calcium phosphate column of total soluble HSV-2 antigens (21).
Preimmune
408 NOTES and do not become encapsidated into virions; (ii) transforming DNA molecules are identical to those 55S molecules that become encapsidated (15) but their expresion is modified in recipient cells by host- and/or virus-determined regulatory mechanisms; or (iii) transforming molecules are fragments of viral DNA generated in vitro by nonspecific shearing forces or produced in recipient cells by the action of cellular nucleases (9). Presently available data appear to argue against the first interpretation but cannot distinguish between the other two interpretations with any degree of certainty. Thus, the majority of viral DNA molecules synthesized in infected nuclei do not become encapsidated and remain associated with the nuclei throughout infection (28). However, these molecules differ from mature virus DNA in their sedimentation characteristics (28), whereas S-1 DNA sediments as a single band with an s20,w of 54.5S (Fig. 1), a value virtually identical to that determined by other investigators for DNA extracted from purified HSV-2 virions (15). Furthermore, the specific infectivity of S-1 viral DNA is relatively low (12), although it does not differ from that reported by Stow and Wilkie (22) for HSV-1 (Glasgow strain 17) DNA. The observation that calcium depletion significantly enhances the specific infectivity of S-1 DNA (Table 1) suggests that a low specific infectivity is not an intrinsic property of this DNA. Indeed, the specific infectivity of DNA extracted from sucrose-gradientpurified S-1 virions is approximately 100-fold higher than that of the DNA extracted from S1-infected cells. Nevertheless, virion-extracted DNA also transforms SHE cells at dilutions (10-4 ,ug of DNA per dish) lacking infectivity, and the transformants express HSV-2 antigens and cause tumors in inoculated hamsters (manuscript in preparation). Finally, it should be pointed out that hamster cells transformed with UV-irradiated HSV-2 contain only a fraction (8 to 32%) of the viral DNA sequences (10), indicating that transformation may be mediated by DNA fragments. Indeed, Camacho and Spear (7) have reported that a fragment of HSV-1 DNA transforms cells in vitro although these transformants do not cause tumors in animals. Farber (9) has shown that 56S viral DNA is degraded in recipient cells to a 20S predominant fragment, suggesting that transforming fragments may also be generated in vivo. Taken as a whole, these observations suggest that the bulk of the S-1 viral DNA molecules (of cellular or virion extraction) are not capable of initiating infection, possibly due to fragmentation occurring in vitro or within the recipient cells. Such molecules retain transforming poten-
J. VIROL.
tial and become the predominant species upon high dilution of the DNA preparation. The possibility cannot be excluded that the neoplastic potential of the SDNA-177 cells is due to the source (cervix cancer) of the S-1 virus. The HSV-DNA-SHE cell system described in this report is a particularly suitable model for the study of the viral genes and gene products involved in neoplastic transfornation. Studies designed to inquire into this question and to examine the relationship between HSV-induced cellular alterations in vitro and tumorigenicity in vivo are now in progress in our laboratory. This work was supported by Public Health Service grant Ca-16043 from the National Cancer Institute. R.J.J. was supported by a National Research Service Award from the National Cancer Institute. We acknowledge helpful discussions with Mark Manak. We also thank M. F. Smith for help with the complement fixation assay, B. Crawford for work on fibrinolytic activity, and L. Trpis for technical assistance.
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21. Smith, C. C., R. B. Bell, and L Aurelian. 1978. Immune response to antigen (AG-e) in patients with HSV-2 asociated diseases, p. 528. Proceedings of the 4th International Virology Congress. Centre for Agricultural Publishing and Documentation, Wageningen, The Netherlands. 22. Stow, N. D., and N. M. Wilkie. 1976. An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA. J. Gen. Virol. 33: 447458. 23. Studier, W. F. 1965. Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11:373-390. 24. Symposium on Immunological Control of Virus-Associated Tumors in Man: Prospects and Problems. 1976. Cancer Res. 36:783-859. 25. Takahashi, M., and K. Yamanishi. 1974. Transformation of hamster embryo and human embryo cells by temperature sensitive mutants of herpes simplex virus type 2. Virology 61:306-311. 26. Walboomers, J. M. M., and J. T. Schegget. 1976. A new method for the isolation of herpes simplex virus type 2 DNA. Virology 74:256-258. 27. Wigler, M., S. Silverstein, L S. Lee, A. Pellicer, Y. C. Cheng, and R. Axel. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223-232. 28. Wilkie, N. M. 1973. The synthesis and substructure of herpesvirus DNA: the distribution of alkali-labile single strand interruptions in HSV-1 DNA. J. Gen. Virol. 21: 453-467. 29. Wilkde, N. M., J. B. Clements, J. C. M. MacNab, and J. H. Subak-Sharpe. 1974. The structure and biological properties of herpes simplex virus DNA. Cold Spring Harbor Symp. Quant. Biol. 39:657-666.
