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Malignant transformation of rat embryo cells by a herpesvirus isolated from L2C guinea pig leukemia
VIROLOGY
82,
100-110 (1977)
Malignant
Transformation of Rat Embryo Cells by a Herpesvirus Isolated from L,C Guinea Pig Leukemia JOHNG SIK RHIM
Department
of Cancer Research,
Microbiological
Associates,
Inc., Bethesda,
Maryland
20016
Accepted May 24,1977 A guinea pig herpesvirus (GPHV) was isolated from mink lung cells after cocultivation with L$2 leukemic cells. The virus was found to have a relatively narrow host range. The isolate was serologically related to the GPHV isolated by G. D. Hsiung and L. S. Kaplow [(1969). J. Viral. 3, 355-3571 but not to the guinea pig cytomegalovirus as determined by neutralization tests. Complement-fixing antigen for human herpes simplex type 1 virus was detected in cell suspensions prepared from GPHV-infected mink cells. Rat embryo cells infected with a GPHV mink isolate showed morphological transformation. The transformed cells produced neither infectious virus nor virusspecific antigens detectable by either complement fixation or fluorescent antibody tests Virus particles were not detected in the transformed cells. No infectious GPHV could be rescued from the transformed cells by cocultivation with guinea pig cells. The morphologically transformed cells formed large cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities while the normal rat embryo cells did not. The transformed cells produced tumors when transplanted subcutaneously into newborn rata. Herpesvirus complement-fixing antigen was detected in rat tumor cells but infectious virus was not recovered from rat tumor cells. INTRODUCTION
L,C leukemia is an acute lymphocytic leukemia of strain 2 guinea pigs (Congdon and Lorenz, 1954). It is the only leukemia available for study in this species and the only experimental leukemia derived from B lymphocytes (Shevach et al., 1972)available for study in any animal species. The L,C leukemia has been widely employed for a variety of virological (Opler, 1967; Hsiung and Kaplow, 1969; Nayak, 1971; Hsiung, 1972; Nayak and Murray, 1973), chemotherapeutic (Pearson et al., 19751, and immunotherapeutic (Ellman and Green, 1971; Katz et al., 1972) studies. Interest in the virological aspects of guinea pig leukemia stems from the unique nature of this system. The etiology of guinea pig leukemia, like human leukemia, remains to be established, although a viral origin has been proposed for both. Two different types of viruses have been found in leukemia guinea pig cells; retravirus-type RNA virus .(Opler, 1967;
Hsiung, 1972; Nayak and Murray, 19731 and herpes-type DNA virus (Nayak, 1971). Retravirus was found in tissue-cultured cells derived from leukemic or normal guinea pigs but only after treatment with bromodeoxyuridine (BrdU) (Hsiung, 1972; Rhim et al., 1973; Nayak and Murray, 1973). However, attempts to grow these BrdU-induced viruses in cell cultures have not been successful (Rhim et al., 1974). Therefore, we have attempted to isolate viruses associated with leukemic cells by cocultivation with various mammalian cells. During the investigation, a guinea pig herpesvirus (GPHV) was isolated from mink lung cells after cocultivation with BZ-L,C leukemic cells but a guinea pig retravirus was not isolated by this method. To determine if a relationship exists between the GPHV and guinea pig leukemia, the oncogenic potential of the GPHV was further investigated. Fong and Hsiung (1973) reported that GPHV is capable of transforming hamster embryo cells.
100 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
HERPESVIRUS
TRANSFORMATION
Viral antigen and infectious virus were detected in the transformed cells. Tumors were produced when one of the tramsformed hamster cell lines (LK51-19-6) was inoculated into hamsters. However infectious GPHV was not isolated from the tumor cells (Michaliski et al., 1976). This communication describes some biological characteristics of the GPHV which was isolated from mink lung cells after cocultivation with L,C! leukemic cells and reports the in vitro malignant transformation of rat embryo cells by this virus. MATERIALS
AND
METHODS
Fresh circulating lymphoblasts from BZ-L,C leukemic guinea pigs were obtained from Dr. Ira Green, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, Maryland. The BZ-L,C line was previously maintained by serial cell passage in syngeneic animals by Dr. B. Zbar, National Cancer Institute, NIH, Bethesda, Maryland. Tissue culture and media. Cells were grown and maintained in Eagle’s minimum essential medium (EMEM) with 10% fetal bovine serum, (FBS), 2 mM glutamine, and 100 units of penicillin and 100 pg of streptomycin per ml (EMEM + 10% FBS). Primary guinea pig kidney (GPK) cells were prepared from inbred strain 13 guinea pigs (Animal Production Units of NIH, Bethesda, Maryland) by standard trypsinization procedures. The mink lung [CCL-64, Mv 1 Lu (NBL-7)l line was obtained from the American Type Culture Rockville , Maryland. A Collection, Fischer rat embryo (RE) cell line (F-111) was originally established by Freeman et al., (1970). Virus assays. The replication of GPHV in cultures was determined by: (1) examination for the presence of cytopathic effect (CPE); (2) assay for complement-fixing (CF) antigen reactive with GPHV and human herpes simplex virus type 1 (HHSV-1) antiserum; and (3) examination by electron microscopy for the presence of virus particles. Virus infectivity for GPHV was assayed in GPK and mink lung cells. The L&
guinea
pig leukemia.
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infectivity titers were expressed at TCD,, per 0.1 ml. CF test. Cell pack preparations for CF testing were made as previously described (Rhim and Huebner, 1970). CF tests were performed by the microtiter technique described for tumor antigen studies (Huebner et al., 1963). Titers were recorded as reciprocals of the highest dilution giving 3 + to 4+ fixation of 1.8 units of complement. Zmmunofhorescent staining. Fluorescent antibody (FA) tests were performed by Dr. K. Takemoto, NIH, Bethesda, Maryland by the indirect procedure using fluorescein-conjugated goat anti-rabbit globulin and HHSV-1 rabbit antiserum or GPHV rabbit antiserum. Anti-G-al sera. HHVS-1 rabbit antiserum was obtained from Microbiological Associates, Inc., Bethesda, Maryland. GPHV rabbit antiserum and guinea pig cytomegalovirus (GPCMV) rabbit antiserum were gifts from Dr. G. D. Hsiung, Veterans Administration Hospital, West Haven, Connecticut. Neutralization test. Equal amounts of virus and antiserum were combined and the mixture was held at 37” for 30 min. The infectivity titer of the virus-serum mixture was determined on GPK cells. Transformation studies. One-day-old petri dish (60 mm) cultures of RE (F-111) (passage 61) cells were infected with GPHV at an input multiplicity of 10. The infected cultures were incubated for 2 hr at 37”, washed twice with medium, and refed with fresh medium. The cultures were incubated at 37” under a 5% atmosphere with medium changes twice weekly. Twentyone days later, replicate cultures were stained with Giemsa, foci were counted, and transformation rates were then determined. Some transformed foci were isolated by the cloning cylinder technique and propagated as clonal lines. Cell aggregation assay. Formation of cellular aggregates by normal and transformed cells was tested using the method described by Steuer et al. (1976) and Steuer and Ting (1976). Freshly trypsinized viable cells (2 x 105)were seeded into 35mm plastic dishes containing a 2-ml agar base
102
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layer (0.9% Difco agar in growth medium with 20% FBS). The dishes were incubated undisturbed at 37” under 5% CO, in air. Viable cell counts were performed daily for 4 days. Soft agar assay. Cells were suspended in 0.36% agar medium (EMEM containing 20% FBS and antibiotics). This suspension was layered onto a 0.9% agar base layer at concentrations of lo3 to lo4 cells per 35-mm dish. Dishes were incubated in a humidified atmosphere (5% CO,) at 37”. After 2 weeks, colonies greater than 0.125 mm in diameter were counted. Results are expressed as percentage plating efficiency (%PE = number of colonies x lOO/number cells plated). Rescue of virus. A genome-rescue experiment was done by cocultivation of equal numbers of GPHV-transformed RE cells and normal GPK cells. Supernatants from these cultures taken from the 14th to 21st day postinfection were assayed for GPHV on GPK and mink lung cells. Oncogenicity
of transformed
cells. New-
born Fischer rats were inoculated subcutaneously with 1 x lo6 freshly trypsinized cells in order to determine their tumorigenicity. RESULTS
Isolation
of the GPHV
Leukemic cells (1 x lo6 cells/flask) were cocultivated with an equal number of mink lung cells in EMEM + 10% FBS. The medium was changed once a week. In such cultures, most of the leukemic guinea pig cells remained in suspension while the mink lung cells grew in monolayer. However, all of the leukemic cells rounded up and died after 4 weeks in culture. It was at this time that visible CPE (Figs. 1A and B) became apparent in mink lung cells. Herpesvirus particles were also seen using electron microscopy (Fig. 2). No guinea pig retraviruses were isolated. Characterization
of the GPHV
The GPHV mink isolate from BZ-L,C! leukemic cells was further characterized (Table 1). Electron microscopy of the infected mink lung cells revealed morphological characteristics typical of herpestype virus (Fig. 2). There were numerous
S. RHIM
extracellular enveloped particles and many intranuclear nucleocapsids. There were also a few examples of enveloped particles in cytoplasmic vesicles and evidence of envelopment by the cytoplasmic membranes as the particles budded into the vesicles. The virus could be passed serially in mink lung cultures, with CPE developing 3-5 days after infection. The CPE was characterized by round, refractile, and loose cells often clumping together (Figs. 1A and B). A similar CPE was noted in the GPHV-infected GPK cells. Vero and cat embryo cells were highly sensitive to GPHV infection, rat and SIRC cells were moderately sensitive, and human tumor cells and mouse embryo cells were only sightly sensitive to GPHV infection, as indicated by CPE. In mink cells, the maximum titer was lo4 infectious units as determined by endpoint dilution. However, comparative titration of virus in mink and GPK cells showed that mink cells were loo-fold less sensitive than GPK cells. GPHV was sensitive to ether treatment. Four logs of GPHV were inactivated following incubation with ether for 20 hr at 4”.
The virus infectivity titer was reduced lOOO-foldby the antiserum against GPHV isolated by Hsiung and Kaplow (1969) but was not reduced by the antiserum against guinea pig cytomegalovirus. CF antigen for HHSV-1 (MacIntyre strain) was detected in cell suspensions prepared from GPHV-infected mink lung cells. Thus the GPHV mink isolate is serologically related to the GPHV isolated by Hsiung and Kaplow (1969) as determined by neutralization tests and also to human herpes simplex virus as determined by the complement fixation test. In Vitro Rat Embryo by GPHV
Cell Transformation
Rat embryo (F-111) cells infected with GPHV showed a moderate CPE in 5-7 days (Fig. 1C) as compared to uninfected RE cells (Fig. 1D). However, the CPE in infected RE cells disappeared within 1 week, and morphologically altered foci began to develop 14-17 days after infection.
HERPESVIRUS
TRANSFORMATION
OF RAT
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103
FIG. 1. Cytopathic effect (CPE) induced by guinea pig herpesvirus (GPHV) in mink lung and rat embryo cells (unstained, 60 x ). (A) Mink lung cells 5 days after GPHV infection. (B) Mink lung cells 10 days after GPHV infection. (C) Rat embryo cells 5 days after GPHV infection. (D) Uninfected rat embryo cells.
The transformed foci were characterized by loss of contact inhibition, resulting in multilayered and irregular cell growth (Figs. 3A and B), and could be readily
distinguished from normal, nontransformed cells (Fig. 30. The transformed foci, however, did not all have the same gross morphological appearance. The
104
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S. RHIM
FIG. 2. Virus particles in section of mink lung cells infected with GPHV. Note particles with dense and coreless centers enveloped by an inner membrane scattered in the nucleolus. Particles enveloped by a double membrane are also seen in the vesicle (30,000~).
transformation efficiency of GPHV in RE cells determined 21 days after infection was 0.01%. Focal areas of transformed cells were isolated by the cloning cylinder technique and propagated. A number of focus-derived clonal lines were established. Two clonal lines designated as GPHV RE #6 and GPHV RE #7 were further characterized. Properties of GPHV-Transformed Embryo Cell Lines
Rat
Transformed cells in tissue culture generally can be distinguished from their normal counterparts by quantitative differences in growth properties such as contact inhibition, saturation density, soft agar colony-forming efficiency, and cell aggregate properties (Table 2). Saturation densities of transformed clonal lines #6 and #7 were 17.3 x lo5 and 12.4 x lo5 cells/cm2, respectively, which is three- to fourfold more than the control, untransformed
line. Normal rat embryo cells were unable to grow in soft agar while the transformed clonal lines demonstrated high plating efficiencies in soft agar. When suspended above an agar base, the transformed cells formed larger cell aggregates than those formed by normal RE cells (Fig. 4). Viable cell counts of trypsinized aggregates on 4 consecutive days indicated that control, untransformed cells underwent a significant decline in a number of viable cells whereas GPHV-transformed RE cells showed growth in the aggregate form (Fig. 5). The transformed cells, as well as untransformed, control RE cells were tested for their ability to produce tumors in newborn syngeneic animals. All newborn Fishcer rats inoculated subcutaneously with 1 x lo6 transformed cells developed tumors at the site of inoculation within 4 weeks (Table 2). No tumors developed over a period of 10 weeks in newborn Fischer
HERPESVIRUS TABLE
TRANSFORMATION
1
PROPERTIES OF GUINEA PIG HERPESVIRUS (GPHV) ISOLATED FROM MINK LUNG CELJ..SAFTER COCULTIVATION WITH L,C GUINEA PIG LYMPHOBLASTS Morphology
A herpes-type virus: numerous extracellular enveloped particles and many intracellular nucleocapsids
Cytopathic
effect
Round, refractile, and loose cells which often clump together
Infectivity
titer”
104.” in mink cells 1W.Oin guinea pig kidney cells
Sensitive etherb
to
Yes
Host ranges
CF titer HHSV-1 gend
CPE’ + + + +: guinea pig, Vero, mink, and cat cells CPE ++: rat and SIRC cells CPE + or -: human osteosarcoma, human RD, NIH Swiss, and BALB/c mouse cells of anti-
13
-
D TCDJO.1 ml. b Four logs of GPHV were inactivated following incubation with ether for 20 hr at 40”. c CPE (cytopathic effects): + = ll-25%; + + = 2650%; + + + + = >75% of cells affected. d HHSV-1 = human herpes simplex virus type 1.
rats inoculated with normal, untransformed RE cells. The tumors were progressive and transplantable. Cells established from the tumors resembled the transformed cell lines. To detect the presence of GPHV- or HHSV-specific antigen in the transformed cells or the tumor cells, CF and FA tests were used. Repeated attempts to demonstrate GPHV- or HHSV-CF antigens in the transformed cells were unsuccessful. The transformed cells were also negative for intracytoplasmic antigen when tested by FA staining with anti-GPHV serum and HHSV-1 antiserum. The rat tumor cells were negative for GPHV or HHSV antigens by the FA test; however, they were positive for CF antigens for HHSV-1 (CF titer, 1:16).
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Neither herpesvirus nor type C virus particles were found in the transformed cells or in the tumor cells when examined by electron microscopy. In an attempt to rescue infectious GPHV, transformed lines and tumor cell lines at various passage levels were cocultivated with guinea pig cells. Supernatants from these cultures taken at 14 or 21 days were inoculated into fresh guinea pig and mink cells and examined for the presence of CPE. All rescue experiments were so far negative, for GPHV. The transformed cells and the. rat tumor cells were also examined for the presence of supernatant viral RNA-dependent DNA polymerase, which is a highly sensitive method for the detection of replicating type C RNA virus. The results were negative. DISCUSSION
The validity of a general strategy for in vitro isolation of type C virus involving cocultivation of suspected virus-producing tissues with cells from various species to maximize the probability of finding a permissive host has been previously demonstrated (Benveniste et al., 1974). Thus the cocultivation method has been proposed as a general approach to isolate infectious type C viruses from tissues. We have therefore attempted to isolate and grow guinea pig retraviruses in mink lung cells after cocultivation with L$ leukemic cells. The mink lung cell line Mv 1 Lu (CCL-641 was selected for cocultivation since recent reports have demonstrated the in vitro susceptibility of mink lung cells to type C sarcoma viruses (Henderson et al., 1974) as well as to the type B mouse mammary tumor virus (Lasfargues et al., 1976). As a result, GPHV, capable of transforming rat embryo cells, was isolated from mink lung cells after cocultivation with L,C! guinea pig leukemic lymphoblasts. However, guinea pig retraviruses were not isolated in mink cells by the cocultivation method. A GPHV was originally isolated from spontaneously degenerated normal and leukemic strain 2 guinea pig kidney cultures (Hsiung and Kaplow, 1969) and from leukemic lymphoblasts of strain 2 guinea
106
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S. RHIM
FIG. 3. (A) Rat embryo cells showing foci of GPHV-transformed cells 3 weeks after GPHV infection (Giemsa stain). (B) A transformed focus of rat embryo cells 3 weeks after GPHV infection (Giemsa stain, 56x). (C) Uninfected rat embryo cells stained with Giemsa (56x).
pigs (Nayak, 1971). Morphological and biochemical characteristics of the GPHV are very similar to those of EB virus found in Burkitt’s lymphoma but are different from guinea pig CMV. Until now, no antigenic
relationship has been reported between GPHV and other herpesviruses including human CMV, HHSV, EB virus, equine herpesviruses types 1 and 2, Marek’s disease of chicken, and Luke virus of frog
HERPESVIRUS
TRANSFORMATION TABLE
OF RAT
Morphology Saturation density” (x 105/cm2) Cell aggregates* Size Viability of cells (x 105) Plating efficiency (%l in soft agar CF titers of HHSV-1 antigene Virus particles Rescuable infectious GPHV Tumorigenicity in rat&
107
2
PROPERTIES OF GUINEA PIG HERPESVIRUS (GPHV)-TRANSFORMED Properties
CELLS
GPHV-RE cells
#6
RAT EMBRYO (RE) CELLS LINES GPHV-RE cells
#7
RE cells
Transformed 12.2
Transformed 9.4
Normal 3.1
Large 4.0 17.3 <2 Negative Negative Positive 5/5e
Large 2.6 12.4 <2 Negative Negative Positive 515
Small 0.4
n Maximum number of cells obtained after initial plating of 5 x lo3 cells/cm2. * Size and viability of cell aggregates were determined on Day 3 after plating initially with the use of an agar static assay (Steuer et al., 1976). c HHSV = human herpes simplex virus type 1. into each newborn Fischer rat. d Cells (1 x IO') inoculated subcutaneously e Number of tumors formed/number of rats inoculated.
(Hsiung et al., 1971). However, a serological relationship between GPHV and human herpesvirus has been demonstrated in this study by complement fixation. But since the existence of herpes group-specific antigen is still open to question, the antigenie relations should be further studied and carefully evaluated. The host range of the GPHV was found to be rather narrow as shown in this study as well as those by others (Hsiung and Kaplow, 1969; Hsiung et al., 1971; Nayak, 1971; Michalski and Hsiung, 1975)whereas human herpesvirus has a wide host range. GPHV-transformed rat cells had the following properties generally associated with viral transformation: (a) altered morphology; (b) increased growth rate; (c) colony formation in soft agar medium; (d) formation of large cell aggregates and growth in this aggregate form above an agar base; (e) tumorigenicity in syngeneic animals. The transformed cells produced neither viral antigen nor a rescuable infectious virus; however, the rat tumor cells were positive for HHSV-type 1 CF antigen while negative for infectious virus. Our studies are similar to those reported by several herpesviruses in hamster (Duff and Rapp, 1971 and 1973; Albrecht and Rapp, 19; Fong and Hsiung, 1973) and rat embryo cells (MacNab, 1974). With one
2 x lo5 cells per plate
exception, as reported by Fong and Hsiung (1973) in the GPHV-induced hamster cell transformation, infectious virus was not recovered from the transformed cells or the tumor cells. Since inactivated virus was used in most of the transformation experiments, it was assumed that complete infectious herpesvirus was not present. Like GPHV-induced hamster cell transformation (Fong and Hisung, 1973), infectious GPHV was used in our rat cell transformation experiments. It thus seems clear that GPHV is able to transform rodent cells without the need for uv irradiation of the virus. It has recently been reported that infectious GPHV was found only from the transformed hamster cells in early passage levels but not from the hamster tumor cells (Michalski et al., 1976). However, in contrast to the GPHV hamster cell transformation system, infectious virus was not recovered from the transformed rat cells or from the rat tumor cells. Herpesvirus antigen was detected in the rat tumor cells even though the same antigen was not detected in the rat transformed cells. Thus, the relationship between the GPHV and the rat tumors was established. The development of transplantable GPHV-induced rat tumors with this in uitro transformation
system may allow the
108
JOHNG
S. RHIM
FIG. 4. Cell agg regates of rat embryo cells transformed by GPHV. Cells were all owei d to aggregate undistl orbed for 3 days in liquid growth medium above an agar base layer (56) 0. (A) Aggregates of uninfec :ted rat embl ryo cells. (B) Aggregates of GPHV-transformed #6 rat embryo cz!llS (Cl Aggregates of GPHV. .transformed #‘7 rat embryo cells.
HERPESVIRUS
TRANSFORMATION
I
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CELLS
109
DUFF, R., and RAPP, F. (1971). Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultra-violet irradiated herpes simplex virus type 2. J. Virol. 8, 469-477. DUFF, R., and RAPP, F. (1973). Oncogenic transformation of hamster embryo cells after exposure to inactivated herpes simplex virus type 1. J. Viral. 12, 207-209. ELLMAN, L., and GREEN, I. (1971). L,C guinea pig leukemia: Immunoprotection and immunotherapy. Cancer 28, 647-654. FONG, C. K., and HSIUNG, G. D. (1973). In vitro transformation of hamster embryo cells by a RE, control I I I guinea pig herpes-like virus. Proc. Sot. Exp. Biol. 1 2 3 4 Med. 144, 974-978. DAYS FREEMAN, A. E., PRICE, P. J., IGEL, H. J., YOUNG, J. FIG. 5. Short-term growth curves of GPHVC., MARYAK, J. M., and HUEBNER, R. J. (1970). transformed rat embryo cells. Viable cell counts Morphological transformation of rat embryo cells were performed on trypsinized aggregates for 4 coninduced by diethylnitrosamine and murine leukesecutive days (2 x loj viable cells were plated per mia virus. J. Nat. Cancer Inst. 44, 65-78. dish). (0-O) GPHV-transformed #6 cells. HENDERSON, I., LIEBER, M. M., and TODARO, G. J. (0-O) GPHV-transformed #7 cells. (A-A) (1974). Mink cell line Mv 1 Lu (CCL-64). Focus Uninfected rat embryo, control cells. formation and the generation of “nonproducer” transformed cell lines with murine and feline saridentification of a specific T-antigen. Anticoma viruses. Virology 60, 282-287. serum to this antigen should be valuable HSIUNG, G. D. (1972). Activation of guinea pig Ctype virus in cultured spleen cells by 5-bromo-2’for further study of GPHV in other transdeoxyuridine. J. Nat. Cancer Inst. 49, 567-570. formation systems. Transplantable tumors HSIUNG, G. D., and KAPLOW, L. S. (1969). Herpeswould also permit studies on virus-specific like virus isolated from spontaneously degenertransplantation antigens induced by ated tissue culture derived from leukemia-suscepGPHV. tible guinea pigs. J. Virol. 3, 355-357. ACKNOWLEDGMENTS HSIUNG, G. D., KAPLOW, L. S., and Booss, J. (1971). Herpes virus infection of guinea pigs, I. Isolation, This investigation was supported by Contract characterization and pathogenicity. Amer. J. EpiNOI-CP-43519A from the Viral Cancer Program of demiol. 93, 298-307. the National Cancer Institute, NIH, Bethesda, MarHUEBNER, R. J., ROWE, W. P., TURNER, H. C., and yland. I thank Dr. G. D. Hsiung, School of Medicine, LANE, W. T. (1963). Specific adenovirus compleYale University, New Haven, Connecticut, for supment-fixing antigens in virus-free hamster and plying the GPHV and GPCMV antiserum; Dr. M. L. rat tumors. Proc. Nat. Acad. Sci. USA 50, 379Vernon, Microbiological Associates, Inc., Bethesda, 389. Maryland, for electron microscopic examination of KATZ, D. H., ELLMAN, L. PAUL, W. E., GREEN, I., cells; Dr. K. Takemoto, National Institute of Aland BENECERRAF, B. (1972). Resistance of guinea lergy and Infectious Diseases, NIH, Bethesda, Marpigs to leukemia following transfer of immunoyland, for fluorescent antibody assay; and Mr. Paul competent allogenic lymphoid cells. Cancer Res. Hill, National Cancer Institute, NIH, Bethesda, 32, 133-140. Maryland, for the CF assay. LASFARGUES, E. Y., VAIDYA, A. B., LASFARGUES, J. REFERENCES C., and MOORE, D. H. (1976). In vitro susceptibility of mink lung cells to the mouse mammary ALBRECHT, T., and RAPP, F. (1973). Malignant tumor virus. J. Nat. Cancer Inst. 57, 447-449. transformation of hamster embryo fibroblasts folMACNAB, J. C. (1974). Transformation of rat embryo lowing exposure to ultraviolet-irradiated human cells by temperature-sensitive mutants of herpes cytomegalovirus. Virology 55, 53-61. simplex virus. J. Gen. Virol. 24, 143-153. BENVENISTE, R. E., LIEBER, M. M., LIVINGSTON, D. MICHAL~KI, F. J., FONG, C. K., HSIUNG, G. D., and M., SHERR, C. J., TODARO, G. J., and KALTER, S. SCHNEIDER, R. D. (1976). Induction of tumors by a S. (1974). Infectious C-type virus isolated from a guinea pig herpesvirus-transformed hamster cell baboon placenta. Nutare (London) 248, 17-20. line. J. Nat. Cancer Inst. 56, 1165-1170. CONGDON, C. C., and LORENZ, E. (1954). Leukemia MICHAWKI, F., and HSIUNG, G. D. (1975). Malignant in guinea pigs. Amer. J. Pathol. 30, 337-359.
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transformation of hamster cells following infection with a bovine herpes virus (Infectious bovine rhinotracheitis virus). Proc. Sot. Exp. Biol. Med. 148, 891-896. NAYAK, D. P. (1971). Isolation and characterization of a herpesvirus from leukemic guinea pigs. J. Viral. 8, 579-588. NAYAK, D. P., and MURRAY, P. R. (1973). Induction of type C virus in cultured guinea pig cells. J. Viral. 12, 1’77-187. OPLER, S. R. (1967). Observations of a new virus associated with guinea pig leukemia: Preliminary note. J. Nut. Cancer Inst. 38, 797-800. PEARSON, J. W., PERK, K., CHIRICOS, M. A., and TORGERSEN, J. A. (1975). Drug therapy against a transplantable guinea pig leukemia. Cancer Res. 35, 1093-1098. RHIM, J. S., DUH, F. G., CHO, H. Y., Wuu, K. D., and VERNON, M. L. (1973). Activation by 5-bromoZ’-deoxyuridine of particles resembling guinea pig
S. RHIM leukemia virus from guinea-pig nonproducer cells. J. Nat. Cancer Inst. 51, 1327-1331. RHIM, J. S., and HUEBNER, R. J. (1970). Rat embryo cells in complement-fixation test for murine leukemia virus. Nature (London) 226, 646-647. RHIM, J. S., Wuu, K. D., Ro, H. S., VERNON, M. L., and HUEBNER, R. J. (1974). Induction of guinea pig leukemia-like virus from cultured guinea pig cells. Proc. Sot. Exp. Biol. Med. 147, 323-330. SHEVACH, E. M., ELLMAN, L., DAVIE, J. M., and GREEN, I. (19721. L&! guinea pig lymphatic leukemia: A “B” cell leukemia. Blood 39, 1-12. STEUER, A. F., RHIM, J. S., and TING, R. C. (1976). Aggregate properties of human sarcoma cells: Correlation with growth in soft agar and tumorigenicity. Proc. Amer. Assoc. CancerRes. 17,34. STEUER, A. F., and TING, R. C. (1976). Formation of larger cell aggregates by transformed cells: An in vitro index of cell transformation. J. Nat. Cancer Inst. 56, 1279-1280.
82,
100-110 (1977)
Malignant
Transformation of Rat Embryo Cells by a Herpesvirus Isolated from L,C Guinea Pig Leukemia JOHNG SIK RHIM
Department
of Cancer Research,
Microbiological
Associates,
Inc., Bethesda,
Maryland
20016
Accepted May 24,1977 A guinea pig herpesvirus (GPHV) was isolated from mink lung cells after cocultivation with L$2 leukemic cells. The virus was found to have a relatively narrow host range. The isolate was serologically related to the GPHV isolated by G. D. Hsiung and L. S. Kaplow [(1969). J. Viral. 3, 355-3571 but not to the guinea pig cytomegalovirus as determined by neutralization tests. Complement-fixing antigen for human herpes simplex type 1 virus was detected in cell suspensions prepared from GPHV-infected mink cells. Rat embryo cells infected with a GPHV mink isolate showed morphological transformation. The transformed cells produced neither infectious virus nor virusspecific antigens detectable by either complement fixation or fluorescent antibody tests Virus particles were not detected in the transformed cells. No infectious GPHV could be rescued from the transformed cells by cocultivation with guinea pig cells. The morphologically transformed cells formed large cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities while the normal rat embryo cells did not. The transformed cells produced tumors when transplanted subcutaneously into newborn rata. Herpesvirus complement-fixing antigen was detected in rat tumor cells but infectious virus was not recovered from rat tumor cells. INTRODUCTION
L,C leukemia is an acute lymphocytic leukemia of strain 2 guinea pigs (Congdon and Lorenz, 1954). It is the only leukemia available for study in this species and the only experimental leukemia derived from B lymphocytes (Shevach et al., 1972)available for study in any animal species. The L,C leukemia has been widely employed for a variety of virological (Opler, 1967; Hsiung and Kaplow, 1969; Nayak, 1971; Hsiung, 1972; Nayak and Murray, 1973), chemotherapeutic (Pearson et al., 19751, and immunotherapeutic (Ellman and Green, 1971; Katz et al., 1972) studies. Interest in the virological aspects of guinea pig leukemia stems from the unique nature of this system. The etiology of guinea pig leukemia, like human leukemia, remains to be established, although a viral origin has been proposed for both. Two different types of viruses have been found in leukemia guinea pig cells; retravirus-type RNA virus .(Opler, 1967;
Hsiung, 1972; Nayak and Murray, 19731 and herpes-type DNA virus (Nayak, 1971). Retravirus was found in tissue-cultured cells derived from leukemic or normal guinea pigs but only after treatment with bromodeoxyuridine (BrdU) (Hsiung, 1972; Rhim et al., 1973; Nayak and Murray, 1973). However, attempts to grow these BrdU-induced viruses in cell cultures have not been successful (Rhim et al., 1974). Therefore, we have attempted to isolate viruses associated with leukemic cells by cocultivation with various mammalian cells. During the investigation, a guinea pig herpesvirus (GPHV) was isolated from mink lung cells after cocultivation with BZ-L,C leukemic cells but a guinea pig retravirus was not isolated by this method. To determine if a relationship exists between the GPHV and guinea pig leukemia, the oncogenic potential of the GPHV was further investigated. Fong and Hsiung (1973) reported that GPHV is capable of transforming hamster embryo cells.
100 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
HERPESVIRUS
TRANSFORMATION
Viral antigen and infectious virus were detected in the transformed cells. Tumors were produced when one of the tramsformed hamster cell lines (LK51-19-6) was inoculated into hamsters. However infectious GPHV was not isolated from the tumor cells (Michaliski et al., 1976). This communication describes some biological characteristics of the GPHV which was isolated from mink lung cells after cocultivation with L,C! leukemic cells and reports the in vitro malignant transformation of rat embryo cells by this virus. MATERIALS
AND
METHODS
Fresh circulating lymphoblasts from BZ-L,C leukemic guinea pigs were obtained from Dr. Ira Green, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, Maryland. The BZ-L,C line was previously maintained by serial cell passage in syngeneic animals by Dr. B. Zbar, National Cancer Institute, NIH, Bethesda, Maryland. Tissue culture and media. Cells were grown and maintained in Eagle’s minimum essential medium (EMEM) with 10% fetal bovine serum, (FBS), 2 mM glutamine, and 100 units of penicillin and 100 pg of streptomycin per ml (EMEM + 10% FBS). Primary guinea pig kidney (GPK) cells were prepared from inbred strain 13 guinea pigs (Animal Production Units of NIH, Bethesda, Maryland) by standard trypsinization procedures. The mink lung [CCL-64, Mv 1 Lu (NBL-7)l line was obtained from the American Type Culture Rockville , Maryland. A Collection, Fischer rat embryo (RE) cell line (F-111) was originally established by Freeman et al., (1970). Virus assays. The replication of GPHV in cultures was determined by: (1) examination for the presence of cytopathic effect (CPE); (2) assay for complement-fixing (CF) antigen reactive with GPHV and human herpes simplex virus type 1 (HHSV-1) antiserum; and (3) examination by electron microscopy for the presence of virus particles. Virus infectivity for GPHV was assayed in GPK and mink lung cells. The L&
guinea
pig leukemia.
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infectivity titers were expressed at TCD,, per 0.1 ml. CF test. Cell pack preparations for CF testing were made as previously described (Rhim and Huebner, 1970). CF tests were performed by the microtiter technique described for tumor antigen studies (Huebner et al., 1963). Titers were recorded as reciprocals of the highest dilution giving 3 + to 4+ fixation of 1.8 units of complement. Zmmunofhorescent staining. Fluorescent antibody (FA) tests were performed by Dr. K. Takemoto, NIH, Bethesda, Maryland by the indirect procedure using fluorescein-conjugated goat anti-rabbit globulin and HHSV-1 rabbit antiserum or GPHV rabbit antiserum. Anti-G-al sera. HHVS-1 rabbit antiserum was obtained from Microbiological Associates, Inc., Bethesda, Maryland. GPHV rabbit antiserum and guinea pig cytomegalovirus (GPCMV) rabbit antiserum were gifts from Dr. G. D. Hsiung, Veterans Administration Hospital, West Haven, Connecticut. Neutralization test. Equal amounts of virus and antiserum were combined and the mixture was held at 37” for 30 min. The infectivity titer of the virus-serum mixture was determined on GPK cells. Transformation studies. One-day-old petri dish (60 mm) cultures of RE (F-111) (passage 61) cells were infected with GPHV at an input multiplicity of 10. The infected cultures were incubated for 2 hr at 37”, washed twice with medium, and refed with fresh medium. The cultures were incubated at 37” under a 5% atmosphere with medium changes twice weekly. Twentyone days later, replicate cultures were stained with Giemsa, foci were counted, and transformation rates were then determined. Some transformed foci were isolated by the cloning cylinder technique and propagated as clonal lines. Cell aggregation assay. Formation of cellular aggregates by normal and transformed cells was tested using the method described by Steuer et al. (1976) and Steuer and Ting (1976). Freshly trypsinized viable cells (2 x 105)were seeded into 35mm plastic dishes containing a 2-ml agar base
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layer (0.9% Difco agar in growth medium with 20% FBS). The dishes were incubated undisturbed at 37” under 5% CO, in air. Viable cell counts were performed daily for 4 days. Soft agar assay. Cells were suspended in 0.36% agar medium (EMEM containing 20% FBS and antibiotics). This suspension was layered onto a 0.9% agar base layer at concentrations of lo3 to lo4 cells per 35-mm dish. Dishes were incubated in a humidified atmosphere (5% CO,) at 37”. After 2 weeks, colonies greater than 0.125 mm in diameter were counted. Results are expressed as percentage plating efficiency (%PE = number of colonies x lOO/number cells plated). Rescue of virus. A genome-rescue experiment was done by cocultivation of equal numbers of GPHV-transformed RE cells and normal GPK cells. Supernatants from these cultures taken from the 14th to 21st day postinfection were assayed for GPHV on GPK and mink lung cells. Oncogenicity
of transformed
cells. New-
born Fischer rats were inoculated subcutaneously with 1 x lo6 freshly trypsinized cells in order to determine their tumorigenicity. RESULTS
Isolation
of the GPHV
Leukemic cells (1 x lo6 cells/flask) were cocultivated with an equal number of mink lung cells in EMEM + 10% FBS. The medium was changed once a week. In such cultures, most of the leukemic guinea pig cells remained in suspension while the mink lung cells grew in monolayer. However, all of the leukemic cells rounded up and died after 4 weeks in culture. It was at this time that visible CPE (Figs. 1A and B) became apparent in mink lung cells. Herpesvirus particles were also seen using electron microscopy (Fig. 2). No guinea pig retraviruses were isolated. Characterization
of the GPHV
The GPHV mink isolate from BZ-L,C! leukemic cells was further characterized (Table 1). Electron microscopy of the infected mink lung cells revealed morphological characteristics typical of herpestype virus (Fig. 2). There were numerous
S. RHIM
extracellular enveloped particles and many intranuclear nucleocapsids. There were also a few examples of enveloped particles in cytoplasmic vesicles and evidence of envelopment by the cytoplasmic membranes as the particles budded into the vesicles. The virus could be passed serially in mink lung cultures, with CPE developing 3-5 days after infection. The CPE was characterized by round, refractile, and loose cells often clumping together (Figs. 1A and B). A similar CPE was noted in the GPHV-infected GPK cells. Vero and cat embryo cells were highly sensitive to GPHV infection, rat and SIRC cells were moderately sensitive, and human tumor cells and mouse embryo cells were only sightly sensitive to GPHV infection, as indicated by CPE. In mink cells, the maximum titer was lo4 infectious units as determined by endpoint dilution. However, comparative titration of virus in mink and GPK cells showed that mink cells were loo-fold less sensitive than GPK cells. GPHV was sensitive to ether treatment. Four logs of GPHV were inactivated following incubation with ether for 20 hr at 4”.
The virus infectivity titer was reduced lOOO-foldby the antiserum against GPHV isolated by Hsiung and Kaplow (1969) but was not reduced by the antiserum against guinea pig cytomegalovirus. CF antigen for HHSV-1 (MacIntyre strain) was detected in cell suspensions prepared from GPHV-infected mink lung cells. Thus the GPHV mink isolate is serologically related to the GPHV isolated by Hsiung and Kaplow (1969) as determined by neutralization tests and also to human herpes simplex virus as determined by the complement fixation test. In Vitro Rat Embryo by GPHV
Cell Transformation
Rat embryo (F-111) cells infected with GPHV showed a moderate CPE in 5-7 days (Fig. 1C) as compared to uninfected RE cells (Fig. 1D). However, the CPE in infected RE cells disappeared within 1 week, and morphologically altered foci began to develop 14-17 days after infection.
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FIG. 1. Cytopathic effect (CPE) induced by guinea pig herpesvirus (GPHV) in mink lung and rat embryo cells (unstained, 60 x ). (A) Mink lung cells 5 days after GPHV infection. (B) Mink lung cells 10 days after GPHV infection. (C) Rat embryo cells 5 days after GPHV infection. (D) Uninfected rat embryo cells.
The transformed foci were characterized by loss of contact inhibition, resulting in multilayered and irregular cell growth (Figs. 3A and B), and could be readily
distinguished from normal, nontransformed cells (Fig. 30. The transformed foci, however, did not all have the same gross morphological appearance. The
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FIG. 2. Virus particles in section of mink lung cells infected with GPHV. Note particles with dense and coreless centers enveloped by an inner membrane scattered in the nucleolus. Particles enveloped by a double membrane are also seen in the vesicle (30,000~).
transformation efficiency of GPHV in RE cells determined 21 days after infection was 0.01%. Focal areas of transformed cells were isolated by the cloning cylinder technique and propagated. A number of focus-derived clonal lines were established. Two clonal lines designated as GPHV RE #6 and GPHV RE #7 were further characterized. Properties of GPHV-Transformed Embryo Cell Lines
Rat
Transformed cells in tissue culture generally can be distinguished from their normal counterparts by quantitative differences in growth properties such as contact inhibition, saturation density, soft agar colony-forming efficiency, and cell aggregate properties (Table 2). Saturation densities of transformed clonal lines #6 and #7 were 17.3 x lo5 and 12.4 x lo5 cells/cm2, respectively, which is three- to fourfold more than the control, untransformed
line. Normal rat embryo cells were unable to grow in soft agar while the transformed clonal lines demonstrated high plating efficiencies in soft agar. When suspended above an agar base, the transformed cells formed larger cell aggregates than those formed by normal RE cells (Fig. 4). Viable cell counts of trypsinized aggregates on 4 consecutive days indicated that control, untransformed cells underwent a significant decline in a number of viable cells whereas GPHV-transformed RE cells showed growth in the aggregate form (Fig. 5). The transformed cells, as well as untransformed, control RE cells were tested for their ability to produce tumors in newborn syngeneic animals. All newborn Fishcer rats inoculated subcutaneously with 1 x lo6 transformed cells developed tumors at the site of inoculation within 4 weeks (Table 2). No tumors developed over a period of 10 weeks in newborn Fischer
HERPESVIRUS TABLE
TRANSFORMATION
1
PROPERTIES OF GUINEA PIG HERPESVIRUS (GPHV) ISOLATED FROM MINK LUNG CELJ..SAFTER COCULTIVATION WITH L,C GUINEA PIG LYMPHOBLASTS Morphology
A herpes-type virus: numerous extracellular enveloped particles and many intracellular nucleocapsids
Cytopathic
effect
Round, refractile, and loose cells which often clump together
Infectivity
titer”
104.” in mink cells 1W.Oin guinea pig kidney cells
Sensitive etherb
to
Yes
Host ranges
CF titer HHSV-1 gend
CPE’ + + + +: guinea pig, Vero, mink, and cat cells CPE ++: rat and SIRC cells CPE + or -: human osteosarcoma, human RD, NIH Swiss, and BALB/c mouse cells of anti-
13
-
D TCDJO.1 ml. b Four logs of GPHV were inactivated following incubation with ether for 20 hr at 40”. c CPE (cytopathic effects): + = ll-25%; + + = 2650%; + + + + = >75% of cells affected. d HHSV-1 = human herpes simplex virus type 1.
rats inoculated with normal, untransformed RE cells. The tumors were progressive and transplantable. Cells established from the tumors resembled the transformed cell lines. To detect the presence of GPHV- or HHSV-specific antigen in the transformed cells or the tumor cells, CF and FA tests were used. Repeated attempts to demonstrate GPHV- or HHSV-CF antigens in the transformed cells were unsuccessful. The transformed cells were also negative for intracytoplasmic antigen when tested by FA staining with anti-GPHV serum and HHSV-1 antiserum. The rat tumor cells were negative for GPHV or HHSV antigens by the FA test; however, they were positive for CF antigens for HHSV-1 (CF titer, 1:16).
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Neither herpesvirus nor type C virus particles were found in the transformed cells or in the tumor cells when examined by electron microscopy. In an attempt to rescue infectious GPHV, transformed lines and tumor cell lines at various passage levels were cocultivated with guinea pig cells. Supernatants from these cultures taken at 14 or 21 days were inoculated into fresh guinea pig and mink cells and examined for the presence of CPE. All rescue experiments were so far negative, for GPHV. The transformed cells and the. rat tumor cells were also examined for the presence of supernatant viral RNA-dependent DNA polymerase, which is a highly sensitive method for the detection of replicating type C RNA virus. The results were negative. DISCUSSION
The validity of a general strategy for in vitro isolation of type C virus involving cocultivation of suspected virus-producing tissues with cells from various species to maximize the probability of finding a permissive host has been previously demonstrated (Benveniste et al., 1974). Thus the cocultivation method has been proposed as a general approach to isolate infectious type C viruses from tissues. We have therefore attempted to isolate and grow guinea pig retraviruses in mink lung cells after cocultivation with L$ leukemic cells. The mink lung cell line Mv 1 Lu (CCL-641 was selected for cocultivation since recent reports have demonstrated the in vitro susceptibility of mink lung cells to type C sarcoma viruses (Henderson et al., 1974) as well as to the type B mouse mammary tumor virus (Lasfargues et al., 1976). As a result, GPHV, capable of transforming rat embryo cells, was isolated from mink lung cells after cocultivation with L,C! guinea pig leukemic lymphoblasts. However, guinea pig retraviruses were not isolated in mink cells by the cocultivation method. A GPHV was originally isolated from spontaneously degenerated normal and leukemic strain 2 guinea pig kidney cultures (Hsiung and Kaplow, 1969) and from leukemic lymphoblasts of strain 2 guinea
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S. RHIM
FIG. 3. (A) Rat embryo cells showing foci of GPHV-transformed cells 3 weeks after GPHV infection (Giemsa stain). (B) A transformed focus of rat embryo cells 3 weeks after GPHV infection (Giemsa stain, 56x). (C) Uninfected rat embryo cells stained with Giemsa (56x).
pigs (Nayak, 1971). Morphological and biochemical characteristics of the GPHV are very similar to those of EB virus found in Burkitt’s lymphoma but are different from guinea pig CMV. Until now, no antigenic
relationship has been reported between GPHV and other herpesviruses including human CMV, HHSV, EB virus, equine herpesviruses types 1 and 2, Marek’s disease of chicken, and Luke virus of frog
HERPESVIRUS
TRANSFORMATION TABLE
OF RAT
Morphology Saturation density” (x 105/cm2) Cell aggregates* Size Viability of cells (x 105) Plating efficiency (%l in soft agar CF titers of HHSV-1 antigene Virus particles Rescuable infectious GPHV Tumorigenicity in rat&
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2
PROPERTIES OF GUINEA PIG HERPESVIRUS (GPHV)-TRANSFORMED Properties
CELLS
GPHV-RE cells
#6
RAT EMBRYO (RE) CELLS LINES GPHV-RE cells
#7
RE cells
Transformed 12.2
Transformed 9.4
Normal 3.1
Large 4.0 17.3 <2 Negative Negative Positive 5/5e
Large 2.6 12.4 <2 Negative Negative Positive 515
Small 0.4
n Maximum number of cells obtained after initial plating of 5 x lo3 cells/cm2. * Size and viability of cell aggregates were determined on Day 3 after plating initially with the use of an agar static assay (Steuer et al., 1976). c HHSV = human herpes simplex virus type 1. into each newborn Fischer rat. d Cells (1 x IO') inoculated subcutaneously e Number of tumors formed/number of rats inoculated.
(Hsiung et al., 1971). However, a serological relationship between GPHV and human herpesvirus has been demonstrated in this study by complement fixation. But since the existence of herpes group-specific antigen is still open to question, the antigenie relations should be further studied and carefully evaluated. The host range of the GPHV was found to be rather narrow as shown in this study as well as those by others (Hsiung and Kaplow, 1969; Hsiung et al., 1971; Nayak, 1971; Michalski and Hsiung, 1975)whereas human herpesvirus has a wide host range. GPHV-transformed rat cells had the following properties generally associated with viral transformation: (a) altered morphology; (b) increased growth rate; (c) colony formation in soft agar medium; (d) formation of large cell aggregates and growth in this aggregate form above an agar base; (e) tumorigenicity in syngeneic animals. The transformed cells produced neither viral antigen nor a rescuable infectious virus; however, the rat tumor cells were positive for HHSV-type 1 CF antigen while negative for infectious virus. Our studies are similar to those reported by several herpesviruses in hamster (Duff and Rapp, 1971 and 1973; Albrecht and Rapp, 19; Fong and Hsiung, 1973) and rat embryo cells (MacNab, 1974). With one
2 x lo5 cells per plate
exception, as reported by Fong and Hsiung (1973) in the GPHV-induced hamster cell transformation, infectious virus was not recovered from the transformed cells or the tumor cells. Since inactivated virus was used in most of the transformation experiments, it was assumed that complete infectious herpesvirus was not present. Like GPHV-induced hamster cell transformation (Fong and Hisung, 1973), infectious GPHV was used in our rat cell transformation experiments. It thus seems clear that GPHV is able to transform rodent cells without the need for uv irradiation of the virus. It has recently been reported that infectious GPHV was found only from the transformed hamster cells in early passage levels but not from the hamster tumor cells (Michalski et al., 1976). However, in contrast to the GPHV hamster cell transformation system, infectious virus was not recovered from the transformed rat cells or from the rat tumor cells. Herpesvirus antigen was detected in the rat tumor cells even though the same antigen was not detected in the rat transformed cells. Thus, the relationship between the GPHV and the rat tumors was established. The development of transplantable GPHV-induced rat tumors with this in uitro transformation
system may allow the
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S. RHIM
FIG. 4. Cell agg regates of rat embryo cells transformed by GPHV. Cells were all owei d to aggregate undistl orbed for 3 days in liquid growth medium above an agar base layer (56) 0. (A) Aggregates of uninfec :ted rat embl ryo cells. (B) Aggregates of GPHV-transformed #6 rat embryo cz!llS (Cl Aggregates of GPHV. .transformed #‘7 rat embryo cells.
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DUFF, R., and RAPP, F. (1971). Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultra-violet irradiated herpes simplex virus type 2. J. Virol. 8, 469-477. DUFF, R., and RAPP, F. (1973). Oncogenic transformation of hamster embryo cells after exposure to inactivated herpes simplex virus type 1. J. Viral. 12, 207-209. ELLMAN, L., and GREEN, I. (1971). L,C guinea pig leukemia: Immunoprotection and immunotherapy. Cancer 28, 647-654. FONG, C. K., and HSIUNG, G. D. (1973). In vitro transformation of hamster embryo cells by a RE, control I I I guinea pig herpes-like virus. Proc. Sot. Exp. Biol. 1 2 3 4 Med. 144, 974-978. DAYS FREEMAN, A. E., PRICE, P. J., IGEL, H. J., YOUNG, J. FIG. 5. Short-term growth curves of GPHVC., MARYAK, J. M., and HUEBNER, R. J. (1970). transformed rat embryo cells. Viable cell counts Morphological transformation of rat embryo cells were performed on trypsinized aggregates for 4 coninduced by diethylnitrosamine and murine leukesecutive days (2 x loj viable cells were plated per mia virus. J. Nat. Cancer Inst. 44, 65-78. dish). (0-O) GPHV-transformed #6 cells. HENDERSON, I., LIEBER, M. M., and TODARO, G. J. (0-O) GPHV-transformed #7 cells. (A-A) (1974). Mink cell line Mv 1 Lu (CCL-64). Focus Uninfected rat embryo, control cells. formation and the generation of “nonproducer” transformed cell lines with murine and feline saridentification of a specific T-antigen. Anticoma viruses. Virology 60, 282-287. serum to this antigen should be valuable HSIUNG, G. D. (1972). Activation of guinea pig Ctype virus in cultured spleen cells by 5-bromo-2’for further study of GPHV in other transdeoxyuridine. J. Nat. Cancer Inst. 49, 567-570. formation systems. Transplantable tumors HSIUNG, G. D., and KAPLOW, L. S. (1969). Herpeswould also permit studies on virus-specific like virus isolated from spontaneously degenertransplantation antigens induced by ated tissue culture derived from leukemia-suscepGPHV. tible guinea pigs. J. Virol. 3, 355-357. ACKNOWLEDGMENTS HSIUNG, G. D., KAPLOW, L. S., and Booss, J. (1971). Herpes virus infection of guinea pigs, I. Isolation, This investigation was supported by Contract characterization and pathogenicity. Amer. J. EpiNOI-CP-43519A from the Viral Cancer Program of demiol. 93, 298-307. the National Cancer Institute, NIH, Bethesda, MarHUEBNER, R. J., ROWE, W. P., TURNER, H. C., and yland. I thank Dr. G. D. Hsiung, School of Medicine, LANE, W. T. (1963). Specific adenovirus compleYale University, New Haven, Connecticut, for supment-fixing antigens in virus-free hamster and plying the GPHV and GPCMV antiserum; Dr. M. L. rat tumors. Proc. Nat. Acad. Sci. USA 50, 379Vernon, Microbiological Associates, Inc., Bethesda, 389. Maryland, for electron microscopic examination of KATZ, D. H., ELLMAN, L. PAUL, W. E., GREEN, I., cells; Dr. K. Takemoto, National Institute of Aland BENECERRAF, B. (1972). Resistance of guinea lergy and Infectious Diseases, NIH, Bethesda, Marpigs to leukemia following transfer of immunoyland, for fluorescent antibody assay; and Mr. Paul competent allogenic lymphoid cells. Cancer Res. Hill, National Cancer Institute, NIH, Bethesda, 32, 133-140. Maryland, for the CF assay. LASFARGUES, E. Y., VAIDYA, A. B., LASFARGUES, J. REFERENCES C., and MOORE, D. H. (1976). In vitro susceptibility of mink lung cells to the mouse mammary ALBRECHT, T., and RAPP, F. (1973). Malignant tumor virus. J. Nat. Cancer Inst. 57, 447-449. transformation of hamster embryo fibroblasts folMACNAB, J. C. (1974). Transformation of rat embryo lowing exposure to ultraviolet-irradiated human cells by temperature-sensitive mutants of herpes cytomegalovirus. Virology 55, 53-61. simplex virus. J. Gen. Virol. 24, 143-153. BENVENISTE, R. E., LIEBER, M. M., LIVINGSTON, D. MICHAL~KI, F. J., FONG, C. K., HSIUNG, G. D., and M., SHERR, C. J., TODARO, G. J., and KALTER, S. SCHNEIDER, R. D. (1976). Induction of tumors by a S. (1974). Infectious C-type virus isolated from a guinea pig herpesvirus-transformed hamster cell baboon placenta. Nutare (London) 248, 17-20. line. J. Nat. Cancer Inst. 56, 1165-1170. CONGDON, C. C., and LORENZ, E. (1954). Leukemia MICHAWKI, F., and HSIUNG, G. D. (1975). Malignant in guinea pigs. Amer. J. Pathol. 30, 337-359.
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transformation of hamster cells following infection with a bovine herpes virus (Infectious bovine rhinotracheitis virus). Proc. Sot. Exp. Biol. Med. 148, 891-896. NAYAK, D. P. (1971). Isolation and characterization of a herpesvirus from leukemic guinea pigs. J. Viral. 8, 579-588. NAYAK, D. P., and MURRAY, P. R. (1973). Induction of type C virus in cultured guinea pig cells. J. Viral. 12, 1’77-187. OPLER, S. R. (1967). Observations of a new virus associated with guinea pig leukemia: Preliminary note. J. Nut. Cancer Inst. 38, 797-800. PEARSON, J. W., PERK, K., CHIRICOS, M. A., and TORGERSEN, J. A. (1975). Drug therapy against a transplantable guinea pig leukemia. Cancer Res. 35, 1093-1098. RHIM, J. S., DUH, F. G., CHO, H. Y., Wuu, K. D., and VERNON, M. L. (1973). Activation by 5-bromoZ’-deoxyuridine of particles resembling guinea pig
S. RHIM leukemia virus from guinea-pig nonproducer cells. J. Nat. Cancer Inst. 51, 1327-1331. RHIM, J. S., and HUEBNER, R. J. (1970). Rat embryo cells in complement-fixation test for murine leukemia virus. Nature (London) 226, 646-647. RHIM, J. S., Wuu, K. D., Ro, H. S., VERNON, M. L., and HUEBNER, R. J. (1974). Induction of guinea pig leukemia-like virus from cultured guinea pig cells. Proc. Sot. Exp. Biol. Med. 147, 323-330. SHEVACH, E. M., ELLMAN, L., DAVIE, J. M., and GREEN, I. (19721. L&! guinea pig lymphatic leukemia: A “B” cell leukemia. Blood 39, 1-12. STEUER, A. F., RHIM, J. S., and TING, R. C. (1976). Aggregate properties of human sarcoma cells: Correlation with growth in soft agar and tumorigenicity. Proc. Amer. Assoc. CancerRes. 17,34. STEUER, A. F., and TING, R. C. (1976). Formation of larger cell aggregates by transformed cells: An in vitro index of cell transformation. J. Nat. Cancer Inst. 56, 1279-1280.