Cell, Vol. 12,973-982,
December
1977. Copyright
Virus Infection Cell Lines
0 1977 by MIT
of Murine Teratocarcinoma
Natalie M. Teich and Robin A. Weiss Imperial Cancer Research Fund Laboratories P.O. Box 123 Lincoln’s Inn Fields London WC2A 3PX, England Gail R. Martin* Department of Anatomy and Embryology University College London Gower Street London WC1 E 6BT, England Douglas R. Lowy Dermatology Branch National Cancer Institute Bethesda, Maryland 20014
Summary Three clonal murine teratocarcinoma stem cell lines were studied for susceptibility to infection by a variety of different viruses. When in the undifferentiated state, these embryonal carcinoma stem cells are as sensitive to infection by encephalomyocarditis, Sindbis, vaccinia and vesicular stomatitis viruses as are embryo fibroblasts derived from the same mouse strain. The undifferentiated teratocarcinoma cells, however, unlike the fibroblasts, are entirely refractory to infection with murine leukemia virus. If the stem cells are allowed to differentiate to form a variety of differentiated cell types, they become permissive for murine leukemia virus replication at a low level. Several techniques were used to elucidate the block to murine leukemia virus infection. In the one nullipotent cell line which does not differentiate in vivo or in vitro, virus adsorption and penetration are not restricted (as measured by infection with murine leukemia virus pseudotypes of vesicular stomatitis virus), but an integrated proviral DNA copy cannot be detected. In a pluripotent stem cell line, proviral DNA sequences are detected, but neither transcription into viral specific RNA nor viral specific protein synthesis is observed. These findings suggest that control of murine leukemia virus replication in the cells is a function of the stage of differentiation and perhaps also of the genetic composition of the teratocarcinoma stem cells. Introduction The replication of animal viruses in undifferentiated cells has received little attention due to the dearth of suitable experimental systems. It is well * Present Institute,
address: University
Department of Anatomy and Cancer Research of California, San Francisco, California 94143.
Stem
known that some viruses show fastidious requirements for replication; some require propagation in vivo or may grow only in organ cultures that maintain well defined tissue structure and differentiation in vitro. Many viruses, however, will replicate in a variety of- cell types and are conveniently propagated in fibroblasts in vitro. Although fibroblastic monolayer cultures are often regarded as undifferentiated cells, they are, in fact, mesenchymal cells that are committed to the synthesis of specific differentiated products such as collagen, hyaluronic acid and fats which may be produced in vitro (Daniel, Dingle and Lucy, 1961; Goldberg, Green and Todaro, 1963; Hamerman, Todaro and Green, 1965; Green and Kehinde, 1974). The most obvious source of undifferentiated cells is the embryo itself. A useful alternative to the cells of the early mouse embryo has recently become available. Embryonal carcinoma cells, which are the stem cells of teratocarcinomas, are pluripotent cells that can be maintained in the undifferentiated state or can differentiate to a wide variety of cell types, including derivatives of all three primary germ layers (reviewed by Pierce, 1967; Damjanov and Solter, 1974; Martin, 1975). The similarity of such cells to the undifferentiated cells of the normal early mouse embryo has been emphasized by recent experiments that demonstrate the ability of teratocarcinoma stem cells to be incorporated into the blastocyst and participate in normal embryonic development (Brinster, 1974; Mintz and Illmensee, 1975; Papaioannou et al., 1975; lllmensee and Mintz, 1976). The availability of pure cultures of undifferentiated teratocarcinoma cells and their differentiated descendents therefore allows examination of the relationship between the differentiative stage of a cell and its ability to support replication of a variety of animal viruses in comparison to fibroblastic embryo cell cultures derived from the same mouse strain. The embryonal carcinoma cell lines isolated by Martin and Evans (1975a) are capable of differentiating in vitro in a distinct series of steps which parallel normal embryogenesis. These teratocarcinoma stem cells are therefore useful for studies of virus replication at various stages of differentiation. Three distinct clonal lines of tumorigenic embryonal carcinoma stem cells derived from strain 129 mice were used in this study: nullipotent (nulli) embryonal carcinoma cells which apparently do not differentiate in vivo or in vitro; pluripotent (S2) cells which under certain conditions form simple embryoid bodies composed of the stem cells surrounded by a single layer of endodermal cells; and pluripotent (PSA4) cells which under the same conditions form simple embryoid bodies which continue to develop into cystic embryoid bodies
Cell 974
which are fluid-filled cysts containing stem cells and a variety of differentiated cells. The latter two cell lines have the capacity to differentiate further into many cell types in vitro (Evans and Martin, 1975; Martin and Evans, 1975b). In addition, we have used the PYS endodermal cell line derived by Lehman et al. (1974) from a teratocarcinoma as a representative differentiated culture containing a single cell type; upon in vivo inoculation, the PYS cells give rise to parietal yolk sac carcinomas only (Swartzendruber and Lehman, 1975). All types of virus we tested, representing both RNA and DNA animal virus groups, replicate with high efficiency in the undifferentiated teratocarcinoma cells with the exception of the murine leukemia viruses (MuLV), for which no replication was detectable. When the teratocarcinoma stem cells were allowed to differentiate, however, we found that they became permissive for MuLV replication. These results led us to examine more fully the nature of the block to virus replication in the undifferentiated cells in terms of the ability of the virus to penetrate the cell surface, to integrate into the host genome and to express proviral functions.
Results Infection with Various Viruses To assess the capacity of undifferentiated teratocarcinoma ceils to support virus multiplication, we selected representative viruses from five different virus groups for study. The sample included the DNA-containing virus, vaccinia and RNA-containing viruses, encephalomyocarditis (EMC) virus, Sindbis virus, vesicular stomatitis virus (VSV) and murine leukemia virus (MuLV). Undifferentiated cultures of the nulli, S2 and PSA4 cells, as well as the PYS endodermal teratocarcinoma cells and strain 129 mouse embryo fibroblasts as differentiated cell controls, were infected with each virus and assayed for virus production by the appropriate techniques (see Experimental Procedures). A summary of the results obtained is presented in Table
1. Infection
of Teratocarcinoma
Cells with Various Replication
Virus
Table 1. For those viruses that elicit discrete plaques of lysis, there was a one-hit dose-response curve with dilution, and the teratocarcinoma cells generally showed equal or slightly enhanced susceptibility to infection. It is notable that the teratocarcinoma cells (both undifferentiated embryonal carcinoma and endodermal cells) showed a marked resistance to infection with murine leukemia virus. We investigated this finding further and discuss it in the following sections.
Infection of Teratocarcinoma Cells at Various Stages of Differentiation with Murine Leukemia Virus (MuLV) The refractory nature of the undifferentiated teratocarcinoma stem cells to MuLV infection in comparison to fibroblasts of the same mouse strain posed the question whether the teratocarcinoma cells become permissive for MuLV replication when they differentiate. To answer this question, we infected cultures of teratocarcinoma cells with MuLV at various stages of differentiation. The undifferentiatiated pure stem cell cultures, embryonal carcinoma, the simple differentiated embryoid bodies (embryonal carcinoma plus endodermal cells) and the fully differentiated cultures (containing a motley of cell types) were infected and then assayed for virus production either directly (by XC plaque assay and infectious center assay) or indirectly (by titration of supernatant harvests). The results of such experiments are shown in Table 2. Neither the undifferentiated cells nor the endodermal cells appear to support MuLV replication. The fully differentiated cultures, however, showed a low but easily detectable level of virus production. These findings were confirmed with a number of MuLV strains: the N tropic AKR-Gross MuLV and the NB tropic Friend leukemia virus complex (lymphoid leukemia virus and helper-dependent spleen focus-forming virus), as well as the NB tropic Moloney MuLV shown in the table. We did not observe the replication of a B tropic MuLV in
Viruses
in:
Teratocarcinoma Endodermal Cells PYS
Undifferentiated Teratocarcinoma
Type
Group
Strain 129 Mouse Embryo Fibroblasts
Vaccinia
Poxvirus
+
+
+
+
+
EMC
Picornavirus
+
+
+
+
+
Sindbis
Togavirus
+
+
+
+
+
vsv
Rhabdovirus
+
+
+
+
+
MuLV
Retrovirus
+
-
-
-
-
Nulli
Cells 52
PSA4
Virus 975
Infection
Table
2. Infection
of Teratocarcinoma
of Teratocarcinoma
Stage of Differentiation Undifferentiated
Cells
Cells
Cells with Mol-MuLV
at Various
Log,,, PFU/ml ___--XC Plaque
Nulli
10.4
co.4
52
10.4
co.4
PSA4
97 8 5 7 7 5
5 4 NIY 6 ND
’ Not determined.
The second assay procedure involved fusion of the infected PSA4 cells to permissive mouse embryo fibroblasts mediated by Sendai virus. Once again, there was no detectable activation or rescue of the MuLV genome from the PSA4 cells. Finally, we attempted to activate endogenous or exogenous integrated proviral genomes by treatment with the halogenated pyrimidine 5-iodo-2’deoxyuridine (IdU). IdU has been shown to be an efficacious inducer of latent proviruses (Lowy et al., 1971) and can be used to answer two questions. Are there inducible endogenous MuLV genomes in strain 129 fibroblasts or teratocarcinoma cells? After nonproductive infection of teratocarcinoma cells with Mol-MuLV, can the infecting NB tropic MuLV be induced? We treated teratocarcinoma cells, 129-ME cells and the highly inducible AKR-2B cell line with 20 pglml IdU for 24 hr. One day later, we added SC-1 mouse cells or SIRC cells to each culture to amplify the replication of any induced mouse-tropic (ecotropic) or xenotropic virus (Levy, 1973). We passaged the co-cultivated cells at weekly intervals, periodically assayed supernatant fluids for reverse transcriptase activity and monitored the cells for ecotropic MuLV by the XC plaque assay. Although virus was consistently detected from the AKR-2B cell line by 4 days after IdU treatment, there was no virus activity in any of the 129-ME or teratocarcinema cells during 6 weeks of monitoring postIdU treatment. It is known, however, that all mouse cells, including those derived from 129 mice, contain endogenous virus sequences (Lowy et al., 1974; Table 5 above), although no infectious virus has ever been isolated from 129 mice. These data suggest that the endogenous 129 viruses are not readily inducible and are perhaps under more stringent host repression in both fibroblast and teratocarcinoma cells derived from this mouse strain compared to other strains. To determine whether the NB tropic MuLV used for infection could be induced from the teratocarcinema cells, cultures which had been infected in the undifferentiated state were allowed to form
fully differentiated cultures and were then treated with IdU and co-cultivated as described above. We then monitored the subsequent co-cultures weekly for 1 month. In four experiments of this nature, we were able to detect production of MuLV only once. This virus isolate manifested NB tropic host range properties; presumably it represented the activation of the exogenously infecting Mol-MuLV, since no endogenous NB tropic virus has yet been isolated from any inbred mouse strain. The infrequency of activation (one of four attempts), however, raises the possibility of accidental laboratory contamination; this cannot be excluded, although we have no evidence for such contamination in other cell cultures maintained in the laboratory. On the other hand, as we have demonstrated the presence of proviral Mol-MuLV sequences in PSA4 teratocarcinoma cellular DNA by nucleic acid hybridization (Table 5), and considering the difficulty in inducing endogenous viral sequences from strain 129 mouse cells in general, it is not surprising that the frequency of viral activation is low.
Discussion To assess the capacity of teratocarcinoma cells to support viral multiplication, we selected representative viruses from five virus groups for study. The sample included one DNA-containing virus (vaccinia) and four RNA-containing viruses (encephalomyocarditis virus, Sindbis virus, vesicular stomatitis virus and murine leukemia virus). With the exception of the murine leukemia virus (MuLV), these viruses were able to replicate in both the undifferentiated embryonal carcinoma stem cells and their differentiated derivatives. These findings further extend the list of viruses which can replicate in embryonal carcinoma cells. Other embryonal carcinoma cell lines have been shown to be susceptible to infection with human adenovirus type 2 (Kelly and Boccara, 1976) and to a lesser extent with Mengo virus (Lehman, Klein and Hackenberg, 1975). Our observation on MuLV is also consistent with the report that these cells are not permissive for the replication of certain viruses. For example, Miller, Ward and Ruddle (1977) have found that synthesis of the parvovirus, minute virus of mice, as measured by fluorescence staining of viral structural proteins, does not occur in embryonal carcinoma cells but does proceed in fibroblasts derived from teratocarcinoma cells. It has also been shown that differentiation of embryonal carcinoma cells is correlated with an alteration in the response to infection with the papovaviruses, polyoma and SV40 (Lehman et al., 1975; Swartzendruber and Lehman, 1975). They found that early T antigens of both viruses and the late V antigen of polyoma virus were expressed only in those
Virus 979
Infection
of Teratocarcinoma
Cells
cells which had undergone some differentiation within a few days after infection. The results described here indicate that the response of teratocarcinoma cells to infection with murine leukemia virus (MuLV) is dependent upon the particular cell line used and on the state of differentiation of the cells. The nullipotent embryonal carcinoma cells which do not differentiate were refractory to MuLV multiplication. Using MuLV pseudotypes of vesicular stomatitis virus (VSV) (that is, VSV genomes with MuLV envelope glycoprotein coat specificities), we have shown that the nulli embryonal carcinoma cells have the specific receptors for MuLV adsorption and penetration. Nucleic acid hybridization experiments using a labeled DNA probe complementary to the infecting Moloney strain of MuLV indicated that neither DNA nor RNA copies of the virus can be detected in MuLV-infected nulli cells. These results suggest that nulli cells exert a post-penetration, pre-integration block to viral replication. We are currently investigating whether a DNA provirus is synthesized after infection of nulli cells. The S2 and PSA4 teratocarcinoma stem cell lines also exhibited resistance to viral multiplication when the cells were undifferentiated or had formed embryoid bodies (embryonal carcinoma cells surrounded by a layer of endoderm). When the cells were allowed to undergo further differentiation to a variety of ceil types before MuLV infection, however, a small proportion of the cells were capable of sustaining MuLV replication. Further studies are now in progress to determine which cells in the differentiated PSA4 cultures are susceptible to MuLV infection and multiplication. The block to viral replication in the undifferentiated S2 and PSA4 embryonal carcinoma cells is not due to an absence of cell surface receptors for MuLV, as demonstrated by the use of MuLV pseudotypes of VSV. Using nucleic acid hybridization techniques, DNA sequences complementary to the infecting viral genome were found in infected PSA4 cells but not in the uninfected cells. The data indicate that most, if not all, of the sequences of the viral genome are found as proviral DNA copies in the MuLV-infected embryonal carcinoma cells of the PSA4 line. The fact that we were unable to detect the presence of viral RNA above the background level, however, suggests that viral specific RNA is not being transcribed or that it is transcribed but rapidly degraded. Analysis of cell protein extracts by sodium dodecylsulfate-polyacrylamide gel electrophoresis following precipitation with anti-MuLV serum did not reveal any virusspecific structural proteins or precursor proteins (data not shown). Thus the results indicate that the block to MuLV replication in the undifferentiated embryonal carcinoma stem cells of the PSA4
line is presumably post-integration and pre-translation. Further experimentation is necessary to decide whether the block is at the transcriptional level. S2 or PSA4 cells which had been infected as undifferentiated cells or as embryoid bodies did not spontaneously produce virus after they had undergone further differentiation. In many attempts to rescue the latent MuLV genome by cocultivation or fusion to susceptible mouse cells or by treatment with the virus inducer IdU, we were able to detect virus production in only one experiment with IdU. This suggests that the infected teratocarcinoma cells exert stringent regulation on virus expression. It is possible that the entire MuLV genome is not integrated into the DNA of all cells, which would account in part for the low frequency of rescue that we have observed. Alternatively, the viral genome may be integrated in a region of a chromosome which is rarely, if ever, transcribed in this particular cell line, or is not transcribed until differentiation to a specific cell type occurs, and this specific differentiation may occur at a very low frequency in these cultures. Our results with the PSA4 line are comparable to those seen with MuLV infection of mouse 4-8 cell embryos in vitro (Jaenisch, Fan and Croker, 1975; Jaenisch, 1976). MuLV replication was not observed in the infected embryos; mice that were born after implantation of these infected embryos into pseudopregnant mothers, however, did contain integrated DNA copies of the MuLV genome. Every organ tested by nucleic acid hybridization contained at least one copy of the MuLV genome, and the progeny of such mice also contained viral sequences homologous to the original infecting virus, indicating that viral sequences were integrated in the germ line as well. Some of these mice succumbed to the classical viral lymphoma, and tissues containing lymphoma cells expressed more viral specific RNA than did uninvolved tissues (Jaenisch et al., 1975). It will be interesting to see whether chimeric mice formed by injection of MuLV-infected embryonal carcinoma cells into preimplantation embryos develop viral lymphoma. Experimental
Procedures
Cells The homogeneous nullipotent embryonal carcinoma cell line “nulli-SCCl” has apparently lost the ability to differentiate; the line originates from a spontaneous testicular teratocarcinoma LS402C-1664 of a strain 129 mouse (Stevens, 1958). The pluripotent cell lines SCC-SP and PSA4 were derived from a teratocarcinoma OTT-5568 formed by the extrauterine implantation of a day 3 embryo of 129 SvSlJCP genotype (Stevens, 1960). All three clonal embryonal carcinoma cell lines were derived from isolated single cells as previously described (Martin and Evans, 1975a). The culture medium used throughout these experiments was Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose and supplemented with 10% heat-inactivated calf serum. Cells were
Cl?11 960
passaged by disaggregation with 0.25% trypsin in Tris-buffered saline EDTA. All cells were maintained at 37°C. Stock cultures of the pluripotent cell lines were maintained in the undifferentiated state by subculturing the cells every 3 or 4 days and seeding them at 5 X lOa cells per confluent feeder layer of mitomycin Ctreated ST0 mouse cells in a 9 cm tissue culture dish (Martin and Evans, 1975a). Stock cultures of the nullipotent cell line were maintained by subculturing the cells every third day and seeding the cells at 7 X 10’ cells per gelatin-coated 9 cm tissue culture dish. To obtain embryoid body formation, the pluripotent cells were seeded at 10’ ceils per 9 cm tissue culture dish in the absence of added feeder cells. 3 or 4 days later, the cells had formed clumps which were removed from the dish by gentle pipetting. The detached clumps were allowed to settle for several minutes, washed in fresh medium and seeded in a bacteriological dish to which they do not adhere. The endodermal cell layer became apparent within 24 hr. The medium was then changed daily. Within 6 days of appearance of the endodermal cell layer, most of the embryoid bodies formed by the clonal PSA4 line had large fluid-filled cysts, whereas the embryoid bodies formed by the 52 cell line remained as simple two-layered structures. Further differentiation of the cells was obtained by transferring embryoid bodies to tissue culture dishes. Between 190-1000 embryoid bodies were seeded per dish: these attached to the dish and were maintained for up to 1 month with medium changes every second or third day. Nullipotent aggregates (pseudo-embryoid bodies) were obtained by seeding 5 X IO6 cells per 9 cm tissue culture dish and manipulating the clumps which formed the same way as for the clumps of pluripotent cells. The PYS endodermal cell line (Lehman et al., 1974) was provided by Dr. J. M. Lehman. Stock cultures of these cells were maintained by subculturing every 4 or 5 days at approximately lOa cells per 9 cm tissue culture dish. Mouse embryo (ME) fibroblast cultures from strain 129 and BALB/c mice were prepared according to Hartley et al. (1965). The NIH/3T3 and SC-1 mouse embryo cell lines, provided by Dr. J. W. Hartley, have previously been described (Jainchill, Aaronson and Todaro, 1969; Hartley and Rowe, 1975). Primary cultures of C/E chick embryo cells were prepared from 11 day old Leghorn embryos according to standard techniques. The rabbit cornea1 cell line SIRC (Leerhoy. 1965) was purchased from the American Type Culture Collection. The ST0 cells are thioguanine-resistant, ouabain-resistant cultures derived from a continuous line of SIM mouse fibroblasts (Ware and Axelrad. 1972) by Dr. A. Bernstein. They were prepared as feeder cells by treatment with fresh mitomycin C (Sigma) at 10 pg/ml for 2 hr. Virus Stocks and Antiserum Moloney NB tropic murine leukemia virus (Mol-MuLV) was originally provided by Dr. J. Hartley. A seed of vesicular stomatitis virus (VSV) strain Indiana was provided by Dr. C. Pringle. The stock of VSV(MuLV) pseudotypes was prepared by establishing chronically infected MuLV-producing BALB/c cells, infecting with VSV at an moi of 1 and harvesting at 16 hr post-infection. Seed cultures of encephalomyocarditis virus (Dr. D. Rekosh), Sindbis virus (Dr. M. Schlesinger) and vaccinia virus (Mr. S. Tymes) were propagated in 129 mouse embryo fibroblasts prior to use in these experiments. Neutralizing antiserum to VSV prepared from a hyperimmunized sheep was provided by Dr. J. Zavada. Neutralizing antiserum to Mol-MuLV was prepared by hyperimmunization of a rabbit with detergent-disrupted whole virus. Assays of MuLV lnfectlon The standard XC plaque assay (Rowe, Pugh and Hartley, 1970) and the infectious center assay on SC-l cells were used for titration of MuLV infection of all fibroblast cultures. Slight modifications were made for the teratocarcinoma cell lines: undifferentiated stem cells were plated without feeder cells at l-2 X 10”
per 3 cm gelatinized plate and infected 1 or 2 days later after a 1 hr DEAEdextran (25 pg/ml) treatment; embryoid bodies were infected by resuspension in the virus inoculum containing 25 pg/ ml DEAE-dextran and washed after a 1 hr adsorption period at 37°C (virus production was monitored by plating the embryoid bodies on SC-1 cells at various times after infection upon which they would adhere and spread out, followed by the UV-XC assay); and differentiated teratocarcinoma cells were prepared as described above and then infected at 1, 2, or 3 weeks following seeding in tissue culture dishes; cell numbers at the time of infection were not determined due to the difficulty of disaggregating such cultures. In addition, supernatants from all cultures at various times after infection were assayed for virus production by inoculation onto susceptible mouse fibroblast cultures and by the reverse transcriptase assay. Plaque Assays of VW and VSV(MuLV) lnfectlon Plaque assays for VSV and VSV pseudotypes have been described previously (Zavada, 1972; Love and Weiss, 1974). In brief, the undifferentiated teratocarcinoma stem cells were seeded at 3-5 X lOa per 3 cm dish, and the fibroblasts were seeded at l-2 X lv per 3 cm dish. 2 days later, appropriate virus dilutions in IO pg/ ml polybrene were added in a 0.2 ml vol and adsorbed for 1 hr at 20°C. The inoculum was then removed, the cells were washed with phosphate-buffered saline and an agar medium overlay was added. After 2 days at 37”c, neutral red was added for macroscopic counting of the plaques. For assay of VSV(MuLV) pseudotypes. aliquots of the undiluted virus stock were first treated with an equal volume of 1:20 dilutions of VSV antiserum for 1 hr at 20°C or 30 min at 2O”c, followed by an overnight treatment at 4°C. Such treatment was sufficient to neutralize 5.5 logs of VSV. The neutralized virus stocks were then used as inocula as described above. Plaque Assays for Other Viruses Tested EMC. vaccinia and Sindbis viruses were assayed by conventional agar overlay techniques. Cells were seeded at densities described above for assays. Virus inocula were adsorbed for 1 hr at 379: and then removed. Cells were examined daily for a period of 2 weeks for microscopic manifestations of cytopathic effects (CPE). Discrete plaques of dead cells were observed with EMC, vaccinia and Sindbis viruses by l-2 days after infection, and final counts were made at 5-6 days. In addition, supernatant fluids were harvested from wells infected with the highest virus dilutions that showed CPE for assay of virus yield on mouse fibroblast cultures. IdU Induction and Reverse Transcriptare Assays To induce endogenous or exogenously infecting MuLV, cells were treated with 20 pg/ml iododeoxyuridine (IdU) for 24 hr; AKR-PB cells (clone 32C) were used as a positive control for induction (Lowy et al., 1971). Assays for virus were performed by the XC plaque technique and by co-cultivating treated cells with mouse or rabbit cells and then testing for reverse transcriptase activity. Reverse transcriptase reaction mixtures of 100 ~1 contained 40 mM Tris (pH 7.6), 15 mM dithiothreitol. 0.5 mM MnCI,. 66 mM KCI, 0.03% Triton X-100, 0.05 mg bovine serum albumin, 6 PM JH-TTP. 0.004 mg poly(rA)-oligo(dT) or poly(dA)-oligo(dT). Preparation of Viral Specific 3H-DNA The single-stranded JH-deoxycytidine-labeled cDNA Moloney murine leukemia virus probe was a gift from Dr. E. M. Scolnick. The virus was grown in NIH/3T3 cells. The probe was prepared from sucrose density-banded virus, using the endogenous reverse transcriptase reaction in the presence of actinomycin D as previously described by Benveniste and Scolnick (1973). The probe has a specific activity of about 2 X 10’ cpm/pg and is approximately 4s in size. Extraction of Cellular RNA and DNA Total cellular RNA and DNA were isolated from the same cells by cesium chloride centrifugation. The procedure of Glison, Crkven-
Virus 981
Infection
of Teratocarcinoma
Cells
jakov and Byus (1974) was used for the isolation of RNA with the addition that DNA was isolated from the viscous region of the cesium chloride supernate. After removal of cesium chloride by dialysis, the DNA was precipitated with ethanol and resuspended in 1% sodium dodecylsulfate, 100 mM NaCl, 1 mfvl EDTA and 50 mM Tris-HCI (pH 7.8). It was sheared through a 27 gauge needle and incubated for 2 hr at 37°C with previously self-digested pronase (Calbiochem) at a final concentration of 150 fig/ml. Protein was then extracted twice with equal volumes of chloroform:isoamyl alcohol (24:l) and aqueous-saturated redistilled phenol. After precipitation in ethanol, the DNA was sheared at 32,000 lb per square inch in a French pressure cell (American Instrument Co.) to obtain fragments of about 4s. The preparation was made 0.5 N with respect to NaOH, dialyzed extensively against water and stored at -20°C. The A,,:A,, was at least 1.85 for all samples. Hybrtdlzetton Procedure Because only limited quantities of cellular DNA were available, DNA-DNA hybridizations could not be carried out in a vast excess of cellular DNA. Each 0.05 ml sample contained 0.02 M Tris-HCI (pH 7.2), 1.5 M NaCl, 0.1% sodium dodecylsulfate. 5 X 1O-5 M EDTA, 1 Kg carrier calf thymus DNA, 5 pg yeast RNA, 1906 cpm 3H-cDNA probe and I-20 pg cellular DNA. After boiling, each sample was reacted under mineral oil at 67°C for 46 hr. The samples were then digested with S, nuclease, precipitated with TCA and counted on filters (Leong et al., 1972). In the absence of cellular DNA, Sl nuclease degraded >95% of the input counts, while the DNA from chronically infected cells protected a maximum of 67% of the input counts from Sl digestion. This value was normalized to lOO%, and Cot analysis was calculated according to Britten, Graham and Neufeld (1974). The conditions for DNA-RNA hybridizations were as for the DNA-DNA hybridizations, except that 3200 cpm 3H-M~LV cDNA were used and l-50 rg of cellular RNA were added instead of cellular DNA. In the absence of cellular RNA, >95% of the input counts were digested by St (Benveniste and Scolnick, 1973). while the RNA from chronically infected cells protected a maximum of 75% of the counts from digestion. This value was normalized to lOO%, and Crt values were calculated (Birnstiel, Sells and Purdom. 1972) and corrected to 0.16 M monovalent salt concentration (Britten et al., 1974). Under our conditions, unique sequence DNA had Cot,,, = 1000.
We thank Janice Rowe for her skillful assistance throughout the course of these experiments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. June
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29,1977;
revised
September
30,1977
Absence cells
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virus
in a rabbit-
Lehman. J. M., Speers, W. C., Swartzendruber, D. E. and Pierce, G. B. (1974). Neoplastic differentiation: characteristics of cell lines derived from a murine teratocarcinoma. J. Cell Physiol. 84, 13-28. Lehman, J. M.. Klein, I. B. and Ha:kenberg, R. M. (1975). The response of murine teratocarcinoma cells to infection with DNA and RNA viruses. In Teratomas and Differentiation, M. I. Sherman and D. Salter. eds. (Academic Press: New York), pp. 289-301. Leong, J.. Garapin, A., Jackson, N.. Fanshier. L.. Levinson. W. and Bishop, J. M. (1972). Virus-specific ribonucleic acid in cells producing Rous sarcoma virus: detection and characterization. J. Virol. 9, 891-902.
Cell 902
Levy, J. A. (1973). Xenotropic virus: murine leukemia viruses associated with NIH Swiss, NZB, and other mouse strains. Science782, 1151-1153. Love, D. N. and Weiss, R. A. (1974). Pseudotypes of vesicular stomatitis virus determined by exogenous and endogenous avian RNA tumor viruses. Virology57, 271-278. Lowy, D. R.. Rowe, W. P., Teich, N. and Hartley, J. W. (1971). Murine leukemia virus: high-frequency activation in vitro by 5iododeoxyuridine and 5-bromodeoxyuridine. Science 174, 155156. Lowy, D. R.. Chattopadhyay, S. K., Teich, N. M., Rowe, W. P. and Levine, A. S. (1974). AKR murine leukemia virus genome: frequency of sequences in DNA of high-, low-, and non-virusyielding mouse strains. Proc. Nat. Acad. Sci. USA 71, 3555-3559. Martin, G. R. (1975). Teratocarcinomas study of embryogenesis and neoplasia.
as a model system Cell 5, 229-243.
Martin, G. R. and Evans, M. J. (1975a). Differentiation lines of teratocarcinoma cells: formation of embryoid vitro. Proc. Nat. Acad. Sci. USA 72, 1441-1445. Martin, clonal mation
for the
of clonal bodies in
G. R. and Evans, M. J. (1975b). Multiple differentiation teratocarcinoma stem cells following embryoid body in vitro. Cell 6, 467-474.
of for-
Miller, R. A., Ward, D. C. and Ruddle, F. Ii. (1977). Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM. which does infect other teratocarcinoma-derived cell lines. J. Cell. Physiol. 97, 393-402. Mintz, B. and Illmensee, K. (1975). mice produced from teratocarcinoma USA 72, 3565-3569.
Normal genetically mosaic cells. Proc. Nat. Acad. Sci.
Nicolas, J. F., Dubois, P., Jakob, l-l.. Gaillard, J. and Jacob, F. (1975). Differentiation en culture d’une lignee de cellules primitives a potentialites multiples. Ann. lnstitut Pasteur 126A, 3-22. Papaioannou, V. E., McBurney, M. W., Gardner, R. L. and Evans, M. J. (1975). Fate of teratocarcinoma cells injected into early mouse embryos. Nature 258, 70-73. Pierce, concept
G. B. (1967). Teratocarcinoma: model for a developmental of cancer. Curr. Topics Dev. Biol. 2, 223-246.
Pincus, T., Hartley, J. W. and Rowe, W. P. (1971). A major genetic locus affecting resistance to infection with murine leukemia viruses. I. Tissue culture studies of naturally occurring viruses. J. Exp. Med. 133, 1219-1233. Rowe, W. P., Pugh, W. E. and Hartley, J. W. (1970). Plaque assay techniques for murine leukemia viruses. Virology 42, 1136-1139. Stern, P. L., Martin, G. R. and Evans, M. J. (1975). Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation. Cell 6, 455-465. Stevens, L. C. (1956). Studies on transplantable testicular mas of strain 129 mice. J. Nat. Cancer Inst., 20, 1257-1276.
terato-
Stevens, L. C. (1960). Embryonic potency of embryoid derived from a transplantable testicular teratoma of the Dev. Biol. 2, 265-297.
bodies mouse.
Swartzendruber, D. E. and Lehman, J. M. (1975). Neoplastic differentiation: interaction of simian virus 40 and polyoma virus with murine teratocarcinoma cells in vitro. J. Cell. Physiol. 85, 179-106. Ware, L. M. and Axelrad, A. A. (1972). Inherited resistance to Nand B-tropic murine leukemia viruses in vitro: evidence that congenic mouse strains SIM and SIM.R differ at the Fv-1 locus. Virology 50, 339-346. Zavada, J. (1972). Pseudotypas of vesicular stomatitis the coat of murine leukemia and of avian myeloblastosis J. Gen. Virol. 15, 183-191. Note Added Peries
virus with viruses.
In Proof
et al. (J. Nat. Cancer
Inst. 59, 463-465,
1977)
have recently
reported that an undifferentiated teratocarcinoma cell resistant to MuLV infection, whereas nontumorigenic tiated cell lines derived from teratocarcinoma embryoid were sensitive to MuLV infection.
line was differenbodies
December
1977. Copyright
Virus Infection Cell Lines
0 1977 by MIT
of Murine Teratocarcinoma
Natalie M. Teich and Robin A. Weiss Imperial Cancer Research Fund Laboratories P.O. Box 123 Lincoln’s Inn Fields London WC2A 3PX, England Gail R. Martin* Department of Anatomy and Embryology University College London Gower Street London WC1 E 6BT, England Douglas R. Lowy Dermatology Branch National Cancer Institute Bethesda, Maryland 20014
Summary Three clonal murine teratocarcinoma stem cell lines were studied for susceptibility to infection by a variety of different viruses. When in the undifferentiated state, these embryonal carcinoma stem cells are as sensitive to infection by encephalomyocarditis, Sindbis, vaccinia and vesicular stomatitis viruses as are embryo fibroblasts derived from the same mouse strain. The undifferentiated teratocarcinoma cells, however, unlike the fibroblasts, are entirely refractory to infection with murine leukemia virus. If the stem cells are allowed to differentiate to form a variety of differentiated cell types, they become permissive for murine leukemia virus replication at a low level. Several techniques were used to elucidate the block to murine leukemia virus infection. In the one nullipotent cell line which does not differentiate in vivo or in vitro, virus adsorption and penetration are not restricted (as measured by infection with murine leukemia virus pseudotypes of vesicular stomatitis virus), but an integrated proviral DNA copy cannot be detected. In a pluripotent stem cell line, proviral DNA sequences are detected, but neither transcription into viral specific RNA nor viral specific protein synthesis is observed. These findings suggest that control of murine leukemia virus replication in the cells is a function of the stage of differentiation and perhaps also of the genetic composition of the teratocarcinoma stem cells. Introduction The replication of animal viruses in undifferentiated cells has received little attention due to the dearth of suitable experimental systems. It is well * Present Institute,
address: University
Department of Anatomy and Cancer Research of California, San Francisco, California 94143.
Stem
known that some viruses show fastidious requirements for replication; some require propagation in vivo or may grow only in organ cultures that maintain well defined tissue structure and differentiation in vitro. Many viruses, however, will replicate in a variety of- cell types and are conveniently propagated in fibroblasts in vitro. Although fibroblastic monolayer cultures are often regarded as undifferentiated cells, they are, in fact, mesenchymal cells that are committed to the synthesis of specific differentiated products such as collagen, hyaluronic acid and fats which may be produced in vitro (Daniel, Dingle and Lucy, 1961; Goldberg, Green and Todaro, 1963; Hamerman, Todaro and Green, 1965; Green and Kehinde, 1974). The most obvious source of undifferentiated cells is the embryo itself. A useful alternative to the cells of the early mouse embryo has recently become available. Embryonal carcinoma cells, which are the stem cells of teratocarcinomas, are pluripotent cells that can be maintained in the undifferentiated state or can differentiate to a wide variety of cell types, including derivatives of all three primary germ layers (reviewed by Pierce, 1967; Damjanov and Solter, 1974; Martin, 1975). The similarity of such cells to the undifferentiated cells of the normal early mouse embryo has been emphasized by recent experiments that demonstrate the ability of teratocarcinoma stem cells to be incorporated into the blastocyst and participate in normal embryonic development (Brinster, 1974; Mintz and Illmensee, 1975; Papaioannou et al., 1975; lllmensee and Mintz, 1976). The availability of pure cultures of undifferentiated teratocarcinoma cells and their differentiated descendents therefore allows examination of the relationship between the differentiative stage of a cell and its ability to support replication of a variety of animal viruses in comparison to fibroblastic embryo cell cultures derived from the same mouse strain. The embryonal carcinoma cell lines isolated by Martin and Evans (1975a) are capable of differentiating in vitro in a distinct series of steps which parallel normal embryogenesis. These teratocarcinoma stem cells are therefore useful for studies of virus replication at various stages of differentiation. Three distinct clonal lines of tumorigenic embryonal carcinoma stem cells derived from strain 129 mice were used in this study: nullipotent (nulli) embryonal carcinoma cells which apparently do not differentiate in vivo or in vitro; pluripotent (S2) cells which under certain conditions form simple embryoid bodies composed of the stem cells surrounded by a single layer of endodermal cells; and pluripotent (PSA4) cells which under the same conditions form simple embryoid bodies which continue to develop into cystic embryoid bodies
Cell 974
which are fluid-filled cysts containing stem cells and a variety of differentiated cells. The latter two cell lines have the capacity to differentiate further into many cell types in vitro (Evans and Martin, 1975; Martin and Evans, 1975b). In addition, we have used the PYS endodermal cell line derived by Lehman et al. (1974) from a teratocarcinoma as a representative differentiated culture containing a single cell type; upon in vivo inoculation, the PYS cells give rise to parietal yolk sac carcinomas only (Swartzendruber and Lehman, 1975). All types of virus we tested, representing both RNA and DNA animal virus groups, replicate with high efficiency in the undifferentiated teratocarcinoma cells with the exception of the murine leukemia viruses (MuLV), for which no replication was detectable. When the teratocarcinoma stem cells were allowed to differentiate, however, we found that they became permissive for MuLV replication. These results led us to examine more fully the nature of the block to virus replication in the undifferentiated cells in terms of the ability of the virus to penetrate the cell surface, to integrate into the host genome and to express proviral functions.
Results Infection with Various Viruses To assess the capacity of undifferentiated teratocarcinoma ceils to support virus multiplication, we selected representative viruses from five different virus groups for study. The sample included the DNA-containing virus, vaccinia and RNA-containing viruses, encephalomyocarditis (EMC) virus, Sindbis virus, vesicular stomatitis virus (VSV) and murine leukemia virus (MuLV). Undifferentiated cultures of the nulli, S2 and PSA4 cells, as well as the PYS endodermal teratocarcinoma cells and strain 129 mouse embryo fibroblasts as differentiated cell controls, were infected with each virus and assayed for virus production by the appropriate techniques (see Experimental Procedures). A summary of the results obtained is presented in Table
1. Infection
of Teratocarcinoma
Cells with Various Replication
Virus
Table 1. For those viruses that elicit discrete plaques of lysis, there was a one-hit dose-response curve with dilution, and the teratocarcinoma cells generally showed equal or slightly enhanced susceptibility to infection. It is notable that the teratocarcinoma cells (both undifferentiated embryonal carcinoma and endodermal cells) showed a marked resistance to infection with murine leukemia virus. We investigated this finding further and discuss it in the following sections.
Infection of Teratocarcinoma Cells at Various Stages of Differentiation with Murine Leukemia Virus (MuLV) The refractory nature of the undifferentiated teratocarcinoma stem cells to MuLV infection in comparison to fibroblasts of the same mouse strain posed the question whether the teratocarcinoma cells become permissive for MuLV replication when they differentiate. To answer this question, we infected cultures of teratocarcinoma cells with MuLV at various stages of differentiation. The undifferentiatiated pure stem cell cultures, embryonal carcinoma, the simple differentiated embryoid bodies (embryonal carcinoma plus endodermal cells) and the fully differentiated cultures (containing a motley of cell types) were infected and then assayed for virus production either directly (by XC plaque assay and infectious center assay) or indirectly (by titration of supernatant harvests). The results of such experiments are shown in Table 2. Neither the undifferentiated cells nor the endodermal cells appear to support MuLV replication. The fully differentiated cultures, however, showed a low but easily detectable level of virus production. These findings were confirmed with a number of MuLV strains: the N tropic AKR-Gross MuLV and the NB tropic Friend leukemia virus complex (lymphoid leukemia virus and helper-dependent spleen focus-forming virus), as well as the NB tropic Moloney MuLV shown in the table. We did not observe the replication of a B tropic MuLV in
Viruses
in:
Teratocarcinoma Endodermal Cells PYS
Undifferentiated Teratocarcinoma
Type
Group
Strain 129 Mouse Embryo Fibroblasts
Vaccinia
Poxvirus
+
+
+
+
+
EMC
Picornavirus
+
+
+
+
+
Sindbis
Togavirus
+
+
+
+
+
vsv
Rhabdovirus
+
+
+
+
+
MuLV
Retrovirus
+
-
-
-
-
Nulli
Cells 52
PSA4
Virus 975
Infection
Table
2. Infection
of Teratocarcinoma
of Teratocarcinoma
Stage of Differentiation Undifferentiated
Cells
Cells
Cells with Mol-MuLV
at Various
Log,,, PFU/ml ___--XC Plaque
Nulli
10.4
co.4
52
10.4
co.4
PSA4
97 8 5 7 7 5
5 4 NIY 6 ND
’ Not determined.
The second assay procedure involved fusion of the infected PSA4 cells to permissive mouse embryo fibroblasts mediated by Sendai virus. Once again, there was no detectable activation or rescue of the MuLV genome from the PSA4 cells. Finally, we attempted to activate endogenous or exogenous integrated proviral genomes by treatment with the halogenated pyrimidine 5-iodo-2’deoxyuridine (IdU). IdU has been shown to be an efficacious inducer of latent proviruses (Lowy et al., 1971) and can be used to answer two questions. Are there inducible endogenous MuLV genomes in strain 129 fibroblasts or teratocarcinoma cells? After nonproductive infection of teratocarcinoma cells with Mol-MuLV, can the infecting NB tropic MuLV be induced? We treated teratocarcinoma cells, 129-ME cells and the highly inducible AKR-2B cell line with 20 pglml IdU for 24 hr. One day later, we added SC-1 mouse cells or SIRC cells to each culture to amplify the replication of any induced mouse-tropic (ecotropic) or xenotropic virus (Levy, 1973). We passaged the co-cultivated cells at weekly intervals, periodically assayed supernatant fluids for reverse transcriptase activity and monitored the cells for ecotropic MuLV by the XC plaque assay. Although virus was consistently detected from the AKR-2B cell line by 4 days after IdU treatment, there was no virus activity in any of the 129-ME or teratocarcinema cells during 6 weeks of monitoring postIdU treatment. It is known, however, that all mouse cells, including those derived from 129 mice, contain endogenous virus sequences (Lowy et al., 1974; Table 5 above), although no infectious virus has ever been isolated from 129 mice. These data suggest that the endogenous 129 viruses are not readily inducible and are perhaps under more stringent host repression in both fibroblast and teratocarcinoma cells derived from this mouse strain compared to other strains. To determine whether the NB tropic MuLV used for infection could be induced from the teratocarcinema cells, cultures which had been infected in the undifferentiated state were allowed to form
fully differentiated cultures and were then treated with IdU and co-cultivated as described above. We then monitored the subsequent co-cultures weekly for 1 month. In four experiments of this nature, we were able to detect production of MuLV only once. This virus isolate manifested NB tropic host range properties; presumably it represented the activation of the exogenously infecting Mol-MuLV, since no endogenous NB tropic virus has yet been isolated from any inbred mouse strain. The infrequency of activation (one of four attempts), however, raises the possibility of accidental laboratory contamination; this cannot be excluded, although we have no evidence for such contamination in other cell cultures maintained in the laboratory. On the other hand, as we have demonstrated the presence of proviral Mol-MuLV sequences in PSA4 teratocarcinoma cellular DNA by nucleic acid hybridization (Table 5), and considering the difficulty in inducing endogenous viral sequences from strain 129 mouse cells in general, it is not surprising that the frequency of viral activation is low.
Discussion To assess the capacity of teratocarcinoma cells to support viral multiplication, we selected representative viruses from five virus groups for study. The sample included one DNA-containing virus (vaccinia) and four RNA-containing viruses (encephalomyocarditis virus, Sindbis virus, vesicular stomatitis virus and murine leukemia virus). With the exception of the murine leukemia virus (MuLV), these viruses were able to replicate in both the undifferentiated embryonal carcinoma stem cells and their differentiated derivatives. These findings further extend the list of viruses which can replicate in embryonal carcinoma cells. Other embryonal carcinoma cell lines have been shown to be susceptible to infection with human adenovirus type 2 (Kelly and Boccara, 1976) and to a lesser extent with Mengo virus (Lehman, Klein and Hackenberg, 1975). Our observation on MuLV is also consistent with the report that these cells are not permissive for the replication of certain viruses. For example, Miller, Ward and Ruddle (1977) have found that synthesis of the parvovirus, minute virus of mice, as measured by fluorescence staining of viral structural proteins, does not occur in embryonal carcinoma cells but does proceed in fibroblasts derived from teratocarcinoma cells. It has also been shown that differentiation of embryonal carcinoma cells is correlated with an alteration in the response to infection with the papovaviruses, polyoma and SV40 (Lehman et al., 1975; Swartzendruber and Lehman, 1975). They found that early T antigens of both viruses and the late V antigen of polyoma virus were expressed only in those
Virus 979
Infection
of Teratocarcinoma
Cells
cells which had undergone some differentiation within a few days after infection. The results described here indicate that the response of teratocarcinoma cells to infection with murine leukemia virus (MuLV) is dependent upon the particular cell line used and on the state of differentiation of the cells. The nullipotent embryonal carcinoma cells which do not differentiate were refractory to MuLV multiplication. Using MuLV pseudotypes of vesicular stomatitis virus (VSV) (that is, VSV genomes with MuLV envelope glycoprotein coat specificities), we have shown that the nulli embryonal carcinoma cells have the specific receptors for MuLV adsorption and penetration. Nucleic acid hybridization experiments using a labeled DNA probe complementary to the infecting Moloney strain of MuLV indicated that neither DNA nor RNA copies of the virus can be detected in MuLV-infected nulli cells. These results suggest that nulli cells exert a post-penetration, pre-integration block to viral replication. We are currently investigating whether a DNA provirus is synthesized after infection of nulli cells. The S2 and PSA4 teratocarcinoma stem cell lines also exhibited resistance to viral multiplication when the cells were undifferentiated or had formed embryoid bodies (embryonal carcinoma cells surrounded by a layer of endoderm). When the cells were allowed to undergo further differentiation to a variety of ceil types before MuLV infection, however, a small proportion of the cells were capable of sustaining MuLV replication. Further studies are now in progress to determine which cells in the differentiated PSA4 cultures are susceptible to MuLV infection and multiplication. The block to viral replication in the undifferentiated S2 and PSA4 embryonal carcinoma cells is not due to an absence of cell surface receptors for MuLV, as demonstrated by the use of MuLV pseudotypes of VSV. Using nucleic acid hybridization techniques, DNA sequences complementary to the infecting viral genome were found in infected PSA4 cells but not in the uninfected cells. The data indicate that most, if not all, of the sequences of the viral genome are found as proviral DNA copies in the MuLV-infected embryonal carcinoma cells of the PSA4 line. The fact that we were unable to detect the presence of viral RNA above the background level, however, suggests that viral specific RNA is not being transcribed or that it is transcribed but rapidly degraded. Analysis of cell protein extracts by sodium dodecylsulfate-polyacrylamide gel electrophoresis following precipitation with anti-MuLV serum did not reveal any virusspecific structural proteins or precursor proteins (data not shown). Thus the results indicate that the block to MuLV replication in the undifferentiated embryonal carcinoma stem cells of the PSA4
line is presumably post-integration and pre-translation. Further experimentation is necessary to decide whether the block is at the transcriptional level. S2 or PSA4 cells which had been infected as undifferentiated cells or as embryoid bodies did not spontaneously produce virus after they had undergone further differentiation. In many attempts to rescue the latent MuLV genome by cocultivation or fusion to susceptible mouse cells or by treatment with the virus inducer IdU, we were able to detect virus production in only one experiment with IdU. This suggests that the infected teratocarcinoma cells exert stringent regulation on virus expression. It is possible that the entire MuLV genome is not integrated into the DNA of all cells, which would account in part for the low frequency of rescue that we have observed. Alternatively, the viral genome may be integrated in a region of a chromosome which is rarely, if ever, transcribed in this particular cell line, or is not transcribed until differentiation to a specific cell type occurs, and this specific differentiation may occur at a very low frequency in these cultures. Our results with the PSA4 line are comparable to those seen with MuLV infection of mouse 4-8 cell embryos in vitro (Jaenisch, Fan and Croker, 1975; Jaenisch, 1976). MuLV replication was not observed in the infected embryos; mice that were born after implantation of these infected embryos into pseudopregnant mothers, however, did contain integrated DNA copies of the MuLV genome. Every organ tested by nucleic acid hybridization contained at least one copy of the MuLV genome, and the progeny of such mice also contained viral sequences homologous to the original infecting virus, indicating that viral sequences were integrated in the germ line as well. Some of these mice succumbed to the classical viral lymphoma, and tissues containing lymphoma cells expressed more viral specific RNA than did uninvolved tissues (Jaenisch et al., 1975). It will be interesting to see whether chimeric mice formed by injection of MuLV-infected embryonal carcinoma cells into preimplantation embryos develop viral lymphoma. Experimental
Procedures
Cells The homogeneous nullipotent embryonal carcinoma cell line “nulli-SCCl” has apparently lost the ability to differentiate; the line originates from a spontaneous testicular teratocarcinoma LS402C-1664 of a strain 129 mouse (Stevens, 1958). The pluripotent cell lines SCC-SP and PSA4 were derived from a teratocarcinoma OTT-5568 formed by the extrauterine implantation of a day 3 embryo of 129 SvSlJCP genotype (Stevens, 1960). All three clonal embryonal carcinoma cell lines were derived from isolated single cells as previously described (Martin and Evans, 1975a). The culture medium used throughout these experiments was Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose and supplemented with 10% heat-inactivated calf serum. Cells were
Cl?11 960
passaged by disaggregation with 0.25% trypsin in Tris-buffered saline EDTA. All cells were maintained at 37°C. Stock cultures of the pluripotent cell lines were maintained in the undifferentiated state by subculturing the cells every 3 or 4 days and seeding them at 5 X lOa cells per confluent feeder layer of mitomycin Ctreated ST0 mouse cells in a 9 cm tissue culture dish (Martin and Evans, 1975a). Stock cultures of the nullipotent cell line were maintained by subculturing the cells every third day and seeding the cells at 7 X 10’ cells per gelatin-coated 9 cm tissue culture dish. To obtain embryoid body formation, the pluripotent cells were seeded at 10’ ceils per 9 cm tissue culture dish in the absence of added feeder cells. 3 or 4 days later, the cells had formed clumps which were removed from the dish by gentle pipetting. The detached clumps were allowed to settle for several minutes, washed in fresh medium and seeded in a bacteriological dish to which they do not adhere. The endodermal cell layer became apparent within 24 hr. The medium was then changed daily. Within 6 days of appearance of the endodermal cell layer, most of the embryoid bodies formed by the clonal PSA4 line had large fluid-filled cysts, whereas the embryoid bodies formed by the 52 cell line remained as simple two-layered structures. Further differentiation of the cells was obtained by transferring embryoid bodies to tissue culture dishes. Between 190-1000 embryoid bodies were seeded per dish: these attached to the dish and were maintained for up to 1 month with medium changes every second or third day. Nullipotent aggregates (pseudo-embryoid bodies) were obtained by seeding 5 X IO6 cells per 9 cm tissue culture dish and manipulating the clumps which formed the same way as for the clumps of pluripotent cells. The PYS endodermal cell line (Lehman et al., 1974) was provided by Dr. J. M. Lehman. Stock cultures of these cells were maintained by subculturing every 4 or 5 days at approximately lOa cells per 9 cm tissue culture dish. Mouse embryo (ME) fibroblast cultures from strain 129 and BALB/c mice were prepared according to Hartley et al. (1965). The NIH/3T3 and SC-1 mouse embryo cell lines, provided by Dr. J. W. Hartley, have previously been described (Jainchill, Aaronson and Todaro, 1969; Hartley and Rowe, 1975). Primary cultures of C/E chick embryo cells were prepared from 11 day old Leghorn embryos according to standard techniques. The rabbit cornea1 cell line SIRC (Leerhoy. 1965) was purchased from the American Type Culture Collection. The ST0 cells are thioguanine-resistant, ouabain-resistant cultures derived from a continuous line of SIM mouse fibroblasts (Ware and Axelrad. 1972) by Dr. A. Bernstein. They were prepared as feeder cells by treatment with fresh mitomycin C (Sigma) at 10 pg/ml for 2 hr. Virus Stocks and Antiserum Moloney NB tropic murine leukemia virus (Mol-MuLV) was originally provided by Dr. J. Hartley. A seed of vesicular stomatitis virus (VSV) strain Indiana was provided by Dr. C. Pringle. The stock of VSV(MuLV) pseudotypes was prepared by establishing chronically infected MuLV-producing BALB/c cells, infecting with VSV at an moi of 1 and harvesting at 16 hr post-infection. Seed cultures of encephalomyocarditis virus (Dr. D. Rekosh), Sindbis virus (Dr. M. Schlesinger) and vaccinia virus (Mr. S. Tymes) were propagated in 129 mouse embryo fibroblasts prior to use in these experiments. Neutralizing antiserum to VSV prepared from a hyperimmunized sheep was provided by Dr. J. Zavada. Neutralizing antiserum to Mol-MuLV was prepared by hyperimmunization of a rabbit with detergent-disrupted whole virus. Assays of MuLV lnfectlon The standard XC plaque assay (Rowe, Pugh and Hartley, 1970) and the infectious center assay on SC-l cells were used for titration of MuLV infection of all fibroblast cultures. Slight modifications were made for the teratocarcinoma cell lines: undifferentiated stem cells were plated without feeder cells at l-2 X 10”
per 3 cm gelatinized plate and infected 1 or 2 days later after a 1 hr DEAEdextran (25 pg/ml) treatment; embryoid bodies were infected by resuspension in the virus inoculum containing 25 pg/ ml DEAE-dextran and washed after a 1 hr adsorption period at 37°C (virus production was monitored by plating the embryoid bodies on SC-1 cells at various times after infection upon which they would adhere and spread out, followed by the UV-XC assay); and differentiated teratocarcinoma cells were prepared as described above and then infected at 1, 2, or 3 weeks following seeding in tissue culture dishes; cell numbers at the time of infection were not determined due to the difficulty of disaggregating such cultures. In addition, supernatants from all cultures at various times after infection were assayed for virus production by inoculation onto susceptible mouse fibroblast cultures and by the reverse transcriptase assay. Plaque Assays of VW and VSV(MuLV) lnfectlon Plaque assays for VSV and VSV pseudotypes have been described previously (Zavada, 1972; Love and Weiss, 1974). In brief, the undifferentiated teratocarcinoma stem cells were seeded at 3-5 X lOa per 3 cm dish, and the fibroblasts were seeded at l-2 X lv per 3 cm dish. 2 days later, appropriate virus dilutions in IO pg/ ml polybrene were added in a 0.2 ml vol and adsorbed for 1 hr at 20°C. The inoculum was then removed, the cells were washed with phosphate-buffered saline and an agar medium overlay was added. After 2 days at 37”c, neutral red was added for macroscopic counting of the plaques. For assay of VSV(MuLV) pseudotypes. aliquots of the undiluted virus stock were first treated with an equal volume of 1:20 dilutions of VSV antiserum for 1 hr at 20°C or 30 min at 2O”c, followed by an overnight treatment at 4°C. Such treatment was sufficient to neutralize 5.5 logs of VSV. The neutralized virus stocks were then used as inocula as described above. Plaque Assays for Other Viruses Tested EMC. vaccinia and Sindbis viruses were assayed by conventional agar overlay techniques. Cells were seeded at densities described above for assays. Virus inocula were adsorbed for 1 hr at 379: and then removed. Cells were examined daily for a period of 2 weeks for microscopic manifestations of cytopathic effects (CPE). Discrete plaques of dead cells were observed with EMC, vaccinia and Sindbis viruses by l-2 days after infection, and final counts were made at 5-6 days. In addition, supernatant fluids were harvested from wells infected with the highest virus dilutions that showed CPE for assay of virus yield on mouse fibroblast cultures. IdU Induction and Reverse Transcriptare Assays To induce endogenous or exogenously infecting MuLV, cells were treated with 20 pg/ml iododeoxyuridine (IdU) for 24 hr; AKR-PB cells (clone 32C) were used as a positive control for induction (Lowy et al., 1971). Assays for virus were performed by the XC plaque technique and by co-cultivating treated cells with mouse or rabbit cells and then testing for reverse transcriptase activity. Reverse transcriptase reaction mixtures of 100 ~1 contained 40 mM Tris (pH 7.6), 15 mM dithiothreitol. 0.5 mM MnCI,. 66 mM KCI, 0.03% Triton X-100, 0.05 mg bovine serum albumin, 6 PM JH-TTP. 0.004 mg poly(rA)-oligo(dT) or poly(dA)-oligo(dT). Preparation of Viral Specific 3H-DNA The single-stranded JH-deoxycytidine-labeled cDNA Moloney murine leukemia virus probe was a gift from Dr. E. M. Scolnick. The virus was grown in NIH/3T3 cells. The probe was prepared from sucrose density-banded virus, using the endogenous reverse transcriptase reaction in the presence of actinomycin D as previously described by Benveniste and Scolnick (1973). The probe has a specific activity of about 2 X 10’ cpm/pg and is approximately 4s in size. Extraction of Cellular RNA and DNA Total cellular RNA and DNA were isolated from the same cells by cesium chloride centrifugation. The procedure of Glison, Crkven-
Virus 981
Infection
of Teratocarcinoma
Cells
jakov and Byus (1974) was used for the isolation of RNA with the addition that DNA was isolated from the viscous region of the cesium chloride supernate. After removal of cesium chloride by dialysis, the DNA was precipitated with ethanol and resuspended in 1% sodium dodecylsulfate, 100 mM NaCl, 1 mfvl EDTA and 50 mM Tris-HCI (pH 7.8). It was sheared through a 27 gauge needle and incubated for 2 hr at 37°C with previously self-digested pronase (Calbiochem) at a final concentration of 150 fig/ml. Protein was then extracted twice with equal volumes of chloroform:isoamyl alcohol (24:l) and aqueous-saturated redistilled phenol. After precipitation in ethanol, the DNA was sheared at 32,000 lb per square inch in a French pressure cell (American Instrument Co.) to obtain fragments of about 4s. The preparation was made 0.5 N with respect to NaOH, dialyzed extensively against water and stored at -20°C. The A,,:A,, was at least 1.85 for all samples. Hybrtdlzetton Procedure Because only limited quantities of cellular DNA were available, DNA-DNA hybridizations could not be carried out in a vast excess of cellular DNA. Each 0.05 ml sample contained 0.02 M Tris-HCI (pH 7.2), 1.5 M NaCl, 0.1% sodium dodecylsulfate. 5 X 1O-5 M EDTA, 1 Kg carrier calf thymus DNA, 5 pg yeast RNA, 1906 cpm 3H-cDNA probe and I-20 pg cellular DNA. After boiling, each sample was reacted under mineral oil at 67°C for 46 hr. The samples were then digested with S, nuclease, precipitated with TCA and counted on filters (Leong et al., 1972). In the absence of cellular DNA, Sl nuclease degraded >95% of the input counts, while the DNA from chronically infected cells protected a maximum of 67% of the input counts from Sl digestion. This value was normalized to lOO%, and Cot analysis was calculated according to Britten, Graham and Neufeld (1974). The conditions for DNA-RNA hybridizations were as for the DNA-DNA hybridizations, except that 3200 cpm 3H-M~LV cDNA were used and l-50 rg of cellular RNA were added instead of cellular DNA. In the absence of cellular RNA, >95% of the input counts were digested by St (Benveniste and Scolnick, 1973). while the RNA from chronically infected cells protected a maximum of 75% of the counts from digestion. This value was normalized to lOO%, and Crt values were calculated (Birnstiel, Sells and Purdom. 1972) and corrected to 0.16 M monovalent salt concentration (Britten et al., 1974). Under our conditions, unique sequence DNA had Cot,,, = 1000.
We thank Janice Rowe for her skillful assistance throughout the course of these experiments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. June
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29,1977;
revised
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have recently
reported that an undifferentiated teratocarcinoma cell resistant to MuLV infection, whereas nontumorigenic tiated cell lines derived from teratocarcinoma embryoid were sensitive to MuLV infection.
line was differenbodies
