Friend murine leukemia virus and spleen focus-forming virus expression in highly malignant interferon-sensitive and interferon-resistant friend leukemia cells

VIROLOGY

150,390-401

(1986)

Friend Murine Leukemia Virus and Spleen Focus-Forming Expression in Highly Malignant Interferon-Sensitive and Interferon-Resistant Friend Leukemia Cells C. OPPI, G. FIORUCCI, L&oratory

M. FERRANTINI,

of Virdogy,

Istituta

Received

Super&e

June

A. BATTISTINI,

di Saniki,

6, 1985; accepted

Vial

Regina

December

AND Elena

Virus

F. BELARDELLI’

299, Rome

00161, It&

11, 1985

Analysis of expression of the Friend murine leukemia virus (F-MuLV) and of the spleen focus forming virus (SFFV) has been undertaken in highly malignant interferon (IFN)sensitive (745) and IFN-resistant (3Cl-8) Friend leukemia cells (FLC), serially passaged intraperitoneally in DBA/B mice. In &JO passaged 745 cells, as well as the clones derived thereof, did not release Friend virus (FV). Western blot analysis of the plasma membrane fractions of the virus nonproducer 745 cells revealed the lack of gp69/70 glycoprotein expression. At least 10 intraperitoneal passages of virus producer in vitro passaged 745 cells were necessary to obtain the selection of the virus nonproducer phenotype. In contrast in tivo passaged 3Cl-8 cells continued to produce FV even after 100 in tivo passages and expressed gp69/70 antigens to a similar extent as the original in vitro passaged FLC. The expression of F-MuLV and SFFV RNAs in virus producer and virus nonproducer FLC clones has been investigated by means of Northern blot technique using probes specific for either F-MuLV or SFFV. No F-MuLV specific RNA sequences were detected in virus nonproducer 745 clones. SFFV specific RNA transcripts and gp52/55 glycoprotein production could be revealed in all the FLC tested. Southern blot analysis showed the presence of F-MuLV specific sequences in the cellular DNA of virus nonproducer 745 clones. As both in tiwo passaged F-MuLV producer 3Cl-8 and F-MuLV nonproducer 745 cells were equally barely immunogenic and highly malignant when injected into syngeneic DBA/2 mice, these results indicate that F-MuLV expression does not result per se in a high immunogenic potential of tumor cells. For the time being, as a specific property of 3Cl-8 versus 745 cells is the interferon-resistant phenotype, it is tempting to speculate that the selection of virus nonproducer cell variants after in W&JOpassages of interferon-sensitive 745 cells could depend on the presence of low levels of endogenous interferon in normal young mice. 8 1986 Academic

Press, Inc.

INTRODUCTION

Murine Friend leukemia cells (FLC) are derived from committed erythroid stem cells infected with FV. These cells were isolated and cultivated in vitro after repeated passage of virus-infected spleen cells as subcutaneous solid (Friend et al, 1966, 1971; Ostertag et al, 1972) or ascitic (Ikawa and Sugano, 1966) tumors in mice. FLC are chronically infected with FV, which is generally shed into the culture supernatants. In previous studies we had observed that cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) FLC (Affabris et

The Friend erythroleukemia virus complex (FV) (Friend, 1957) induces a rapidly fatal erythroleukemic disease syndrome in genetically susceptible mice. FV consists of two major components: (1) the replication-competent ecotropic Friend virus, i.e., Friend murine leukemia virus (F-MuLV), and (2) the replication-defective spleen focus-forming virus (SFFV) (Axelrad and Steeves, 1964). 1 To whom dressed. 0042~6322/86 Copyright All rights

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reprints

$3.00

Q 1986 by Academic Press, Inc. of reproduction in any form reserved.

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390

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VIRUS

AND

FRIEND

a& 1982), continuously passaged in vitro, were not very tumorigenic when first injected ip into syngeneic DBA/Z mice, although they did form solid tumors when injected SC(Belardelli et al, 1984a). By serially passaging FLC (either 745 or 3Cl-8 cells) ip in DBA/B mice we obtained two different FLC lines capable of growing ip and inducing tumor ascites. The SCinjection of DBA/Z mice with these in viva passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated SC with in vitro passaged FLC. Moreover clones derived from in wivo passaged FLC maintained the highly malignant phenotype even after several in vitro subcultivations (Belardelli et al, 1984a). The acquisition of the highly malignant phenotype of the in viva passaged FLC paralleled specific changes in cell surface glycoproteins of tumor cells (Amici et cd, 1984). Retrovirus expression in tumor cells has been generally associated with increased immunogenicity and decreased tumorigenicity of tumor cells transplanted in syngeneic animals (Silagi et al, 1972; Bauer, 1974; Levy and Leclerc, 1977; Old and Stockert, 1977). In particular, Reading and co-workers (1980) demonstrated an inverse correlation between the highly malignant phenotype of murine lymphosarcoma cells (obtained by in viva selection of metastatic variants) and expression of RNA tumor virus envelope glycoprotein gp70. It seemed of interest, therefore, to study F-MuLV and SFFV expression in highly malignant clones derived from in viva passaged FLC. In preliminary experiments we had observed that clones derived from in wivo passaged 745 cells did not release any detectable amount of FV as determined by reverse transcriptase assay in the culture supernatants (Belardelli et cd, 1984a). In the present study we analyzed the expression of F-MuLV and SFFV genomes in virus nonproducer clones derived from in tivo passaged interferon-sensitive 745 cells. This type of study has been extended to FLC derived from in viva passaged interferon-resistant 3Cl-8 cells and the results obtained are also reported in this article.

TUMOR

CELL

VARIANTS

MATERIAL

AND

391 METHODS

Mice. Male and female 6- to 8-week-old DBA/B mice were obtained from Charles River Italia S.p.A. (Milan). Tuwwr cells. Interferon-sensitive (745) and interferon-resistant (3Cl-8) FLC (Affabris et al, 1982), as well as clones derived from in tivo passaged FLC (745-E7, 745C2,745-D3, and 3Cl-8-F8) (Belardelli et al, 1984a), were grown in RPM1 medium (Flow, Irvine, Scotland) supplemented with 10% fetal calf serum (FCS). Clones derived from in viva passaged FLC maintained the highly malignant phenotype of the in viva passaged cells even after several in vitro subcultivations. The in vitro growth curves of these clones were identical to those of the original in vitro passaged FLC. In tivo passaged FLC were maintained by weekly ip injections in 5- to 8week-old DBA/Z mice. Determimtian of sensitivity ar resistunce to interfertm FLC were seeded at 5 X lo5 cells/ml at 37” with or without 200 U/ml mouse (Y/P interferon (prepared from mouse sarcoma C243 cells inoculated with Newcastle disease virus (Mechti et uL, 1984), sp act 2 X 10’ reference units/mg of protein). Twenty hours later cultures were centrifuged and cell pellets were infected with 1 PFU/cell of vesicular stomatitis virus (VSV) (1 hr at 37’ in water bath). Cells were then washed three times with culture medium and reseeded at 5 X lo5 cells/ml. Each culture was harvested 12 hr after infection and supernatants were titrated on confluent L929 cell monolayers for evaluation of virus yields. Reversd trunscriptuse activity assay. The assay was performed in a final volume of 100 ~1 containing 20 mMTris-HCl, pH 7.8, 60 mM NaCl, 1 mM MnC12, 5 mM dithiothreitol, 0.015% Nonidet-P40, 50 pg/ml poly(A) (Sigma Chemical Co., St. Louis, MO.), 5 pg/ml oligo (dt)12-18(P-L Biochemicals, Inc., Milwaukee, Wise.), 2 $lf TTP, 20 &i/ml [aHITTP (Radiochemical Centre, Amersham, UK). Samples of clarified supernatants, ranging from 10 to 30 ~1, with additional medium containing 10% fetal calf serum to a total volume of 30 ~1, were added to the reaction mixtures. To rule out

392

OPPI ET AL.

artifactual results due to the presence of inhibitors in the fluids, the enzymatic activity was derived from the linear portion of the curves. Values were expressed as TTP picomoles incorporated into acid-insoluble materials per lo6 cells. Analysis of FV antigens on FLC membranes bj.4 indirect immuni&oresc4mce technique. Conventional monospecific goat serum against gp69-70 (the major envelope glycoprotein of FV), was obtained from Dr. D. P. Bolognesi (Duke University Medical Center). One milliliter of the immune serum was absorbed three times at +4’ for 1 hr with 2 X 10’ spleen lymphocytes from normal DBA/Z mice. Aliquots corresponding to 2 X lo6 FLC were centrifuged, washed, and incubated at 0” for 30 min with 200 ~1 of anti-gp69/70 serum, diluted 1:lOO in phosphate-buffered saline (PBS). After further washing, cells were incubated with 100 ~1 of fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat immunoglobulin (Nordic, Tilburg, The Netherlands), diluted 1:20 in PBS. After 30 min at 0”, cells were washed, placed on a microscope slide, covered with a coverslip, and examined. Fluorescence microscopy and photography were performed with a Leitz fluorescence microscope equipped with a Ploem illuminator and a Leitz automatic microscope camera. Detection of FV protein expression b western blot analysis. Partially purified plasma membrane fractions were obtained as described by Gazitt and Friend (1981). Briefly, cell pellets were lysed in cold 5 mM Tris-HCl, pH 7.4,2.5 mM MgClz for 10 min at 4”. After centrifugation at 100 g for 5 min, pellets (nucleated ghosts) were incubated for 5 min in 0.25 ml of 0.1% (v/v) Nonidet-P40 in the same buffer at 4” and then homogenized to disrupt plasma membranes. The homogenates were centrifuged (100 g for 5 min) and the intact nuclei were separated from the supernatants (containing the plasma membrane fractions). Plasma membrane fractions were processed for electrophoresis according to the method described by Laemmli (1970). Aliquots of samples corresponding to equal number of cells (2 X 106) were prepared with a final concentration of 1% SDS, l-

2% mercaptoethanol, 1:lO electrode buffer, 20% ethylene glycol, 0.001% bromophenolblue, boiled 2 min, and analyzed on 8.5% polyacrylamide slabs gels. Molecular weight standards were 14C-methylated markers (Radiochemical Centre). After electrophoresis proteins were transferred into nitrocellulose membrane (Bio-Rad) as described by Towbin and coworkers (1979). Nitrocellulose membrane was saturated by overnight incubation at room temperature in PBS containing 2% bovine serum albumine (BSA). Western blot analysis was performed using goat serum against gp69/70 (obtained from Dr. D. P. Bolognesi), diluted 1:lOOin PBS containing 2% BSA (incubation at room temperature for 6 hr under gentle shaking). After several washings with PBS, rabbit serum against goat Ig was added. After 4 hr at room temperature, nitrocellulose membrane was extensively washed in PBS and protein A (l%I-labeled) (New England Nuclear) was added (1 X lo5 cpm/ml, diluted in PBS containing 2% BSA). After 1 hr at room temperature nitrocellulose membrane was further washed in PBS (last washing in PBS containing 1% Triton X100), dried, and exposed to Kodak X AR 5 Xray film at -80”. Preparations of nucleic acids and gel blotting. Total cellular RNA was purified by means of guanidine hydrochloride extraction procedure (Adams et d, 1977). Ten micrograms of total RNA (denatured in 2.2 M formaldehyde, 50% (v/v) formamide at 60”) was applied to each lane of a 1% w/v agarose gel containing 2.2 Mformaldehyde (Rave et aL, 1979). Electrophoresis was carried out for 18 hr at 30 V. Transfer of RNA to Genescreen membrane (NEN) was accomplished at 10 V overnight using a BioRad apparatus. The filters were baked for 2 hr at 80” in a vacuum oven and hybridized according to Thomas (1980). Ten micrograms of high molecular weight DNA, prepared as described (Eva et c& 1983), were digested with BamHI (Boehringer-Mannheim) under the conditions suggested by the manufacturer, electrophoresed in horizontal agarose (0.7% w/ v) gels (18 hr at 30 V), and blotted to nitrocellulose according to the technique de-

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AND

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scribed by Southern (1975). Hybridization was carried out under stringent conditions (50% formamide and fivefold-strength standard saline citrate at 42“) for 48 hr. RNA and DNA filters were both hybridized with 2 X lo6 cpm/ml of hybridization buffer, of nick translated (Rigby et a& 1977) =P-labeled gel eluted (Chouith et al, 1979) viral DNA plasmid inserts. DNA probes. The probe specific for ecotropic sequences was a 0.4-kb BumHISum1 cloned fragment of the F-MuLV env gene, not hybridizing with xenotropic and/ or MCF viral genomes. The probe specific for xenotropic, MCF, and SFFV env genes was a 0.4-kb BamHI-SmuI fragment of the cloned genome of SFFVp. Both these probes (the clone E57BSl and the clone S23BS4,respectively) were kindly provided by Dr. F. Moreau-Gachelin (Moreau-Gachelin et al, 1983). The genome of F-MuLV clone 201, molecularly cloned in pBR 322 (clone 57 (Oliff et aL, 1980)) recognizing both F-MuLV and SFFV sequences, was kindly provided by Dr. A. I. Oliff. RESULTS

Stability of the Phenotype of Sensitivity or Resistance to Interferon in Highly Malignant Clones Derived from in vivo Passaged FLC Table 1 shows the results of a representative experiment in which different types of FLC (either passaged in vitro or clones derived from in vivo passaged cells) were tested for sensitivity or resistance to the inhibitory effect of interferon on virus (i.e., VSV) production. Treatment of in vivo passaged 745 cells with 200 units/ml of mouse interferon CJ p resulted in a lOOO-fold inhibition of VSV yield with respect to the control untreated cultures, whereas no significant reduction of virus production was detected in in vivo passaged 3Cl-8 cells incubated with the same amount of interferon. Furthermore, clones derived from in vivo passaged FLC retained the original phenotype of sensitivity or resistance to the inhibitory effect of interferon on virus production. The phenotype of sensitivity or resistance to the inhibitory effect of interferon on cell mul-

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393

VARIANTS TABLE

1

STABILITY OF SENSITIVP~YOR RESISTANCE TO THE bHIBITORY EFFECTS OF MOUSE INTERFERON 018ON VSV MULTIPLICATION

In vitro

Cell type

treatment with 200 U/ml of interferon

Mean VSV yield/O.2 ml (log,,) k SE’

745, passaged in vitro

No

Yes

8.5 * 0.3 5.3 f 0.4

745, passaged in viva (~68)”

No Yes

7.7 f 0.3 4.7 f 0.1

745-E7

No Yes

8.3 + 0.2 5.0 + 0.2

3Cl-8, passaged

No

Yes

8.0 + 0.1 8.3 + 0.4

3Cl-8, passaged in tivo (p69) a

No Yes

7.5 + 0.4 7.3 k 0.1

3Cl-8-F8

No Yes

8.3 + 0.2 8.3 -+ 0.3

in vitro

“FLC recovered from mice injected with in vivo passaged FLC were tested for sensitivity or resistance to the inhibitory effect of interferon on VSV production after two passages in vitro. b There were three virus-infected cultures for each experimental condition.

tiplication was also conserved in the clones derived from in uivo passaged FLC (data not shown). As previously demonstrated (Belardelli et cd, 1984a), 745-E7, 745-C2, 745-D3, and 3Cl-8-F8 cells induced hemorrhagic ascites when injected ip and metastasized to the liver and the spleen when injected SCin DBA/Z mice. Friend Virus Release and Virus Protein Expression The results reported in Table 2 indicate that in vivo passaged 745 cells, as well as the clones derived thereof, did not release any detectable reverse transcriptase activity in the culture supernatants, whereas marked levels of reverse transcriptase activity were detected in the supernatants of the original in vitro passaged 745 cells (Experiment 1). On the contrary both in vivo

OPPI ET AL.

394

TABLE

2

FRIEND VIRUS RELEASE IN 745 AND 3C1-8 CELL CLONES AS DETERMINED BY REVERSE TRANSCRIPTASE ACTIVITY ASSAY Reverse transcriptase Cell type

Expt 1

745, passaged in vitro 745, passaged in vivo (p92) b ‘745-E7 745-C2 745-D3 3Cl-8, passaged in vitro 3Cl-8, passaged in viva (p92) b 3Cl-S-F8

31.5 Below detection Below detection Below detection

activity

(TTP pmol/106 cells)’

Expt 2 53.0 Below detection Below detection

44.2 76.3 25.3

Expt 3 66.2 Below detection Below detection

53.8 79.1 57.7

‘Different types of FLC were seeded (5 X 106 cells/ml) in 35-mm petri dishes (Falcon) in RPM1 medium supplemented with 10% FCS. After 24 hr at 37” cells were counted, centrifuged, and supernatants were clarified (10,OOOgfor 10 min). Aliquots of culture supernatants were tested for reverse transcriptase activity as described under Methods. b Tested for RT activity after two passages in vitro.

passaged 3Cl-8 cells, as well as 3Cl-8-F8 cells, released FV in the culture medium (as determined by reverse transcriptase activity assay) in similar amounts to the original in w&o passaged 3Cl-8 cells, (Table 2, Experiments 2 and 3). These results indicate that serial in viva passages of interferon-sensitive 745 cells had resulted in the selection of virus nonproducer 745 cells, whereas serial intraperitoneal passages of interferon-resistant 3Cl-8 cells had not determined any significant change of FV production. Figure 1 shows FV antigen expression on cell membrane of the original in vitro passaged 745 cells (Fig. 1A) and of 745-E7 cells (Fig. 1B) as determined by indirect immunofluorescence technique using a goat serum against gp69/70 (the major envelope glycoprotein of the F-MuLV). 745-E7 cells expressed very low levels of gp69/70-like antigens on the cell membrane as compared to the original in vitro passaged 745 cells. Similar immunofluorescence patterns have been obtained using other virus nonproducer clones derived from in vivo passaged 745 cells (data not shown). As the low expression of gp69/70-like antigens on the cell membrane of virus nonproducer 745 clones could depend on the expression of SFFV gp52/55 glycoprotein

(sharing some similar antigenic determinants with gp69/70 F-MuLV glycoprotein), we performed Western blot analysis using antibody to gp69/70 on plasma membrane fractions of the different FLC types. As can be seen in Fig. 2, in vivo passaged 745 cells (lane 2), as well as 745-E7 cells (lane 3), did not express gp69/70 antigens, which were clearly detected on plasma membrane fractions of the original virus producer in vitro passaged 745 cells (lane 1). On the contrary 3Cl-8 cells passaged in vitro (lane 4), 3C18cells passaged in viva (lane 5) and 3Cl-8-F8 cells (lane 6) expressed similar amounts of gp69/70 glycoprotein on the cell membrane. No significant differences were revealed between all the cell types with respect to the expression of the SFFV gp52/ 55 glycoprotein, also recognized by antibody to gp69/70 antigen. As 745-E7,745-C2,745-D3, and 3Cl-8-F8 cells had been cloned from in viva passaged FLC after 29 ip passages in DBA/B mice, it seemed of interest to determine the exact number of ip passages required to obtain the virus nonproducer phenotype of 745 cells. We started, therefore, a new series of ip passages of 745 cells, determining the capacity of tumor cells to release FV after different ip passages. Although a decrease in the FV release was already detected af-

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395

Expression of F-MuL V and SFF V RNAs in Virus Producer and Virus Nonproducer FLC Clones In order to discern whether the absence of FV release in virus nonproducer 745 clones was due either to a transcriptional defect or to a translational block in the expression of FV proteins, we performed Northern blot analysis with the following %P-labeled probes: (1) a probe specific for ecotropic sequences (0.4 kb BarnHI-SmaI cloned sequences of F-MuLV), recognizing the F-MuLV transcripts but not the SFFV ones; (2) a probe specific for xenotropic, MCF, and SFFV sequences (0.4 kb BarnHISmaI fragment of the cloned genome of SFFVp (Moreau-Gachelin et c& 1983), not hybridizing with F-MuLV transcripts; (3) the cloned genome of F-MuLV (8.5 kb) hybridizing with both F-MuLV and SFFV (Oliff et al, 1980). As can be seen in Fig. 3A, RNA blot analysis of in vitro passaged either 745 (lane 1) or 3Cl-8 (lane 4) cells, using the ecotropic specific probe, revealed the expression of 38 S and 24 S F-MuLV transcripts, whereas no ecotropic-related sequences were detected in virus nonproducer 745 clones (lanes 2 and 3). In contrast, 3Cl8-F8 cells (clone derived from in vivo pas-

FIG. 1. Expression of FV antigens on the cell membrane of virus producer 745 and virus nonproducers 745-E7 cells as determined by indirect immunofluorescence technique using anti gp69-70 serum. (A) 745 cells (original in vitro passaged cells). (B) 745-E7 cells. 3500X.

ter 2 ip passages, 10 in tivo passages were necessary to get the appearance of the virus nonproducer phenotype (data not shown). On the contrary even 100 ip passages of interferon-resistant 3C1-8 cells in DBA/Z mice did not result in any significant change in the capacity of 3Cl-8 cells to release FV, as determined by reverse transcriptase activity assay in the culture supernatants (data not shown).

gp69/70

e

9P=-

FIG. 2. Western blot analysis of FV proteins on FLC plasma membrane fractions using anti gp69-70 serum. Lane 1: 745 cells (passaged in vitro). Lane 2: 745 cells (60 passages in tivo). Lane 3: 745-E7 cells. Lane 4: 3 Cl-8 cells (passaged in vitro). Lane 5: 3 Cl-8 cells (60 passages in viva). Lane 6: 3 Cl-&F8 cells.

OPPI ET AL.

396 123456

123456

-32s

FIG. 3. Detection of F-MuLV and SFFV RNAs in virus producer and virus nonproducer FLC clones. Ten micrograms of total RNA extracted from different cell types were electrophoresed on agarose gels and the RNAs were hybridized to nick-translated q-labeled ecotropic-specific probe (A) or to xenotropic-specific probe (B). Molecular weights were determined using ethidium-bromide stained endogenous 18 and 28 S mRNAs and Esc&richia coli 23 and 16 S rRNAs as markers. (A) Lane 1: RNA from in vitro passaged 745 cells. Lane 2: RNA from 745-E7 cells. Lane 3: RNA from 745-C2 cells. Lane 4: RNA from in vitro passaged 3Cl-8 cells. Lane 5: RNA from 3Cl-8-F8 cells. Lane 6: RNA from NIH 3T3 cells. (B) Lane 1: RNA from NIH 3T3 cells. Lane 2: RNA from in vitro passaged 745 cells. Lane 3: RNA from 745-E7 cells. Lane 4: RNA from 745-C2 cells. Lane 5: RNA from in vitro passaged 3Cl-8 cells. Lane 6: RNA from 3Cl-8-F8 cells.

saged interferon-resistant FLC) (lane 5) did show F-MuLV RNA expression in similar amounts to the original in vitro passaged 3Cl-8 cells. 12

34

c = FIG. 4. Expression of F-MuLV and SFFV RNAs in FLC clones, detected using the entire cloned genome of F-MuLV. Northern blot was performed as described under Methods. Molecular weight standards are those described in the legend to Fig. 3. Lane 1: total RNA from in vitro passaged 745 cells. Lane 2: total RNA from 745-E7 cells. Lane 3: total RNA from 745X2 cells. Lane 4: total RNA from NIH 3T3 cells.

Figure 3B shows the Northern blot analysis of the same FLC clones using the xenotropic-specific probe. No significant differences were detected in the expression of SFFV related RNA sequences between the different FLC clones. The two major bands corresponded to 32 S genomic RNA and 21 S env-related RNA transcripts. Figure 4 shows the Northern blot analysis of RNAs from in vitro passaged 745 cells (lane 1) and from 745E7 (lane 2) and 745X2 (lane 3) cells, hybridized with the entire cloned genome of F-MuLV. Neither 38 S F-MuLV genomic RNA nor 24 S subgenomic F-MuLV RNA expression could be detected in both 745-E7 and 745-C2 cells, further indicating that these virus nonproducer cells did not express any F-MuLV related normal transcript. RNAs of 32 and 21 S in 745-E7 and 745-C2 cells are likely to represent SFFV genomic and subgenomic transcripts, respectively. Detection of F-MuLV DNA Sequences in Vims Producer and Virus Nonproducer FLC Clones In order to analyze whether F-MuLV sequences were present in the cellular DNA

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of virus nonproducer 745 cell clones, Southern blot analysis was performed using the ecotropic-specific probe to avoid hybridization with SFFV and endogenous virus related sequences. Figure 5 shows that the DNAs isolated from virus producer 745 (lane l), 3Cl-8 (lane 3), and 3Cl-8-F8 (lane 4) cells, as well as from virus nonproducer 745E’7 cells (lane 2) (digested with BarnHI), revealed a O-9-kb band, which was absent in the DNAs isolated from NIH 3T3 cells (lane 5) and spleen cells from DBA/B mice (lane 6). According to a previously published restriction map (Moreau-Gachelin et aL, 1983), the 0.9-kb BumHI F-MuLV specific fragment corresponds to the internal envrelated BumHI fragment of the cloned FMuLV (Oliff et &, 1980), whereas F-MuLV sequences adjacent to this fragment do not hybridize with the ecotropic-specific probe (Moreau-Gachelin et d, 1983). These data indicate that F-MuLV specific DNA sequences are integrated in the genome of virus nonproducer 745 clones. As shown in Table 3. treatment of virus 123456

0.9 kb

FIG. 5. Detection of F-MuLV DNA sequences in FLC clones. High molecular weight DNAs (10 pgcg)from in vitro passaged 745 cells (lane l), ‘745E7 cells (lane 2), in vitro passaged 3Cl-8 cells (lane 3), 3Cl-8-F8 cells (lane 4), NIH 3T3 cells (lane 5), and spleen cells (DBA/ 2 mice) (lane 6) were digested with BarnHI, electrophoresed in 0.7% agarose gels, blotted and hybridized to 8ep-labeled nick translated ecotropic-specific probe. Hind111 fragments of phage EDNA served as molecular weight standards.

TUMOR

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VARIANTS TABLE

397 3

5-A!ZACYTKnNE (5 AZACYD) &DUCFION OF RETR~VIRUS PRODUCTION IN VIRUS NONPRODUCER 745 CLONES’

Cell type 745 (Original in vitro

passaged cells) 745-E7

745-D3

Treatment None 5 azacyd, 0.1 k&ml 5 azacyd, 0.4 a/ml

Reverse transcriptase (TTP pm011 lo6 cells) 155.0 124.5 113.5

None 5 azacyd, 0.1 &ml 5 azacyd, 0.4 dml 5 azacyd, 1.0 fig/ml

Below detection 2.7

None 5 azacyd, 0.1 Irsdml 5 azacyd, 0.4 dml 5 azacyd, 1.0 &ml

Below detection 1.5

11.6 8.4

5.7 24.0

DDifferent types of 745 cells (lo6 cells/ml) were cultivated in RPM1 medium supplemented with 10% FCS in the presence of different doses of 5-Azacytidine (Sigma, St. Louis, MO.) as indicated. After 20 hr at 37”, cells were washed twice and seeded in fresh medium (5 X 106 cells/ml). After 24 hr at 37”, cells were counted, centrifuged and supernatants were clarified (10,000 Q. for 10 min). Aliquots of culture supernatants were tested for reverse transcriptase activity as described under Methods.

nonproducer 745 clones with 5-Azacytidine (5 azacyd), a potent inducer of type C virus (Niwa and Sugahara, 1981), resulted in the induction of virus production, as determined by reverse transcriptase activity in the culture supernatants. No increase of virus production was observed after treatment of virus producer 745 cells with the corresponding doses of 5 azacyd. These data seem to support the hypothesis of a repression mechanism of F-MuLV expression in virus nonproducer 745 cells.

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tential of the in titro passaged FLC. (2) As SFFV RNAs appeared to be expressed to The results reported in this article in- a similar extent in all the FLC studied, it seems likely that SFFV transcription and, dicate that serial ip passages of interferonin particular, gp52/55 glycoprotein sensitive 745 cells in normal young DBA/ 2 mice resulted in the selection of virus expression, represent inherent properties nonproducer highly malignant 745 cells of FLC, and that differences in the FLC lacking the expression of F-MuLV. Like- malignant phenotype do not result in any wise clones derived from in vivo passaged significant change in SFFV expression. Host immune response to tumor-asso745 cells, which maintained the highly maciated antigens has been widely studied in lignant phenotype when injected in DBA/ 2 mice (Belardelli et c& 1984a), did not re- type C RNA virus-induced murine tumor lease FV (Table 2). Ecotropic specific se- systems. These tumors tend to be quite anquences of the F-MuLV could be detected tigenic and evoke humoral and cell-mein the cellular DNAs of virus nonproducer diated immunity to tumor associated an745 clones (Fig. 5, lane 2), but no F-MuLV tigens and/or viral antigens (reviewed by RNA transcripts were revealed in these Bauer, 1974; Levy and Leclerc, 1977; Old cells by Northern blot technique using two and Stockert, 1977). In particular some exdifferent 32P-labeled F-MuLV probes (Fig. perimental data indicate that gp69/70 an3A, lanes 2 and 3; Fig. 4, lanes 2 and 3). In tigens can be immunogenic in mice. For incontrast, highly malignant in viva pas- stance, some mouse strains can be immusaged interferon-resistant 3Cl-8 cells, as nized against gp69/70 (Callahan et al, 1979; well as cloned 3Cl-8-F8cells, were still ca- Boiocchi and Nowinski, 1979). However, pable of expressing F-MuLV sequences although some authors indicated a possible (Fig. 3A, lane 5) and of releasing FV (Table relationship between expression of gp70 2, Experiments 2 and 3) in similar amounts antigens on tumor cells and immunogenicto the original in vitro passaged cells. SFFV ity (Silagi et ak, 1972; Reading et uL, 1980), RNA transcripts (Fig. 4) and gp52/55 gly- our finding that gp69/70 antigens are norcoprotein (Fig. 2) were expressed to a sim- mally present in highly malignant 3Cl-8 ilar extent in all FLC tested. cells suggests that expression of gp69/70 We have previously demonstrated that glycoprotein does not simply result in a in vitro passaged FLC (either 745 or 3Cl-8 high immunogenic potential of tumor cells. cells) were not capable of forming ascites Moreover, although antibodies to FLC when first injected ip in DBA/B mice (Be- could be detected using Western blot techlardelli et aL, 1984a). These cells appeared nique in sera from mice injected with virus to be highly immunogenic; high titers of producer in vitro passaged FLC, no specific antibodies to FLC were detected in serum antibodies to gp69/70 could be found in from mice injected ip with these cells, these sera (our unpublished observations). whereas antibodies to FLC were barely re- The apparent lack of specific immune reacvealed in sera from mice injected with in tivity to gp69/70 may be due to tolerance vivo passaged FLC (our unpublished data). induced by endogenous xenotropic MuLV Two major conclusions can be drawn gp69/70 expression. In fact this antigen is from our results: (1) As both in viva pas- normally expressed in thymus, spleen, and saged F-MuLV nonproducer 745 and F- other tissues in several strains of mice MuLV producer 3Cl-8 cells are equally tu- (Lerner et aL, 1976; McClintock et aL, 1977; morigenic (Belardelli et aL, 1984a) and Morse et aL, 1979). It is also possible to enpoorly immunogenic when injected in visage that the lack of gp69/70 reactivity DBA/B mice, we can assume that the may be due to specific suppressor cells expression of F-MuLV does not play a sig- (Caulfield and Cerny, 1980). Several articles have been published on nificant role per se in the appearance of the highly malignant phenotype of in viva the isolation and characterization of virus passaged FLC or in the immunogenic po- nonproducer FLC. Virus nonproducer FLC DISCUSSION

FRIEND

VIRUS

AND

FRIEND

(HFL/d) have been isolated by Freedman and co-workers (Freedman and Lilly, 1975; Freedman et al, 1978) after in vitro cultivation of tumor cells from BALB/c (H-p) mice infected with FV. These cells were reported to be capable of releasing both infectious SFFV and F-MuLV in early passages in culture. A shutdown in the expression of the 5’ portion of the F-MuLV genome has been demonstrated in virus nonproducer clones derived from these cells. One of these clones did not express gp’70 antigens (Anand et ab, 1981). Although other virus nonproducer FLC clones have been obtained in a number of laboratories by cloning FLC under different in vitro conditions, the isolation of virus nonproducer 745 cells described in this article represents, to our knowledge, the first example of an in vivo selection of FLC variants occurring after serial ip passages of interferon-sensitive tumor cell clones in syngeneic mice. It is rather intriguing, however, that 10 ip passages of virus-producer interferonsensitive 745 cells result in the appearance of a virus nonproducer phenotype, whereas even 100 ip passages of interferon-resistant 3Cl-8 cells do not result in any significant change in the expression of F-MuLV. For the time being we are left with the following question: are the different FLC phenotypes resulting from in vivo passages in some way related to the sensitivity versus resistance to interferon of the tumor cells? In particular in vitro treatment of 745 cells with a/p mouse interferon results in a marked block of virus production as well as in an increase of virus antigen expression on the cell surface, whereas interferon does not exert any effect on virus production in interferon-resistant 3Cl-8 cells (Affabris et al, 1982). Moreover interferon does increase H-2 (class I) antigen expression on interferon-sensitive 745 cells, whereas it is not effective in increasing H2 antigens on interferon-resistant 3Cl-8 cells (our unpublished data). Although interferon was not detected in the peritoneal washings or sera of mice injected with virus producer FLC, an ensemble of results has recently indicated that low doses of

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spontaneous interferon could be present in the peritoneal cavity of normal young mice (Belardelli et a!, 1984b; Gresser et al, 1985). In particular, in vitro passaged FLC can form ascites when first injected ip in DBA/ 2 mice pretreated with antibody to mouse interferon a//3 (Gresser et d, 1983). At the present time, as the only difference that we know between 745 and 3Cl-8 cells is the fact that 3Cl-8 cells have been isolated and characterized as an interferon-resistant cell clone (Affabris et c& 1982), it is tempting to speculate that the selection of virus nonproducer cell variants after in vivo passages of interferon-sensitive 745 cells could depend on the presence of low levels of endogenous interferon in normal young mice. As an H-Z/viral protein complex on the cell surface has been described as the basis for the H-2 mediated T cell cytotoxicity (Blank and Lilly, 1977), it is possible to envisage that an interferon-induced increase in the expression of H-2 and FV antigens on the cell membrane may be an important step in triggering a T cell response, which could lead to the selection of virus nonproducer interferon-sensitive 745 cells after several in vivo passages. ACKNOWLEDGMENTS We thank Dr. I. Gresser, Dr. G. B. Rossi, and Dr. P. Tambourin for helpful discussion. We are indebted to Dr. F. Moreau-Gachelin and Dr. J. Robert-L&&es (Faculte de Medecine Lariboisibre-St. Louis, Paris) for gifts of the 0.4-kb BumHI-Sam1 cloned fragment of the F-MuLV env gene and of the 0.4-kb BornHISmaI fragment of the cloned genome of SFFV; and to Dr. A. I. Oliff for gift of the cloned genome of FMuLV (clone 201). This work was supported in part by grants from Consiglio Nazionale delle Ricerche (Progetto Finalizzato Controllo delle Malattie da Infezione 63.02916.52 and Progetto Finalizzato Oncologia 84.00498.44). REFERENCES ADAMS, S. L., SOBEL, M. E., HOWARD, K., YAMADA,

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