Prmted in Sweden Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4X27/79/040253-l2$02.M)/0

Experimental

ENHANCED

Cell Research I19 (1979) 253-264

EXPRESSION

MESSENGER

OF VIRAL

RNA IN DIMETHYL

POLYPEPTIDES SULFOXIDE

BROMODEOXYURIDINE-TREATED ERYTHROLEUKEMIC GIULIA

Chair Institute

COLLETTA,

of Viral

Oncology

of General

AND AND

FRIEND CELLS

FRANCESCO FRAGOMELE, MARIA LUISA SANDOMENICO and GIANCARLO VECCHIO and CNR

Pathology,

Center

2nd Faculty I-80131

for Experimental

of Medicine Naples,

Endocrinology and Surgery,

and Oncology,

University

of Naples,

Italy

SUMMARY The intracellular virus-specific macromolecular changes induced by dimethyl sulfoxide (Me,SO) and/or bromodeoxyuridine (BUdR) have been analysed in the Friend erythroleukemic cell clone 745 A 19 (FLC). These cells, which were transformed in vivo by the Friend virus, are arrested in an undifferentiated state but can be induced to differentiate in vitro by Me$O. We have shown that treatment of FLC with 2% Me,!30 for 4 davs enhances the extracellular virus oroduction and. concomitantly, increases by about 4-fold the~nttacellular virus-specific polyribos&nal RNA. The simultaneous addition to FLC of MeSO and BUdR (the latter is known to inhibit the MeSO-induced differentiation) brings about an even greater increase in both viral polypeptides and viral messenger RNA (mRNA). As a consequence of treatment with both drugs, new viral nucleotide sequences are expressed. as revealed by nucleic acid hybridization studies. Moreover, it appears that two p30-like virus-specific intracellular polypeptides, instead of one single ~30, a& expressed in FLC treated with Me,SO and BUdR. These data also sunnort the idea that gene sequences different from those present in the original Friend virus are expressed as a cc&equence of the combined treatment. It is concluded that the main site of action of both drugs in this cell system, is at the level of transcription of viral RNA and it is suggested that the drugs may act upon cell differentiation by modulating differently the expression of integrated viral genomes.

Friend erythroleukemic cells (FLC) represent a very suitable system for studying the expression of specialized cellular functions which are markers of erythroid differentiation [l-4], the expression of integrated viral genomes [5, 61 and the possible viral involvement in the process of cell differentiation [7, 81. Recently it has become clear that this cell system offers unique opportunities for studying the modulation of the various cell functions outlined above through the use of a variety of chemical substances [9]. The majority of the 17-791805

studies thus far reported in the literature have concerned the expression of functions related to differentiation and, in particular, the effects of substances which are capable of inducing cell differentiation, such as dimethyl sulfoxide (Me&SO) [l, 31; other studies have examined substances which exert an inhibitory effect upon cell differentiation, such as bromodeoxyuridine (BUdR) [IO], interferon at high doses [ll] and tumour-promoting agents [ 12, 131. While the effects of such substances on cell differentiation (either stimulatory or inExp Cell Res 119 (1979)

254

Colletta et al.

hibitory) have been well characterized, relatively few investigations have focused on the possibility of modulating viral genome expression [ 14, 151. We have analysed the intracellular concentration of viral mRNAs and of viral proteins synthesized in the presence of drugs which are known to enhance extracellular virus production. Moreover, since there have been recent suggestions that the expression of the different components of the Friend virus complex play a role in triggering the process of differentiation [7, 81, we have analysed the expression of the viral genome in the Friend cell system under conditions in which both virus induction and differentiation induction occurred (treatment with Me&SO alone) and under conditions in which the virus was induced but the differentiation was inhibited (treatment with both Me&SOand BUdR). The results reported here clearly demonstrate that Me&SO by itself enhances the’ intracellular concentration of virus-specific macromolecules, probably by stimulating the transcription of virus-specific mRNA. Moreover, they show that under all the conditions of enhanced viral expression studied by us there is also the induction of new viral RNA sequences. Both new viral RNA sequences and new viral polypeptides, which are not present in the uninduced state, are found in cells treated simultaneously with Me,SO and BUdR.

MATERIALS

AND METHODS

Cell culture and virus purification The 745A 19 clone of the Friend erythroleukemic cells, derived from the 745 A clone, was obtained from Dr Dinha Singer, Columbia University, New York, N.Y. These cells were obtained after in vivo injection of the Friend virus. The original Friend virus isolate is the “anemic strain”, FV-A [16]. The other available strain is the “polycythemic strain”, FV-P, which consists of the lymphatic leukemia virus (LLV) and a Exp Cell Res 119 (1979)

defective virus, the spleen focus-forming virus (SFFV) [17]. The 745A clone produces, in the unstimulated state, low amounts of the LLV and undetectable amounts of the SFFV component [ 141. The cells were cultured in suspension either in 75 cm% Falcon plastic bottles in 40 ml of Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (FCS) in 5% CO, in air, or in spinners, in the same medium to which a final concentration of 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer, pH 7.0, was added. The cells were usually seeded at a concentration of 5~ 104/ml. When treated, the cells were seeded at 2~ 105/mIand grown continuously in the presence of the drugs (2% v/v Me,SO and/or 20 pg/ml BUdR). Untreated cells reached maximal concentrations of about 2x106/ml and had a doubling time of about 16 h. However, untreated cells could be grown for several days in spinners if kept at lower concentrations by diluting them with fresh medium. Me,SO-treated cells were grown in the same conditions for 4-5 days. Cells treated only with BUdR or with Me,SO plus BUdR grew up to 8x 105/ml and had a doubling time of approx. 24 h. They could be maintained in culture by dilution with fresh medium up to 4 days after which time an increasing proportion of cells began to die. Cultivation of the 78 A 1 clone of rat embryo libroblasts, a cell line infected by and producing the Moloney murine sarcoma-leukemia virus (M-MSVMuLV) complex and the procedures for virus purification have been described previously [18]. When reverse transcriptase assays were to be performed, the clarified cell culture supematants were directly pelleted over 2 ml of 20% glycerol in IO mM Tris-HCI, pH 7.4, 100 mM NaCl, 7 mM EDTA (NTE buffer) in the SW41 rotor of the Spinco-Beckman ultracentrifuge at 36000 rpm for 30 mitt at +4”C, and the virus pellets were resuspended in 200-300 ~1 of 10 mM TrisHCl, pH 7.4, 100 mM NaCl.

Labelling

ofcells

The 745 A 19 cells from logarithmically growing cultures, either untreated or continuously treated with 2 % Me,SO or with 2 % Me,S0+20 pg/ml BUdR, were taken from Falcon bottles or from spinners, centrifuged at 2000 rpm with the HL-8 rotor of the Sorvall RC-3 centrifuge at 30°C for 15 min and then resuspended in minimal Eagle’s essential medium without L-leucine containing 10% dialysed FCS and 20-50 rCi/ ml [3H]leucine (spec. act. 4tl-60 Cilmmole). Cell concentration during labelling ranged from 3 to 6x 106/ml and the times of labelling and chase were as indicated.

Immunoprecipitation

experiments

The antiserum against the M-MSV-MuLV was obtained by injecting the virus produced by the 78 A 1 cells into rabbits as previously described [18]. The antiserum against p30 of the Rauscher leukemia virus was obtained through the OfBce of Resources and Logistics, Virus Cancer Program, NCI, Bethesda, Md. Immunoprecipitation and polyacrylamide gel electrophoresis in sodium dodecylsulfate (SDS) were performed essentially as described [18].

Virus-specific Preparation

of complementary

[3H]DNA

Complementary [3H]DNA was prepared from gradient purified M-MSV-MuLV by the endogenous reverse transcriptase reaction carried out in the presence of actinomycin D. The complementary [aH]DNA of the AKR virus was a generous gift of Dr Nobuo Tsuchida, The Wistar Institute, Philadelphia, Pa. The procedure for the preparation of the cDNAs has been previously described [19, 201. These cDNAs contain at least 80-90% of their respective genomic sequences. The complementary [3H]DNA of Rauscher MuLV RNA was a generous gift of Dr S. A. Aaronson, NCI, Bethesda, Md. It was prepared as described [21].

Preparation of total polyribosomes and extraction of mRNA Logarithmically growing cells were first Douncehomogenized five times with an A pestle in 50 mM Tris-HCI, pH 7.2, 25 mM KCI, 5 mM MgCI, (TKM buffer) and then treated with final concentrations of 1% (v/v) Triton X-100 and 1% Na-deoxycholate (w/v). The lysed extracts were then centrifuged for 5 mm at 27 000 g at 4”C, 500 I.Lglml sodium heparin were added to the supematants which were then layered onto a double sucrose cushion (2.5 ml of 2.0 M sucrose and 2.5 ml of 1.5 M sucrose both in TKM and both containing 500 wg/ml sodium hepatin) [22]. This was then centrifuged at 70000 rpm for 2 h at 4°C in a 75 Ti rotor in a Spinco-Beckman preparative ultracentrifuge. After treatment of polyribosomes with 2 mglml proteinase K for 30 mitt at 25”C, the polyribosomal RNA was extracted with phenol-chloroform-isoamyl alcohol. RNA was then precipitated overnight with 95 % ethanol and then resuspended in H,O.

Hybridization

experiments

The RNA extracted from virions or from polyribosomes was serially diluted in H,O and 5 ~1 from each diluted sample were mixed with 5 ~1 of [3H]cDNA (1000 cpm) from M-MSV-MuLV, AKR or Rauscher MuLV dissolved in 4~ annealing buffer (1.2 M NaCI, 0.1 M Tris-HCI, pH 7.4, 0.04 M EDTA). The mixtures were taken up into 20 ~1 disposable capillaries and hybridized at 68°C for 20 h. Hybridization was, therefore, performed in 2x annealing buffer (final concentration). At the end of the incubation the hybrids were assayed by Sl nuclease essentially as described previously [23].

Reverse transcriptase

assay

This was performed with 100 ~1 of a reaction mixture which contained 50 ul of the oelleted and resuspended virus and 50 ~1 containing 20 mM Tris-HCI, pH 8.0, 100 mM NaCl, 1 mM MnCI,, 10 mM dithiotreitol, 20 pg/ml poly-rC and 4.5 be/ml oligo-dG, 0.015 % v/v Nonidet P-40, 150@Zi/ml [aH]dGTP (spec. act. 11 CilmMole). Incubation was for 1 h at 37°C and the reaction was stopped by adding to each sample 15 LLIof lo-* M ATP in 0.1 M EDTA. Each sample was then spotted onto Whatman DE-81 filter paper

macromolecules

in Friend cells

255

discs and then washed several times with 5 % Na,HPO, at 0°C. The filters were then washed once with HzO, once with 95% ethanol. dried and counted in 10 ml toluene containing 4 g/l of 2,5 his-2-(5-terr-butylbenzoxazoIyl)thiophene (BBOT).

RESULTS Analysis of mRNA

Our analysis of intracellular virus-specific products has focused on the polyribosomal RNA and the polypeptides of treated and untreated cells. For the analysis of the virus-specific RNA we have used three different cDNA probes prepared from three different murine leukemia viruses, the Rauscher MuLV, the Moloney MSV-MuLV and the AKR MuLV. The Rauscher MuLV cDNA is an appropriate probe for analysing Friend virus RNA-specific sequences produced by the 745 A 19 cell clone, since the Rauscher virus, as the virus produced by the 745 A 19 cell line, consists mainly of the LLV component and has very little SFFV, and since Friend & Rauscher virus genomes have been reported to be almost 100% homologous [24]. Moreover, we have also performed some experiments with a Friend LLV cDNA probe and the results were identical to those obtained with the Rauscher cDNA [38]. Since the Rauscher cDNA probe was available in larger amounts, we have used it throughout the present study. The Moloney and the AKR cDNAs were used in order to detect any additional viral sequences induced under our experimental conditions but not related to the Friend virus. Our data show that up to about 70 % of the Rauscher cDNA hybridized to polysomal RNA from untreated cells at a ratio of RNA to cDNA of about 10000/l. The Moloney cDNA hybridized to about 40% with the RNA extracted from FLV. Since the efficiency of the hybridization (obtained Exp Cd Res 119 (1979)

256

Colletta et al.

Fig. I. Abscissa: C,t (molesx secx I-‘); ordinate: % hybridiza-

tion. Hybridization of polysomal RNA from MeSO-treated FLC to the PH]cDNA from Rauscher MuLV. Total polyribosomes were prepared from FLC untreated (e- - -0) or continuously treated with 2 % Me,SO for (A) 1, (B) 2, (C)3 and (D) 4 days (O-O). The RNA was phenol extracted and serial dilutions of it were hybridized to 1000 cpm of [3H]cDNA from Rauscher MuLV. Hybridization was for 20 h at 68°C. All other details are given under Materials and Methods.

by using the cDNA and the RNA from MMSV-MuLV virions) is about 80%, it follows that this cDNA probe recognizes about half the sequences of the FLV. This is in good agreement with data concerning homologies between Moloney and Friend leukemia viruses (Scolnick, E, personal communication). It is important to point out that the M-MSV-MuLV mixtures contain a large excess of the MuLV virions over the MSV pseudotypes; therefore the cDNA probe used in our experiments can be considered essentially as having been obtained with MuLV only. We have analyzed the polyribosomal RNA from FLC treated either with Me,SO or with BUdR or with both drugs simultaneously for quantitating the virus-specific mRNA content with respect to the untreated cells. In fig. 1 are reported the results obtained with cells treated with Me,SO alone. The results of such analysis, Exp Cell Res II9 (1979)

performed with the cDNA from Rauscher MuLV and expressed as C,t curves, show a clear shift in the half C, t values as a function of time, thus demonstrating that increasing amounts of virus-specific mRNA

I

I

I 10D

I 10’

I lo2

0

0,

I

ld

CJ; ordinate: % hybridization. Hybridization of polysomal RNA from Me,SO+ BUdR-treated FLC to the [3~]~~~~ from Rauscher MuLV. Total polyribosomes were prepared from untreated (O- - -0) or from 4 days Me,SO+BUdR-treated (O-O) FLC. The RNA was extracted and hybridized to the Rauscher MuLV PH]cDNA as described in the caption to fig. 1.

Fig. 2. Abscissa:

Virus-specific

L

1 100

10'

, Id

, IO'

1 Id

macromolecules

in Friend cells

251

I

Fig. 3. Abscissa:

C,t (molesxsecx 1-l); ordinate: % hybridization. Hybridization of polysomal RNA from BUdRtreated FLC to the [3H]cDNA from Rauscher MuLV. Total polyribosomes were prepared from untreated (O---O) or from 4 days BUdR-treated FLC (O-O). The RNA was extracted and hybridized to the Rauscher MuLV PHIcDNA as described in the caption to fig. 1.

Fig. 4. Abscissa:

C, t; ordinate: % hybridization. Hybridization of polysomal RNA from Me,SO+ BUdR-treated FLC to the [3H]cDNA from M-MSVMuLV. Total polyribosomes were prepared from untreated (O- - -0) or from 4 days Me,SO+BUdR-treated FLC (O-O). The RNA was extracted and hybridized to the [3H]cDNA from the M-MSV-MuLV as described in the caption to fig. 1.

are present on polyribosomes of the cells as the time of treatment increases. The greatest shift in the half C,t value, corresponding to approximately a 4-fold increase in viral mRNA, occurs with the RNA from cells treated for 4 days. When the cells were treated for 4 days with both Me,SO and BUdR, under conditions in which there is a block in cell differentiation [lo], an even greater increase of virus-specific mRNA is observed. In this case the increase is approx. 56fold as compared with untreated FLC (fig. 2). We have also analysed the role played by BUdR alone in the virusspecific RNA induction process. The result of this experiment is shown in fig. 3, where it can be seen that BUdR alone is also capable of inducing an increased expression of virus-specific mRNA. However, the increment is less than that found with Me,SO alone (fig. ID) or with the two drugs used in combination (fig. 2). From figs 1-3 it appears that drug treatment causes an increase in the maximal hybridization value as well. This increase

is best visualized in the case of cells treated with Me,SO for 4 days and cells treated with both rugs (figs 1D and 2, respectively). Since “h t e implication of such a finding is that new viral RNA sequences are expressed as a consequence of the induction, we investigated whether or not this result could be repeated with the use of probes different from the Rauscher cDNA. Such latter cDNA, in fact, being very closely related to the Friend virus, is less suitable for detecting sequences different from the Friend virus. We have therefore used a Moloney MSV-MuLV probe and a probe from AKR virus. The result obtained with the Moloney cDNA probe is reported in fig. 4. Also with this probe it is clear that the Me,SO+ BUdR-treated cells contain higher proportions of virus-specific RNA than untreated cells. The half C, t values show an acceleration of the hybridization reaction of approx. 2-fold for the treated cells. From the hybridization curve shown in fig. 4, it can also be seen that the maximal hybridizaExp Cell Res 119 (1979)

258

Colletta et al.

Table 1. Hybridization of polysomal RNA from Friend cells with different [3H]cDNA probes -

% Hybridization at saturation with cDNA from AKR

Rauscher

37.5

38.25

-

45.7

68.0 77.65

45.0

50.0

77.0

Treatment

Maloney

None 2 % Me,SO (2 days) 2 % Me,S0+20 rglml

BUdR (4 days)

tion obtained with the RNA from treated cells is higher than that obtained from untreated cells. Even though the differences are not very large, they have been consistently observed in different experiments (table 1). It is evident from table 1 that the phenomenon of increment in the hybridization found is even more evident with the Moloney and with the AKR probes than with the Rauscher cDNA. It can be calculated that the increment in percentage hybridization at saturation is 12% with the Rauscher probe, 17% with the Moloney probe and 24% with the AKR probe, respectively, with the RNA from cells treated for 4 days with Me&SOand BUdR. Analysis of extracellular virus The available data on the increase in virus production in Me&SO-treated Friend cells deals primarily with the quantitation of the extracellular virus measured by different criteria [5, 6, 141. These analyses did not give data regarding the intracellular virusspecific products made under the same experimental conditions. Since the inducing drugs, especially Me,SO, may have more than one site of action (for example on virus release as well as on the transcription of integrated viral genes), we have examined the levels of extracellular virions in treated Exp Cell Res 119 (1979)

and untreated cells as a function of the number of days of treatment with the inducing drug in order to ascertain whether the effect of Me,SO on extracellular virus production was higher than that observed on intracellular virus-specific RNA. The extracellular virions have been quantitated by means of the reverse transcriptase reaction. The results of such an experiment, reported in fig. 5, show that the levels of extracellular virus in the Me&SO-treated cultures are increased about 3.5-fold over the levels in the untreated cultures by the 4th day. A smaller increase is found earlier. Therefore the increase in virus production found by us is of the same magnitude and shows the same time course as the increase found with the intracellular virus RNA and virus proteins (see below).

1

I .

3-

2/[y

1-

I

1

2

3

4

5

Fig. 5. Abscissa: time of treatment with Me&SO(days); ordinate: [3H]dGMP incorporation (dpmx 10-*x 105 cells x ml-’ sup fluid). Extracellular virus production in the supematant fluids from FLC treated with 2 % Me,SO as a function of time. FLC were seeded either in the presence (0-O) or in the absence (O-O) of 2% Me,SO at different initial cell concentrations, so that, at the time of medium harvesting, all cell counts ranged between 5~10~ and I~lO~/rnl, i.e. both the control and the Me,SO-treated cells were in the logarithmic phase of growth. The cell supematants were then analysed for reverse transcriptase activity as described under Materials and Methods.

Virus-specific

macromolecules

in Friend cells

259

shown). However, this effect is diffkult to quantitate due to the inhibition of protein synthesis exerted by Me,SO. The combined treatment with Me,SO+BUdR, instead, clearly gives rise to a twofold increase in the percentage of protein precipitable with the anti-M-MSV-MuLV serum, as shown in fig. 6. In this case the proportion of virusspecific protein rises from 6 to 11% of the 3 4 1 2 total cellular protein after 4 days of treatment. The more pronounced effect of the Fin. 6. Abscissa: time of treatment with Me240 and BGdR (days); ordinate: % immunoprecipitation. Me,SO-BUdR treatment reflects well the Immunoprecipitation of [3H]Ieucine-IabeIled cell extracts. Constant amounts of cell lysates (lOOOO- situation found with the virus-specific 100000 cpm, corresponding to 60 pg of protein) were mRNA described above. The cell extracts immunoprecipitated with increasing amounts of either obtained from untreated and Me,SO-BUdRanti-M-MSV-MuLV or anti-u30 sera. Immunourecipitation was carried out for the cell extracts frdm each treated FLC were also precipitated with the day of the Me,SO+BUdR treatment and the values of maximal immunoprecipitation (corresponding to the monospecific antiserum against the Rauequivalence points of the resmctive immunoprecipitascher ~30. The degree of immunoprecipitat&n curves) were plotted against the time of treatment tion observed with this antiserum was of the cells with the two drugs. O-O, Anti-M-MSVMuLV serum; O---O, anti-Rauscher p30 serum. slightly lower than that obtained with the anti-M-MSV-MuLV serum but the percentage of immunoprecipitation obtained lmmunoprecipitation of virus-specific with the proteins extracted from treated polypeptides FLC is also increased twofold with respect Throughout this study we have used two to untreated cells (see fig. 6). types of antisera. The first was a polyvalent antiserum prepared against whole disrupted SDS gel analysis Moloney MSV-MuLV virions, and the sec- In order to analyse the protein changes ocond was a monospecific antiserum prepared curring in the Friend cells during induction against Rauscher MuLV ~30. The antise- of virus, we have examined the immunorum against M-MSV-MuLV recognized precipitates obtained from control and inequally well M-MSV-MuLV and FLV, duced cells by SDS gel electrophoresis. since about 80% of the trichloroacetic acid The structural proteins of murine viruses (TCA) precipitable radioactivity of both are not synthesized directly but, in analogy viruses can be immunoprecipitated with 300 to the avian system [26], are translated as ~1 of the antiserum [25]. The two antisera high molecular weight precursors which are were used for the immunoprecipitation of then cleaved [27-291. For this reason we intracellular proteins from FLC treated have performed pulse-chase experiments. with MezSO alone or with Me$O and The results of typical pulse-chase experiBUdR for different periods of time. The ments obtained with the immunoprecipiproportion of the proteins immunoprecipitates from untreated FLC are shown in fig. tated from cells treated with Me,SO alone 7, At least three polypeptides having molincreases when tested with the p30 antise- ecular weights higher than p30 have been rum after 4 days of treatment (result not detected with the anti-M-MSV-MuLV seExp Cell Res 119 (1979)

260

Colletta et al.

polypeptide found by Jamjoom et al. with the Rauscher virus [30] and to the 180 K polypeptide found in different erythroleukemic cell lines [31]. The 70-80 K polypeptides are definitely gag precursors (Pr 80gaa) in the Rauscher system [32]. The 40-50 K polypeptide may also represent an intermediate product in the processing pathway of gag proteins, and components having similar molecular weights have also been described recently by Racevskis & Koch with different as well as with the same cell line used by us [3 1, 331. Our analysis of the induced cells has focused on the virus-specific polypeptide patterns obtained from FLC treated for 4 days with Me,SO+BUdR, since the maximal increment in virus-specific polypeptides has been found in the cells treated with both drugs. Moreover, under these conditions the synthesis of hemoglobin is highly reduced compared to the synthesis of the virus-specific proteins. As shown in fig. 8, the pattern of labelling of immunoprecipitates from Me,SO-BUdR treated FLC is Fig. 7. Abscissa: slice no.; ordinate: (left) [3H]leucine cpmx lo-* (O-O); (right) [Ylleucine cpmx lo-* quite different from that of untreated cells. (---). The proportion of radioactivity present in SDS polyacrylamide gel electrophoresis of immunoprecipitates of labeled FLC extracts. FLC were either the high molecular weight peaks, especiallabelled for (A) 15 mm or (B) labelled for 15 mitt and ly in the 140 K polypeptide, is quite reduced then chased for (B) 3 h, or(C) 6 h. The cell extracts, corresponding to approx. 200000 cpm and to 750 pg in these cells after 6 h of chase, compared of protein, were immunoprecipitated with 400 ~1 antiwith the extracts obtained from untreated M-MSV-MuLV serum and the immunoprecipitates analysed by SDS gel electrophoresis as previously de- cells (see fig. 7C). The 40 and 50 K peaks, scribed [18]. O-O, [3H]leucine-labelled cellular exwhich are resolved in the pattern shown in tracts; ---, [WJeucine-labelled M-MSV-MuLV marker. fig. W, are, instead, still present after 6 h of chase in the treated FLC. The 40 K polypeptide, which is present almost in the same proportion as the ~30, probably represents rum in untreated FLC pulse labeled for 15 a new intermediate in the processing of the min (see fig. 7A). Since the antiserum used gag precursor polypeptide as also noted by contains antibodies mainly against the gag Racevskis & Koch with a different inducer polypeptides [ 181,it is reasonable to assume and a different erythroleukemic cell line that the 140 K polypeptide represents an [31]. The most interesting difference, howincomplete read-through product of the gag ever, between untreated and treated FLC is and pol genes, analogous to the Pr 2Wag-Po1 that treated cells show the presence of a Exp Cell Res 119 (1979)

Virus-specific

macromolecules

in Friend cells

261

DISCUSSION The Friend cell clone used by us produces, in the unstimulated state, low amounts of virus, mainly the LLV component, and can be induced to differentiate with l-2% Me&SO [l]. Dimethyl sulfoxide also induces, in most Friend cell clones and in the same concentration range, changes in the expression of virus. An increase in budding virus particles, [6] as well as an increase in the production of biologically active virus [5], has been described as a result of Me,SO treatment. The present experiments with the 745 A 19 cell clone show an approx. 4fold increase in the extracellular virus (as measured by the reverse transcriptase assay) at the 4th day of treatment with Me,SO alone. The results obtained with the production of extracellular virus under the same inducing conditions are in agreement with those found in the intracellular virusspecific macromolecules. The immunopreFig. 8. Abscissa: slice no.; ordinate: (left) [3H]leucine cipitable proteins, in fact, show the highest (right) [‘Tlleucine cpmx lo-* cpmx IO-* (O-O); (---). increase at the 4th day of treatment (unSDS polyacrylamide gel electrophoresis of immunopublished results). The kinetic analysis of precipitates of labeled Me,SO+BUdR-treated FLC extracts. FLC were treated with Me,SO+BUdR for 4 the mRNA shows also a progressive indavs and the cell extracts immunonrecioitated and crease in the accumulation of the mRNA analysed as described in the caption to-fig. 7. (A) Extract from cells oulse-labelled for 15mitt: (B) extract with the time of the Me,SO treatment. The from cells pulse-labelled for 15 min and then chased for highest levels of RNA (about 4-fold com6 h. (O-O) [3H]1eucine-labelIed cell extracts; (---) [Tlleucine-labelled M-MSV-MuLV marker. pared with control cells) are reached also at the 4th day of treatment. This is at variance with the data obtained by Pragnell et al. [15] for some of their cell clones where the double peak in the p30 region, one peak maximal increase in the intracellular viral with a mobility slightly lower and one with RNA was found at earlier times. On the a mobility slightly higher than the marker other hand, a situation somewhat similar to ~30. These results indicate that the pro- the one described here has been found with other erythroleukemic cell clones which, in cessing of viral gag precursor polyprotein in induced cells may be different from the analogy to the 745 A 19 cells, are low or processing in control cells. In addition, the negative for virus production [34]. appearance of a new peak in the p30 region It has been postulated [14], on the basis with a mobility different from p30 is highly of results obtained with the 745A clone of suggestive of the induction of a new viral Friend cells as well as with different erythpolypeptide. roleukemic cell lines, that the effect of Exp Cd/ Res 119 (1979)

262

Colletta et al.

Me&SO on virus production is an indirect result of its action on cellular growth inhibition and that this effect largely disappears when Me,SO-treated growing cultures are compared with growing and not with stationary untreated cultures. It is important to point out that, in the experiments performed by us, the Me,SOtreated cells were not compared with control stationary cultures, but with cells which were approximately in the same phase of growth as the treated ones (see caption to fig. 5). In the light of the findings of Sherton et al. [14], it is equally important to point out that also the results concerning the intracellular concentrations of viral components were obtained by us using logarithmically growing cells and therefore the differences found with the time of treatment with the drugs cannot be due to the possibility that some of the cells examined by us were arrested in the GO stage of the cell cycle. Furthermore, Sherton et al. [14] were unable to find differences in the concentration of the p30 between growing and GO arrested Friend cells, whereas we did find an increase in the p30 concentration as a result of treatment with the drugs. Our results, therefore, confirm the data obtained by Dube et al. [S] and by Pragnell et al. [15] who claim that viral induction in their Friend cell clones is Me,SO or differentiation dependent. The results presented here, therefore, clearly indicate that Me,SO has a site of action at the level of viral mRNA transcription or that it inhibits viral mRNA degradation and does not act merely as an inducer of virus release. After Me&SO treatment we have also noted that new nucleotide sequences are expressed compared with the unstimulated cells. The presence of these new sequences is evinced by the increased percentage of Exp Cell Res I19 (1979)

hybridization obtained at saturation levels of the C,.t curves presented in fig. I. This increase, although small, has been found consistently in our experiments and with all probes tested. The new nucleotide sequences may represent either sequences from an induced endogenous virus unrelated to the Friend virus, or SFFV-related sequences which, as stated before, are not expressed in the 745A 19 cells in the unstimulated state. The latter possibility would represent a situation analogous to that found by Pragnell et al. [15] who also found an increase in SFFV-related sequences in some of their cell clones upon induction. Further analyses, with the use of the SFFV-specific cDNA probe, will clarify this point. In the cells treated simultaneously with Me,SO and BUdR we have also found an increase in the intracellular concentration of virus-specific polypeptides and mRNA. The kinetics of the increase found with the virus-specific polypeptides show that the maximal increment is reached at the 4th day of continuous treatment (see fig. 6). The degree of the increment found both for virus-specific polypeptides and viral mRNA is higher than that found with the use of Me&SO alone. Also with the combined Me,SO+BUdR treatment, new nucleotide sequences are expressed. The increment in the hybridization found with the Moloney MuLV probe and the polysomal RNA from the treated cells is even higher than that found with the Rauscher probe (see figs 2, 4 and table 1). This observation, which is strengthened by the even higher increase in hybridization found by using the cDNA probe made with the AKR MuLV (see table l), is indicative of induction of viral sequences not expressed in the untreated FLC. Whether the new sequences expressed in the cells treated with Me,SO+

Virus-specific

BUdR are the same or different from those expressed by treatment with Me&SO alone, cannot be established on the basis of the experiments reported here. It is important to point out, however, that the treatment with BUdR alone results in an increased viral RNA concentration and, probably also in the expression of new nucleotide sequences (see fig. 3). On the other hand, it is well known that BUdR induces endogenous viral sequences in murine systems [35, 361. The possibility that at least part of the new nucleotide sequences found in the cells treated with Me,SO+BUdR represents endogenous sequences not related to the Friend virus is strengthened by the SDS gel experiments. The protein patterns obtained with untreated cells have confirmed that the viral protein synthesis occurs, in the Friend virus system, with modalities that closely resemble those observed with other murine RNA tumor virus systems [27-291. The protein patterns obtained with cells treated with Me,SO+BUdR seem to indicate a more rapid processing of the 140 K polypeptide, whereas the 40 and 50 K proteins accumulate for longer times. The most striking observation, however, is that of the presence of two closely migrating polypeptides in the p30 region. Such observation could be related to the expression of an endogenous virus and/or of a newly activated Friend virus having a p30 with different electrophoretic mobility. It is interesting to point out, in this respect, that viruses having different tropisms (B versus NB) have been previously shown to possess ~30’s which differ in their electrophoretie mobilities [37]. Evidence is accumulating for the idea that Friend virus plays a role in the process of erythroid differentiation. It has been proposed that an Me,SO-induced product

macromolecules

in Friend cells

263

(RNA) of erythroid cells interacts with the Friend virus (SFFV) genome during differentiation. This may confer erythroid specificity to SFFV [8]. In the light of this model it is tempting to speculate that the inhibition of differentiation found in FLC treated simultaneously with Me,SO and BUdR is due to a virus induction pattern which is different from that found with cells treated with Me,SO alone. Future work will establish whether or not the endogenous viral sequences which seem to be induced in our Me,SO+BUdR-treated cells interfere with the process of erythroid differentiation. This work was supported by the Progetto Finalizzato Virus of the Consinlio Nazionale delle Ricerche (CNR), contracts no. 17600698 84 and 77 00315 84. We are grateful to Natalie Teich, Harold Varmus and Benoit de Crombrugghe for critically reviewing the manuscript and to Stuart Aaronson and Charlotte Friend for the advices and help received during the progress of the work. The skillful technical assistance of Mr Mario Esposito is gratefully acknowledged. We are also grateful to Giuliana de Angelis for typing the manuscript.

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12. Yamasaki, H, Fibach, E, Nudel, U, Weinstein, I B, Rifkind, R A & Marks, P A, Proc natl acad sci US 74 (1977) 3451. 13. Rovera, G, O’Brien, T G & Diamond, L, Proc natl acad sci US 74 (1977) 2898. 14. Sherton, C C, Evans, L H, Polonoff, H & Kabat, D, Virology 19(1976) 118. 15. Pragnell, 1 B, Ostertag, W & Paul, J, Exp cell res 108 (1977) 269. 16. Friend, C, Sher, W, Tsuei, D, Haddad, J, Holland, J G, Szrajer, N & Haubenstock, V, Oncogenic viruses and host cell genes (ed Y Ikawa). Academic Press, New York (1977). 17. Steeves, R A, J natl cancer inst 45 (1975) 289. 18. Shanmugam, G, Vecchio, G, Attardi, D & Green, M, J virol 10 (1972) 447. 19. Tsuchida, N, Robin, M S & Green, M, Science 176 (1972) 148. 20. Vecchio, G, Tsuchida, N, Shanmugam, G & Green, M, Proc natl acad sci US 70 (1973) 2064. 21. Cabradilla, C D, Robbins, K C & Aaronson, S A, Proc natl acad sci US 73 (1976) 4541. 22. Palmiter, R D, Biochemistry 13 (1974) 3606. 23. Shanmugam, G, Bhaduri, S & Green, M, Biochem biophys res commun 56 (1974) 697. 24. East, J L, Knesek, J E, Chan, J C & Dmochowski, L, J virol 15 (1975) 13%. 25. Vecchio, G, Fragomele, F, Colletta, G, Laurenza, M & Sandomenico, M L, Xth Meeting of European tumour virus group, Grindelwald (1976) 165. 26. Vogt, V M, Eisenman, R & Diggelmann, H, J mol biol% (1975) 471.

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27. Shapiro, S 2, Strand, M & August. J T, J mol biol 107 (1976) 459. 28. Van Zaane, D, Dekker-Michielsen, M J A & Bloemers, P J, Virology 75 (1976) 763. 29. Arcement, L J. Karshim, W L, Naso, R B & Arlinghaus, R B, Virology 69 (1976) 763. 30. Jamjoom, G A, Naso, R B & Arhnghaus, R B, Virology 78 (1977) 11. 31. Racevskis, J & Koch, G, J virol. In press. 32. Jamjoom, G A, Ng, V L & Arlinghaus, R B, J virol 25 (1978) 408. 33. Racevskis, J & Koch, G, J virol 21 (1977) 328. 34. Ostertag, W, Krieg, C J, Cole, T, Pragnell, I B, Swetly, P, Weimann, B J & Dube, S K, Exp cell res 116(1978)31. 35. Lowy, D R, Rowe, N P, Teich, N & Hartley, J W, Science 174 (1971) 155. 36. Ostertag, W, Roesler, G, Krieg, C J, Kind, J, Cole, T, Crozier, T, Gaedicke, G, Steinheider, G, Kluge, N & Dube, S K, Proc natl acad sci US 71 (1974) 4980. 37. Hopkins, N, Schindler, J & Hynes, R, J virol 21 (1977) 309. 38. Dolei, A, Colletta, G, Capobianchi, M, Rossi, G B & Vecchio, G. Submitted for publication.

Received August 15, 1978 Revised version received October 30, 1978 Accepted November 1, 1978