Vol. 31, No. 1

JOURNAL OF VIROLOGY, July 1979, p. 178-183 0022-538X/79/07-0178/06$02.00/0

Virus Production and Hemoglobin Synthesis in Variant Lines of Dimethyl Sulfoxide-Treated Friend Erythroleukemia Cells DEANE TSUEI,'* HARRIETTE HAUBENSTOCK,t ROBERTO REVOLTELLA,2 AND CHARLOTTE FRIEND' The Mollie B. Roth Laboratory, The Center for Experimental Cell Biology, Mt. Sinai School of Medicine, City University of New York, New York, New York 10029,1 and Laboratorio di Biologia Cellulare, Consiglio Nazionale delle Richerche, Rome, Italy 001962 Received for publication 3 January 1979

Variant Friend erythroleukemia cell clones were compared in regard to their response to dimethyl sulfoxide and in their abilities to synthesize virus and hemoglobin. Clear evidence was obtained that cellular growth is required for virus production. The effects of dimethyl sulfoxide on virus production were not observed in cell lines that were resistant to growth perturbation by the compound. Studies of cell variants that were defective in either hemoglobin or virus synthesis indicate that these activities are independently regulated.

Lines of established Friend erythroleukemia (FL) cells are chronically infected with virus (11). Treatment of the cultures with dimethyl sulfoxide (DMSO) stimulates erythroid differentiation accompanied by increased hemoglobin synthesis (12), as well as virus production (15, 27, 33). Thus, this system provides a model for the study of the mechanisms controlling both hemoglobin and virus synthesis. The fact that the mechanisms controlling these two functions were separate was suggested by the electron microscopic observations that cells grown in the presence of DMSO (12) or with bromodeoxyuridine, which inhibits differentiation, had increased numbers of budding viruses on their membranes (32). In fact, budding was further enhanced on the cells growing in medium containing both DMSO and bromodeoxyuridine. Subsequently, it was found that differentiation could be stimulated in the treated cells of some lines whether or not they were producing virus (16, 37, 41). Variant clones which differ in their response to DMSO were studied to determine whether or not there was a relationship between the mechanisms regulating hemoglobin and virus synthesis. In the present report, some of the properties of three FL clones which are phenotypically different from the parent cultures are described. They were compared to the prototype cells on the basis of their ability to synthesize hemoglobin and virus in response to DMSO treatment. Among the variants were clones that were DMSO resistant either for hemoglobin synthet Present address: Research Institute of the Hospital for Joint Diseases and Medical Center, Mount Sinai School of Medicine, City University of New York, New York, NY 10029.

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sis, for viral production, or for both functions. Our data show no correlation between hemoglobin and virus synthesis, indicating that each of these functions is regulated independently of the other. MATERIALS AND METHODS Tissue cultures. The four FL lines used here were: (i) clone 5-86, a subclone of line 745A, the origin and properties of which have been described (11); (ii) clone H-19, a DMSO-resistant line that was derived from line 5-86 (by H.H.) by two subsequent long-term cultivations in medium with 1.9% DMSO but which is now maintained in DMSO-free medium (periodically, cells are checked for DMSO resistance and have been found to be stable in resistance); (iii) clone 707M, developed by J. Paul from our original line 707 (14); and (iv) clone F4-1, derived from one of W. Ostertag's lines and kindly provided by H. Eisen. Cultures were seeded at 105 cells per ml in 250-ml Falcon flasks containing 30 ml of medium. Dehydrated Eagle basal medium (GIBCO, Grand Island, N.Y.), diluted with Earle balanced salt solution was used for lines 5-86 and H-19. Medium RPMI 1640 (Flow Laboratories, Inc. Rockville, Md.), supplemented with 2 mM glutamine, was used for lines 707M and F4-1. Both media contained 15% fetal bovine serum (Reheis Chemical Co., Kankakee, Ill.), 250 U of penicillin per ml, and 0.2 mg of streptomycin per ml. The cultures were grown at 37°C in a humidified incubator containing 5% CO2 in air. Certified reagent-grade DMSO (Fisher Scientific Co., Fair Lawn, N.J.) was added to the medium to make a final concentration of 1.9% (vol/vol) for all the lines except 707M. For optimal hemoglobin induction with line 707M, 1.0% DMSO was used. The number of cells was determined by counting in a hemacytometer with 0.1% trypan blue added to estimate the ratio of living to dead cells. Benzidine staining. The hemoglobin-positive cells were determined by a modification of the method of

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Orkin et al. (26) as described by Scher and Friend (34). RT assay. Cultures were clarified by centrifuging at 200 x g, followed by another centrifugation at 10,000 rpm in a Sorvall SS34 rotor at 40C. The resulting supernatant fluid was then spun at 27,000 rpm in a Spinco 30 rotor at 4°C. The virus pellet was resuspended into 0.2 ml of 0.1 M NaCl-0.01 M Tris (pH 7.6)-l mM EDTA (NTE buffer). Exogenous reverse transcriptase (RT) activity associated with virus was assayed by a modification of the method of Mayer et al. (22). The reaction mixture in a final volume of 50 pl contained: 50 mM Tris (pH 8.0), 30 mM NaCl, 2 mM dithiothreitol, 0.1 mM dATP, 0.8 mM MnCl2, 0.02% (vol/vol) Nonidet P40 (Shell Oil Co., New York, N.Y.), 10jug of poly(rA dTjo) (Collaborative Research, Inc., Waltham, Mass.) per ml, 1.0 I&Ci of [3H]dTTP (New England Nuclear Corp., Boston, Mass.; 51 Ci/ mmol), 0.01 mM dTTP, and 25 p1 of concentrated virus suspension. The mixture was incubated at 37°C for 30 min, stopped with 10 pl of 0.2 M EDTA, and chilled in ice. A volume of 50 pl was spotted on a 25mm disk of DE-81 filter paper (Whatman Inc., Clifton, N.J.), which was washed six times with 5% Na2HPO4, twice with water, twice with 95% ethanol, and twice with ethyl ether (1). Filters were dried and counted in a toluene-based scintillation fluid. Competitive radioimmunoassay. Purified viral gp71 and p30 from Friend leukemia virus (FLV)-infected Eveline STU and specific goat antisera to FLV proteins gp7l or p30 were generous gifts from D. Bolognesi. Virus protein tyrosine residues were labeled with "I by the lactoperoxidase (21) or the chloramineT iodination procedures (13). The procedures for the purification of viral antigens and the antisera (25) have been described. The method for the competitive radioimmunoassay has been described also (R. Revoltella, C. Friend, and D. Bolognesi, manuscript in preparation). Briefly, 200 Ad of fluid from each of the four FL cultures was removed daily for assay. They were clarified and centrifuged at 10,000 rpm in a Sorvall SS34 rotor. To the supernatant fluid, 50 pl of anti-FLV p30 or anti-FLV gp7l sera was added (1:1,000 to 1: 250), and the mixture was first incubated at 370C for 30 min, then overnight at 4°C, after which 50 pl of I25labeled gp7l or p30 was added. The incubation was continued at 37°C for 1 h and at 4°C for 4 h. Donkey gamma globulin, a generous gift of the National Institutes of Health Resources through J. Gruber, was added to precipitate the antigen-antibody complex. The mixture was further incubated at 37°C for 1 h and then at 4°C for 1 h. The precipitate was collected and washed with NTE buffer, and l"I-labeled antigen present in the precipitate was measured in a gamma counter.

RESUJLTS Cell growth. The growth curves of each of the four FL lines in culture with or without DMSO are shown in Fig. 1. Over the 6-day period of the experiment, the growth pattems of all untreated cultures were comparable, with a log-phase growth in the first 3 days and an average doubling time of 12 to 15 h. With DMSO included in the medium, the DMSO-resistant

179

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variant H-19 showed the early lag in growth characteristic of the response ofthe parental line 5-86 and reached stationary phase after 4 days. In the DMSO-resistant line 707M and inducible line F4-1, however, the patterns of cell growth were not affected by the presence of DMSO in the medium, and no lag in growth of the treated cells was observed. Hemoglobin synthesis. Hemoglobin synthesis was assayed daily by benzidine staining. The percentage of benzidine-positive (B+) cells at each time interval is shown in Fig. 2. In cultures without DMSO, all four lines had low levels of B+ cells, ranging from 0 to 3%. When grown in medium with DMSO, the inducible lines 5-86 and F4-1 showed a significant increase of B+ cells starting 2 to 3 days after treatment and reached a level of 85 to 90% B+ cells by day 6. In comparison, lines H-19 and 707M responded poorly to DMSO stimulation and had less than 5% B+ cells by day 6. Virus release. Daily accumulation of viral RT activity in the culture fluid was measured as an indication of virus release from FL cells (Fig. 3). The same pattern of virus release was found in line 5-86 and its DMSO-resistant derivative

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the four cell lines by competitive radioimmunoassay for FLV gp7l and p30 with monospecific sera. Determinations are plotted as the percent displacement of "2I-labeled viral antigen by 200 pcl of culture fluid. The culture medium of all four lines contained appreciable amounts of gp71 whether or not the lines were releasing virus (Fig. 4). The level of gp7l detected in the culture fluid of lines 707M and F4-1, which produce little or no virus, was comparable to that detected in the 5-86 and H-19 virus-producing cultures. There was little difference between the amount of antigen released in the control and DMSO-treated cultures. In the assay for viral p30, the two virus-producing lines, 5-86 and H-19, as well as the low virus producer 707M, each shed approximately the same amount of antigen into the medium. No p30 was detected in F4-1 culture fluid (Fig. 5). The levels of p30 in all control and DMSOtreated cultures were similar except for F4-1, where antigen was only detectable after DMSO

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2 15~~~~~~~~I line, H-19. The control cultures of these two lines showed an exponential increase in virus 10~~~~~~I o release during the early log phase of cell growth. Virus accumulation peaked on day 3 and decx :0 creased sharply after the cells reached stationary phase. When treated with DMSO, virus release z in both cultures was stimulated. There was an initial lag in virus release, which paralleled the .11 cell growth. The virus release peaked on day 4 aand decreased after the cultures reached stationF4 -1 707 M I-| ary phase. The virus is attenuated for leukemoo I, genicity in DBA/2 and Swiss mice and does not Control 15 DM SO -Treated K I~~~ form syncytia on XC cells. There is no spleen L_,tm 1 0'ZA r n 0-I Ifocus-forming virus activity detectable in the 10 spleens of BALB/c mice, although on rare occasions a few foci have been observed on the spleen of a DBA/2 mouse inoculated intravenously with 0.5 ml of undiluted culture fluid. The virus, however, is effective in protecting I e- 5 'O U e S ' D b ) b mice against challenge with virulent leukemoDAY genic strains of FLV (9). FIG. 3. Accumulation of viral RTactivity. Medium Both the noninducible line 707M and the inthe cultures was harvested daily for RT assay ducible line F4-1 produced very low amounts of from as described in the text. Each point represents the virus. DMSO treatment slightly enhanced virus average three reaction mixtures after zero-time release in line 707M, but had no effect on line reaction of had been subtracted. Each reaction tube F4-1. contained virus concentrated from 3.75 ml of medium Release of viral antigen. The presence of fluid. About 2,000 cpm equals I pmol of [3H]TMP viral antigens was assayed in the culture fluid of incorporation. E

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181

86 is highly indmucible for hemoglobin synthesis, and virus production is enhanced by DMSO treatment (Fig. 2 and 3). Clone H-19 is DMSO resistant in that the cells are not inducible for hemoglobin synthesis but have the same pattern of virus production as the parental clone 5-86. On the other hand, clone 707M is minimally inducible for both hemoglobin and virus. The third variant, clone F4-1, is inducible for hemoglobin but produces no significant amount of

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Differences in the ability to synthesize hemoglobin (31) and other erythrocyte-associated products (7) had been previously noted in other FL clones. Our data indicate that clonal lines of FL cells are also variable in their viral properties. Several lines of evidence indicate that virus production and erythroid differentiation are under separate controls: (i) studies with interferon showed that virus release can be inhibited without affecting the synthesis of hemoglobin in DAY DMSO-treated cells, depending on the concenFIG. 4. Competitive radioimmunoassay for gp7l. tration of interferon used (18, 20, 30, 38); (ii) Medium (0.2 ml) from the cultures was sampled daily. bromodeoxyuridine, when added to cultures toAfter clarification, it was used to compete with the gether with DMSO, inhibits DMSO-stimulated binding ofgoat anti-FLVgp71 to purified "LI.labeled FLV gp71 (see the text). Each point represents the erythrodifferentiation but increases virus budding and extracellular RT activity (10, 32); (iii) average of two reaction mixtures. steroids, such as dexamethasone and hydrocorI I I I I II tisone, inhibit DMSO-stimulated erythrodifferDO 0 5-86 H - 19 entiation, but virus release is doubled (35). In addition, the fact that lines F4-1 and 707M show 30 *- Control no lag in growth in response to the inducer x---x DMSO-Treoted that arrest of G-1, which may not be a suggests O 6 prerequisite for differentiation (8), may be ixnplicated in virus synthesis. Evidence that line B8-3 does not release virus after DMSO stimulation, although entire virus-specific sequences 2 are found in the polysomes of the cells, indicates .0 z -J (I)

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that some lines may have a defect that affects virus assembly but does not interfere with their ability to differentiate (28). a. Although cell replication may (19, 23) or may (n not 0 (17, 40) be a prerequisite for the stimulation 630 _ 0of erythroid differentiation in FL cells, it appears to be necessary for virus production (10, 37). Our data (Fig. 3) showed that viral RT activity in culture fluid of virus-producing lines 5-86 and 2 I:'., _________ ____ H-19 was exponentially increased during log of cell growth and sharply dropped after phase O0 2 3 4 5 6 0 2 3 4 5 6 the cultures reached stationary phase. The rate DAY FIG. 5. Competitive radioimmunoassay for FLV of viral synthesis was higher when the cells were p30. Samples, procured as described in the legend to in log phase than when they were stationary, as Fig. 4, were used to compete with the binding of goat the results which are the sum of virus accumuanti-FLV p3O to purified '25I-labeled FLV p30 (see lation and degradation indicate. After 4 days of the text). DMSO treatment, there was a one- to fivefold increase in RT activity in the medium as comwere compared in regard to their ability to propared to that from untreated parallel cultures. duce virus and synthesize hemoglobin after The difference in the peak RT activity betreatment with DMSO. The prototype clone 5- tween the responsive lines of DMSO-treated w

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cells and their respective controls is modest as compared to that observed in some of Ostertag's lines (5). The reason for the disparity may be due to the fact that individual FL cell lines have different constitutive levels of virus production. The magnitude of the enhancement in response to the inducer is a reflection of this. The small increase in response to DMSO stimulation is consistent and characteristic of our cell lines. It should be mentioned that, when the growth of the DMSO-treated cultures coincides with that of the untreated controls, the RT activity peaks of each occur on the same day, revealing the readily apparent differences between the two in the level of the enzyme (10). This increase in viral enzyme activity confirms and extends the findings of inducer-stimulated virus production by electron microscopy (32) and by biological assays (5, 15, 37). The report that RT activity was reduced after DMSO treatment in an independently derived FL line, T3C12 (6), is in contrast to the report of Ikawa et al., who had developed that line. These investigators had noted an increase in RT as well as in the titer of the virus recovered from the treated cell cultures (15, 16). The half-lives of the RT in the presence and absence of DMSO did not differ. When viral enzyme was measured in 3-day control and DMSO-treated cultures at 370C, the half-life ranged from 2.5 to 3.5 h for both samples. These data are similar to those obtained with virus in another FL line (37) and other RNA tumor viruses (4, 38). The rate of RT viral inactivation may possibly account for the rapid decrease in the amount of virus RT in the stationary cultures. The question of whether or not the increase in RT activity observed in DMSO-treated 5-86 or H-19 cultures is a reflection of an increase in the synthesis of the virus with which the cells were initially infected, the activation of an endogenous virus, or a mixture of such viruses, remains to be resolved. A difference in the tropism of the spleen focus-forming virus component of FLV recovered after DMSO stimulation of one FL line suggests that an endogenous virus has been activated (5). The virus producer cell lines, 5-86 and H-19, both control and DMSO treated, released approximately the same amount of gp71 and p30. Similar observations were noted when intracellular p30 and gp7l in cells of line 745 were measured (36). The nonproducer cell line F4-1, while still releasing gp7l into the culture fluids, was incapable of releasing p30. It is interesting that p30 determinants that are not processed to form p30 have been detected in Ostertag's nonproducer FL cell line, FSD1-B8 (29). Since the

expression of antigens reacting with antibodies to the major envelope glycoprotein gp7l has been demonstrated on the surfaces of a variety of normal as well as leukemic virus-producing celLs (2, 3, 26, 42), this antigen may be related to nonvirus cell surface antigens, whereas p30 appears to be virus specific. The data on the release of viral gp7l but not of p30 suggest that this antigen may be degraded extracellularly. ACKNOWLEDGMENTMS We thank J. Gilbert Holland for expert technical assistance. This work supported in part by Public Health Service grants CA-10000 and CA-13047 from the National Cancer Institute. LMRATURE CITED 1. Blatti, S. P., C. J. Ingles, T. J. Lindell, P. W. Morris, R. F. Weaver, F. Weinberg, and W. J. Rotter. 1970. Structure and regulatory properties of eukaryotic RNA polymerase. Cold Spring Harbor Symp. Quant. Biol.

35:649-657. 2. Del Villano, B. C., and R. A. Lerner. 1976. Relationship between the oncornavirus gene product gp7O and a major protein secretion of mouse genital tract. Nature (London) 259:497-499. 3. Del Villano, B. C., B. Nave, B. P. Croke, R. A. Lerner, and F. J. Dixon. 1975. The oncomavirus glycoprotein gp69/71: a constituent of the surface of normal and malignant thymocytes. J. Exp. Med. 141:172-187. 4. Dougherty, R. E. 1961. Heat inactivation of Rous sarcoma virus. Virology 14:371-372. 5. Dube, S. K., I. B. Pragnell, N. Kluge, G. Gaedicke, G. Steinheider, and W. Ostertag. 1975. Induction of endogenous and of spleen focus forming viruses during dimethylsulfoxide-induced differentiation of mouse erythroleukemia cells transformed by spleen focus forming virus. Proc. Natl. Acad. Sci. U.S.A. 72:18631867. 6. Ebert, P. S., and D. N. Buell. 1977. Viral reverse transcriptase suppression associated with erythroid differentiation of Friend leukemia cells. J. Natl. Cancer Inst.

58:635-640. 7. Eisen, H., S. Nasi, C. P. Georgopoulos, D. ArndtJovin, and W. Ostertag. 1977. Surface changes in differentiating Friend erythroleukemic cells in culture.

Cell 10:689-695. 8. Friedman, E. A., and C. L Schildkraut. 1978. Lengthening of the GI phase is not strictly correlated with differentiation in Friend erythroleukemia cells.

Proc. Natl. Acad. Sci. U.S.A. 75:3813-3817. 9. Friend, C. 1966. Immunologic relationships among some of the murine leukemia viruses, p. 51-60. In W. J. Burdette (ed.), Viruses inducing cancers. University of Utah Press, Salt Lake City. 10. Friend, C. 1978. The phenomenon of differentiation in murine erythroleukemic cells, p. 253-281. In The Harvey lectures 1976-1977. Academic Press, New York. 11. Friend, C., H. D. Preisler, and W. Scher. 1974. Studies on the control of differentiation of murine virus-induced erythroleukemic cells, p. 81-101. In A. Monroy and A. A. Moscona (ed.), Current topics in developmental biology. Academic Press, New York. 12. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971. Hemoglobin synthesis in murine virus-induced leukemic cells in vivo: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. U.S.A.

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hormone of high specific radioactivity. Biochem. J. 89: 114-123. 14. Harrison, P. R., R. S. Gilmour, N. Affara, D. Conkie, and J. Paul. 1974. Globin messenger RNA synthesis and processing during haemoglobin induction in Friend cells. II. Evidence for post-translational control in clone 707. Cell Differ. 3:23-30. 15. Ikawa, Y., ML Furusawa, and H. Sugano. 1973. Erythroid membrane-specific antigen in Friend virus-induced leukemia cells, p. 956-967. In R. M. Dutcher and L Chieco-Bianchi (ed.), Unifying concepts of leukemia. S. Karger, Basel. 16. Ekawa, Y., Y. Inoue, ML Aide, C. Kameji, C. Shibeta, and IL Sugano. 1976. Phenotypic variants of differentiation-inducible Friend leukemia lines: isolation and correlation between inducibility and virus release, p. 3747. In J. Clemmesen and D. S. Yohn (ed.), Comparative leukemia research. S. Karger, Basel. 17. Leder, A., S. Orkin, and P. Leder. 1975. Differentiation of erythroleukemic cells in the presence of inhibitors of DNA synthesis. Science 190:893-894. 18. Leiberman, D, Z. Voloch, H. Aviv, I. Nudel, and M. Revel. 1974. Effects of interferon on hemoglobin synthesis and leukemia virus production in Friend cells. Mol. Biol. Rep. 1:447451. 19. Levy, J., M. Terada, R. A. Rifkin, and P. A. Marks. 1975. Induction of erythroid differentiation by dimethyl sulfoxide in cells infected with Friend virus. Relation to the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 74:28-32. 20. Luftig, R. B., J.-F. Conscience, A. Skoultchi, P. McMillan, M. ReveL and F. H. Ruddle. 1977. Effect of interferon on dimethyl sulfoxide-stimulated Friend erythroleukemia cells: ultrastructural and biochemical study. J. Virol. 23:799-810. 21. Marchalonis, J. J. 1969. An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299-305. 22. Mayer, R. J., R. G. Smith, and R. Gallo. 1975. Reverse transcriptase in normal Rhesus monkey placenta. Science 185:864-867. 23. MCliHntock, P. R., J. N. Ihie, and D. R. Joseph. 1977. Expression of AKR murine leukemia virus gp71-like and Balb(x) gp71-like antigens in normal mouse tissues in the absence of overt virus expression. J. Exp. Med. 146:422-435. 24. McClintock, P. R., and J. Papaconstantinou. 1974. Regulation of hemoglobin synthesis in a murine erythroblastic leukemic cell. The requirement for replication to induce hemoglobin synthesis. Proc. Nat. Acad. Sci. U.S.A. 71:4551 4555. 25. Metzgar, R., T. Muhanakumar, and D. Bolognesi. 1976. Regulation between membrane antigens of human leukemia cells and oncogenic RNA virus structural components. J. Exp. Med. 143:47-63. 26. Orkin, S. H., F. I. Harosi, and P. Leder. 1975. Differentiation in erythroleukemic cells and their somatic hybrids. Proc. Natl. Acad. Sci. U.S.A. 72:98-102. 27. Ostertag, W., G. Roesler, C. J. Krieg, J. Kind. T. Cole, T. Crozier, G. Gaedicke, G. Steinheider, N. Kluge, and S. Dube. 1974. Induction of endogenous virus and of thymidine kinase by bromodeoxyuridine in cell cultures transformed by Friend virus. Proc. Natl.

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Acad. Sci. U.S.A. 71:4980-4985. 28. Pragnel, I. B., W. Ostertag, and J. Paul. 1977. The expression of viral and globin genes during differentiation of the Friend cell. Exp. Cell Res. 108:269-278. 29. Racevskis, J., and G. Koch. 1977. Viral protein synthesis in Friend erythroleukemia cell lines. J. Virol. 21:328337. 30. Roai, G. B., A. Dolei, L Cioe, A. Benedetto, G. P. Matarese, and F. Belardelli. 1977. Inhibition of transcription and translation of globin messenger RNA in dimethyl sulfoxide-stimulated Friend erythroleukemic cells treated with interferon. Proc. Natl. Acad. Sci. U.S.A. 74:2036-2040. 31. Rovera, G., and J. Bonaiuto. 1976. The phenotype of variant clones of Friend mouse erythroleukemic cells resistant to dimethyl sulfoxide. Cancer Res. 36:40574061. 32. Sato, T., E. de Harven, and C. Friend. 1973. Increased virus budding from Friend erythroleukemic cells treated with dimethyl sulfoxide, dimethyl formamide and/or bromodeoxyuridine in vitro, p. 143-151. In Y. Ito and R. M. Dutcher (ed.), Comparative leukemia research 1973. Leukemogenesis. University of Tokyo Press, Tokyo. 33. Sato, T., C. Friend, and E. de Harven. 1971. Ultrastructural changes in Friend erythroleukemia cells treated with dimethyl sulfoxide. Cancer Res. 31:1402-1407. 34. Scher, W., and C. Friend. 1978. Breakage of DNA and alteration in folded genomes by inducers of differentiation in Friend erythroleukemia cells. Cancer Res. 38: 841449. 35. Scher, W., D. Tsuei, S. Sasma, P. Price, N. Gabelman, and C. Friend. 1978. Inhibition of DMSO-stimulated Friend cell erythrodifferentiation by hydrocortisone and other steroids. Proc. Natl. Acad. Sci. U.S.A. 75: 3851-3855. 36. Sherton, C. C., S. L Dresler, E. Polonoff, M. C. Webb, and D. Kabat. 1978. Synthesis of murine leukemia virus proteins in differentiating Friend erythroleukemia cells. Cancer Rea 38:1426-1433. 37. Sherton, C. C., L H. Evans, E. Polonoff, and D. Kabat. 1976. Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells. J. Virol. 19:118-125. 38. Smith, R. E. 1974. High specific infectivity avian RNA tumor viruses Virology 66:543-547. 39. Swetly, P., and W. Ostertag. 1974. Friend virus release and induction of hemoglobin synthesis in erythroleukemic cells respond differently to interferon. Nature (London) 251:642-644. 40. Tabuse, Y., M. Kawamura, and M. Furusawa. 1976. Induction of hemoglobin synthesis in Friend leukemia cells without the necessity of mitosis. Differentiation 7: 1-5. 41. Tsuei, D., H. Haubenstock, and C. Friend. 1977. Virus production and erythroid differentiation in Friend erythroleukemia cells. In Vitro 13:148. 42. Wecker, E., A. Schimpl, and T. Hunig. 1977. Expression of MuLV gp71-like antigen in normal mouse spleen cells induced by antigenic stimulation. Nature (London) 269:598-600.