Selective induction of murine oncornavirus gene expression in somatic cell hybrids between mouse peritoneal macrophages and SV-40-transformed human cells

VIROLOGY

70, 545-549 (1976)

Selective Induction of Murine Oncornavirus Gene Expression Somatic Cell Hybrids between Mouse Peritoneal Macrophages SV-40-Transformed Human Cells

in and

ME’ITE STRAND, J. T. AUGUST, AND CARLO M. CROCE Department of Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461; and The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104 Accepted November

21, 1975

Somatic cell hybrids of BALB/c and C57BL/6 mouse peritoneal macrophages and human Lesch-Nyhan fibroblasts transformed by infection with simian virus 40 have been examined by radioimmunoassay for synthesis of the viral envelope and major core proteins of the murine endogenous type-C oncomavirus. Little or no detectable viral proteins (approximately 1 nglmg of protein) were present in extracts of the parental cells. In contrast, hybrid clones of BALB/c cells containing human chromosome 7 with the simian virus 40 genome synthesized extremely high concentrations of the murine type-C virus core protein (210 to 6000 nglmg) and also the envelope glycoprotein (83 to 250 ng/mg). The highest concentrations were in cells containing human chromosomes 5 and 11 in addition to 7. There was little or no change in the concentration of viral proteins in the C57BL/6-human cell hybrids. These results suggest that human-cell gene products can be active in the induction of the murine oncornavirus genome.

Peritoneal macrophage cultures derived from C57BL/6 or BALB/c mice were prepared according to the method of Cohn and Benson (13) with some modifications. Human-skin fibroblasts deficient in hypoxanthine - guanine - phosphoribosyl - transferase were derived from a patient with the Lesch-Nyhan syndrome (14) and were obtained from the Institute for Medical Research, Camden, N.J. (LN cells). LN-SV cells were SV-40-transformed LN fibroblasts (15). Mouse peritoneal macrophages were fused with LN-SV cells in the presence of P-propiolactone-inactivated Sendai virus at pH 8.0 (16). The fused cultures were selected by growth in hypoxanthine-aminopterinthymidine (HAT) medium (17). The hybrid cell colonies were picked, propagated in HAT medium, and cloned. Each clone studied was unique and derived from a different fusion event as each hybrid colony originated from a different flask. At the time of this study, hybrid clones derived from the fusion of mouse macro-

Mouse peritoneal macrophages (MPM), normally a nondividing cell Cl), when fused with human Lesch-Nyhan fibroblasts transformed by W-40 (LN-SV), yield clones that grow indefinitely in culture (2, 3). It has been shown that these mouse-human cell hybrids selectively contain copies of the human chromosome 7 carrying the SV-40 genome (2,3). In these studies we have analyzed the effect of human chromosome 7 containing the SV-40 genome on the expression of endogenous virus genome(s) of mouse cells. The type-C DNA provirus is a normal chromosomal constituent of mouse cells, and expression of these viral genes may occur normally in some strains of mice, or may be induced in others (4-12). The concentration of viral proteins in tissues of different strains of mice varies indicating mechanisms markedly (11,12), controlling the level of viral-gene expression, acting either on transcription or translation of viral nucleic acid or on metabolism of viral proteins. 545 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

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phages with LV-SV cells were subcultured for more than 30 passages. Cells grown to subconfluence were washed twice in Ca2+- and Mg2+-free phosphate buffered saline. The flasks were then incubated at 37” for 5 min in 10 ml of TEN buffer (20 mM Tris-hydrochloride, pH 7.6, 1 mM EDTA, 100 mM NaCl). Flasks were agitated vigorously, and the cells were harvested by low-speed centrifugation. Cell pellets were suspended in 10 ml of TEN buffer, enumerated, and concentrated by centrifugation. Vital staining with trypan blue indicated that these cells remain intact and viable throughout this procedure. Cell packs were stored frozen at -20” prior to solubilization. For analysis frozen cells were suspended in approximately 0.2 ml of TEN buffer per 10’ cells. Triton X-100 was added to a final concentration of 0.4%, and the suspension was incubated at 37” for 15 min and then frozen in Dry Ice-methanol and thawed at 37” three times. The resulting suspension was centrifuged at 2000g for 10 min, and the supernatant was collected. Each of the parental cell types (LN-SV, BALB/c MPM and C57BL/6 MPM) were examined for the cellular concentration of murine virus p30 and gp69/71 by competition radioimmunoassay (18). The assay system utilized purified Rauscher murine virus p30 and gp69/71 proteins (19) as labeled antigens and anti-feline virus serum as described previously for analysis of murine viruses and tissues (11, 12, 20). This system has the advantage of measuring conserved interspecies antigenic determinants common to murine viruses (20). Little or no detectable viral proteins were present in extracts of the parental cells (Table 1). The concentrations of viral proteins in mouse peritoneal macrophages were lower than those found in extracts of spleens of these strains, BALB/c mouse spleens containing 4 and
TABLE 1 CONCENTRATIONS OF VIRAL PROTEINS IN EXTRACTS OF PARENTAL CELL@

Cells

Viral protein gp69/71 P30 (ng/mg protein)

LN-SV C57BL/6 MPM BALB/c MPM

co.1 4.0 co.1

co.1 1.0 1.0

’ Protein concentration was measured by radioimmunoassay as described in the text and in Figs. 1 and 2.

cells, the absence of detectable viral protein is as expected for a human cell analyzed with an assay capable of detecting mouse or feline virus proteins. So as to extend this analysis to include the p30 proteins of primate viruses that have been detected in human tissues (211, further analysis of each of the different cells was carried out with an assay system composed of Rauscher murine lzsI p30 antigen and anti-RD 114 virus serum. This system had previously been shown to detect proteins of each of the major groups of mammalian type-C viruses, the murine, feline, RD 114/ baboon, and woolly monkey/gibbon ape (21, 22). The values obtained were not significantly different from those of the murine antigen:anti-feline virus serum assay system; no virus protein was detected in the LN-SV cells at the concentrations tested (data not shown). A large number of independent clones of human-mouse hybrids produced by fusion of peritoneal macrophages from normal C57BL/6 and BALB/c mice with LN-SV cells have been found to exhibit the characteristics of the transformed phenotype (2). These included the lack of densitydependent inhibition of cell growth, a saturation density greatly exceeding that of normal mouse or human cells, and the formation of colonies of soft agar. No significant differences in saturation density or generation time were observed between the C57BL/6- and BALB/c-derived clones (2, 3). The generation time of both was approximately 16 to 18 hr as compared to 26 hr for the LN-SV parental cells. All these hybrid clones were SV-40 T-antigen positive. Karyological analysis was per-

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formed with 67 of these hybrid clones; all 67 contained the human chromosome 7, and in 13, chromosome 7 was the only human chromosome present in the hybrid cells. In the present study, five of these hybrid clones, three of BALB/c and two of C57BL/6 origin, were selected for analysis of the synthesis of type-C virus p30 and gp69/71 proteins. All contained human chromosome 7; two of the BALB/c:LN-SV hybrids, clones 36 and 43, also contained human chromosome 5 in more than 65% of the cells (Table 2). Each of the BALB/c hybrids contained high concentrations of viral p30 (Fig. 1). This was particularly true of clones 36 and 43, where the concentration of viral p30 protein was more than lo4 greater than in the parental cells and, astonishingly, represented almost 1% of the total soluble protein of the cell extract (Table 3). Each also produced high concentrations of the membrane glycopeptide gp69/71. For one of the clones, cl 10, the ratio of p30 to gp69/ 71 was approximately 2:1, the same as the ratio of these proteins in virions, and may represent the coordinate induction of all of the viral proteins. Indeed, these BALB/

d lo-510-410-310-z10-IIO0 PROTEIN (mg)

FIG. 1. Analysis of type-C virus proteins in LN-

SV cells, BALB/c MPM, and hybrid cells of clones 53-87 (3) Cl 10, 53-87 (3) Cl 36, and 53-87 (3) Cl 43. The competition radioimmunoassays were performed as described using (A) 1 ng of L251-labeled Rauscher murine virus p30 (8 x lo4 cpm/ng) and rabbit anti-feline (Theilen strain) virus serum (l:lO,OOOfinal dilution), and (B) 2 ng of *“-“I-labeled Rauscher murine virus gp69/71(7 x lo4 cpm/ngl and rabbit anti-feline (Theilen strain) virus serum (1:3,000 final dilution). TABLE 3

TABLE 2 KARYOL~GIC ANALYSIS OF THE HYBRID CLONES STUDIED

Hybrid clones

Human chromosomes present in the hybrids’ 5

BALB/c MPM:LN-SV 53-87 (3) Cl 10 53-87 (3) Cl 36 53-87 (3) Cl 43 C57BL/6 MPM:LN-SV 53-87 (1) Cl 16 53-87 (1) Cl 21

7

20/20 22122

Cells

o/20 o/22

” Human chromosomes present in the hybrid cells were identified by a modification of the Giemsa banding method described by Seabright (23, 24). The chromosome preparations were treated with a solution of 0.05% trypsin with 0.02% EDTA (GIBCO) for 5 min. They were washed with medium containing 5% fetal calf serum, and then washed with Hanks’ solution. The chromosome preparations were then stained with 2% Giemsa at pH 6.8 for 5 min, rinsed with pH 6.8 buffer (obtained by dissolving one tablet of G. T. Gurr, pH 6.8, buffer in 100 ml of water), and dried with humidified hot air. At least 20 metaphases of each clone were analyzed.

Viral protein gp69/71 P30 (ng/mg protein)

11

O/25 25125 o/25 17125 25125 4125 24/30 30/30 13/30 5/20 o/22

CONCENTRATIONS OF VIRAL PROTEINS IN EXTRACTS OF HYBRID CELL@

BALB/c MPM:LN-SV 53-87 (3) Cl 10 53-87 (3) Cl 36 53-87 (3) Cl 43 C57BL/6 MPM:LN-SV 53-87 (1) Cl 16 53-87 (1) Cl 21

83 250 250

210 6000 6000

0.5 0.5

0.1-I 1

a Protein concentration was measured by radioimmunoassay as described in the text and in Figs. 1 and 2.

c:LN-SV hybrid clones all produce infectious murine leukemia virus (K. Huebner, personal communication). For the other two clones much greater concentrations of p30 than gp69/71 were found. This relative excess of p30 was also found in spleen cells of BALB/c mice (11, 12). At this time it is not known if the the differences between

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clone 10 and clones 36 and 43 are random, or can be attributed to the presence of other human chromosomes (5 and 11) in clones 36 and 43. It is not related to synthesis of a human cell virus, as characterization of the antigenic reactivity of these proteins by appropriate assay systems distinguish the proteins as murine, not primate (data not shown). In contrast to these findings with the BALB/c cells, the hybrids of LN-SV with C57BL/c cells showed little or no significant differences over the parental C57BL/c MPM (Fig. 2). Thus, the mechanisms operative in induction of viral proteins in these somatic cell hybrids appear to be specific to cells of certain strains of mice, and not a general phenomenon. The mechanism of induction of viral gene expression is unknown and appears to operate at many levels of cellular metabolism. Type-C virus or viral protein synthesis in cultured cells has been observed to occur under manv circumstances, such as treatment with a variety of agents, including halogenated pyrimidines (7, 8, 25, 27), inhibitors of protein synthesis (27), physical or chemical carcinogens (28, 29), or lipopolysaccharides (30, 31), or after external infection with leukemia viruses (32), spontaneous or SV40-induced transformation (33,34), mixed

lymphocyte culture (3.51, or even longterm cultivation (3638). Thus, it is not clear whether the induction of BALB/c type-C virus(es) in these hybrid cells is a direct effect of the presence of human chromosome 7 or of the SV-40 genome, or a secondary effect of cell transformation or cell division. Nevertheless, other studies of transformed BALB/c cells, whether spontaneously transformed or transformed by treatment with chemicals, by infection with murine sarcoma viruses, or by hybridization to murine sarcoma virus-transformed rat or mouse cells, indicate that induction of endogenous BALB/c virus proteins was not invariably observed (34, 3942). Moreover, in other studies, SV-40 transformed mouse cell lines of BALB/c or C57BL/6 fibroblasts were found not to produce type-C viruses as detected by XC plaque assay or to express p30 (Huebner and Croce, unpublished observations). Thus, it appears that the induction of viral genes in BALB/c cells might in some way be related to a role of human chromosome 7 or of the human chromosome 7 carrying the SV-40 genome. Whether or not other human chromosomes alone might be operative in the same manner unfortunately cannot be tested as viable hybrids are not obtained by fusion of BALB/c macrophages with normal human cells (2) and with the present hybrids chromosome 7 is selectively retained. However, of particular interest is the extremely high concentration of viral p30 protein in hybrids containing human chromosomes 5 and 11, in addition to 7. The possible role of these chromosomes will be tested by the analysis of segregant subclones of these hybrid cells which have lost human chromosome 5 or 11, or both. ACKNOWLEDGMENTS

IL+

10-5

14’ PROTEIN

10-1

10-z

10-I

100

(mq)

FIG. 2. Analysis of type-C virus proteins in LNSV cells, C57BL/6 MPM, and hybrid cells of clones 53-87 (1) Cl 16, and 53-87 (1) Cl 21. The competition radioimmunoassays were as described in Fig. 1.

This work was supported in part by US Public Health Service Research Grant Nos. CA 10815 and CA 16685 from the National Cancer Institute, No. RR 05540 from the Division of Research Resources, Nos. GM 20700 and 11301 from the Institute of General Medical Sciences, by Contract No. 71-2251 within the Virus Cancer Program of the National Cancer Institute, and by a Basil O’Connor Starter Grant to C.M.C. from the National Foundation. C.M.C. is a recipient of a Research Career Develop-

SHORT ment Award No. CA 00143 from the National Institute.

COMMUNICATIONS Cancer

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