Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2682-2686, July 1975 Cell Biology

Hemoglobin synthesis in somatic cell hybrids: Coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells* (gene induction/Friend cells/erythroblasts/gene regulation/dimethylsulfoxide)

ALBERT DEISSEROTH, JANE BARKER, W. FRENCH ANDERSON, AND ARTHUR NIENHUIS Molecular Hematology Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014

Communicated by Donald S. Fredrickson, April 2,1975

Somatic cell hybrids were derived by fusion ABSTRACT of mouse erythroleukemia cells with fractionated human marrow enriched in erythroblasts, or with chinese hamster fetal liver erythroid cells. Such interspecific hybrid cells, when isolated in suspension culture, had retained nearly all the mouse chromosomes and had lost most of the human or chinese hamster chromosomes. However, two such hybrids (one human, the other hamster) studied 4-6 weeks after fusion, were found to contain several non-mouse chromosomes. RNA extracted from the human marrow X erythroleukemia hybrid annealed equally to both human and mouse globin complementary DNA, indicating that coexpression of the globin genes of each species had occurred in the hybrid cells. Mouse and human mRNA were found to accumulate only after incubation of the cells in 2% dimethylsulfoxide. The chinese hamster X erythroleukemia hybrid appeared to contain a double complement of mouse chromosomes in addition to several chinese hamster chromosomes. After 7 days of incubation in 2% dimethylsulfoxide, [3H]leucine was incorporated into chinese hamster beta-globin and the mouse globin chains. Thus, globin genes from differentiated cells, when introduced into spontaneously proliferating erythroleukemia cells, may be expressed after exposure of the resulting hybrid cells to an agent capable of inducing hemoglobin synthesis in the erythroleukemia cell.

phase, and only mouse globin was produced. In this paper, we report further studies of interspecific somatic cell hybrids derived from fusion of the Friend mouse erythroleukemia cell with hematopoietic cells of human and chinese hamster origin. Two hybrid lines containing several nonmouse chromosomes exhibited co-expression of the mouse and hamster, or mouse and human globin genes on exposure of hybrid cells to Me2SO. METHODS Cell Fusions. A 6-thioguanine-resistant clone of the Friend mouse erythroleukemia cell line (clone 745) was used. This line had a spontaneous reversion frequency of less than 10-8 (7), even under fusion conditions. Cells from human marrow aspirates were enriched in erythroid cells by centrifugation through a discontinuous albumin gradient (15-30%) to reduce the number of nonnucleated cells and then fractionated by gravity sedimentation through a 1-2% albumin gradient (7, 10, 11). Fractions containing at least 60% nucleated erythroid cells, as determined by the benzidine stain, were used for fusion. CH erythroblast populations were obtained from the 13- to 14-day fetal CH liver, a stage at which over 90% of the cells were erythroid. Erythroleukemia cells were fused with CH or human cells in a ratio of 10-20/1 in the presence of 500 hemagglutination units of beta-propiolactone-inactivated Sendai virus (7). After incubation in F-14 medium (GIBCO) for 24 hr, the cells were dispersed into several tissue culture flasks at low concentration and cultured thereafter in HAT medium (12), supplemented with 10% fetal calf serum (GIBCO). Hybrid cell populations arising in one or more of these flasks were then isolated and characterized. Benzidine staining of cells was performed as described (13). Cell extracts were electrophoretically analyzed for glu-

Hybrids between somatic cells provide an experimental system in which the coexpression of specific structural genes of two parent cell types can be examined (1-3). To study the regulation of the globin genes, we have used cell hybrids formed with a murine erythroleukemia line. Induction of mouse hemoglobin synthesis by dimethylsulfoxide (Me2SO) has been observed in a number of these erythroleukemia cell lines (4-6), but fusion of erythroleukemia cells with fibroblasts of mouse or human origin produces hybrid cells in which extinction of globin mRNA synthesis has occurred at the level of transcription or mRNA processing (7-9). We have previously described a hybrid cell line, formed by fusion of human bone marrow to erythroleukemia cells (7), which had a low level of constitutive hemoglobin synthesis that was markedly amplified on exposure of the hybrid cells to Me2SO. This line, when studied 10 weeks after fusion, retained an average of 1 to 3 human chromosomes per meta-

cose-6-phosphate dehydrogenase (G-6-P dehydrogenase) (EC 1.1.1.49; D-glucose-6-phosphate:NADP+ 1-oxidoreductase) or 6-phosphogluconate dehydrogenase (6-P-GA dehydrogenase) [EC 1.1.1.43; 6-phospho-D-gluconate:NAD(P)+ 2-oxidoreductase] as described (14). Metaphase spreads were prepared and stained as described (7). Analysis of Globin Chain Synthesis. Hybrid cells, exposed to 2% Me2SO for 5-10 days, were labeled by incubation in the presence of [3H]leucine (specific activity 30-50 Ci/mmol). One millicurie of isotope was added to 20 ml of

Abbreviations: cDNA, complementary DNA; CM-cellulose, carboxymethyl cellulose; Me2SO, dimethylsulfoxide; HAT medium, F-14 medium supplemented with aminopterin, hypoxanthine, and thymidine as formulated by Littlefield (10); G-6-P dehydrogenase, glucose-6-phosphate dehydrogenase; 6-P-GA dehydrogenase, 6phosphogluconate dehydrogenase; 1S = one diploid set of chromosomes; 2S = two diploid sets of chromosomes; CH, chinese hamster. *This is paper II of a series. Paper I is ref. 7.

F-14 medium with 10% fetal calf serum containing 107 cells. After 20 hr, the cells were washed twice with normal saline and lysed in distilled H20 by multiple freeze-thaw cycles.

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Cell Biology: Deisseroth et al. ['4C]Leucine-labeled CH and mouse marker hemoglobins prepared with phenylhydrazine-induced reticulocytes as described (15, 16). Appropriate labeled carrier hemoglobin (0.3-0.5 mg) was added to 1.0 ml of hybrid cell lysate. The sample was applied to a 0.5 X 2.5 cm column of carboxymethyl (CM)-cellulose in 10 mM phosphate buffer, pH 6.3. The column was rinsed with an additional 10-15 ml of 10 mM phosphate buffer, pH 6.3, and then the hemoglobin was eluted with 10 mM sodium phosphate buffer, pH 9.1. Additional unlabeled carrier hemoglobin (40 mg) was added, an acid acetone procedure was performed, and the globin chains were fractionated on a CM-cellulose column in 8 M urea/phosphate buffer as described (15-17). One-milliliter aliquots of the fractions were added to 10 ml of Riafluor (New England Nuclear), and radioactivity was determined by a double-label liquid scintillation technique. Peak fractions containing the globin chains from the CH X erythroleukemia hybrid were pooled, dialyzed for 24 hr against 2 liters of 0.15% acetic acid, and lyophilyzed. An aliquot was electrophoresed on polyacrylamide gels by the technique of Moss and Ingram (18), as used in this laboratory (11). The gels were sliced, the radioactive globins were eluted, and double-label liquid scintillation counting was performed. Analysis of mRNA Content of Hybrid Cells. Total cellular RNA greater than 7 S was recovered from the cells by phenol extraction and sucrose gradient centrifugation (7, 19). Human and mouse globin mRNAs were prepared from reticulocytes (19) and their complementary DNAs (cDNAs) were synthesized (20-23). RNA was annealed to cDNA and mRNA-cDNA hybrids were detected after exposure to micrococcal nuclease as described (7, 23-25). RNA-dependent DNA polymerase was prepared from avian myeloblastosis virus kindly supplied by Drs. J. and D. Beard, Office of Program Resources and Logistics, Viral Oncology, through the

Proc. Nat. Acad. Sci. USA 72 (1975)

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RESULTS Erythroleukemia Hybrids. Karyological analysis of cells of human marrow X erythroleukemia hybrid no. 1 (Table 1) 6 weeks after fusion disclosed one to three extra biarmed chromosomes per metaphase (Fig. 1A), presumably of human origin, which were not present in any of 50 metaphase spreads analyzed of the mouse erythroleukemia parent (Fig. 1B). Twenty percent and 54% of the metaphase spreads analyzed of hybrid cell populations nos. 3 and 4 contained a double complement of mouse chromosomes (2S erythroleukemia), and the remainder were cells containing a single complement of mouse chromosomes (iS erythroleukemia), as shown in Table 1. Both these populations contained a small number of biarmed chromosomes not present in metaphase spreads of the erythroleukemia parent. The hybrid nature of human marrow X erythroleukemia hybrid cell lines nos. 1-4 was further confirmed by the presence of the heteropolymeric form of G-6-P dehydrogenase (Table 1). On the average, 15% (range 10-20%) of these hybrid cells became benzidinepositive after 10 days of incubation in 2% Me2SO. Me2SOtreated cells from hybrids nos. 2, 3, and 4, analyzed at 6 weeks after fusion by CM-cellulose chromatography, demonstrated synthesis of mouse but not human globin (Table 1). Cells of hybrid population no. 1 (representing at least 27 population doublings) were analyzed 6 weeks after fusion by molecular hybridization reactions with globin cDNA. Mouse globin mRNA, synthesized in erythroleukemia parent cells Human Marrow X Mouse

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^^A A( I0 ^A ^ FIG. 1. (A) Karyotype of human marrow X mouse erythroleukemia cell hybrid no. 1 six weeks after fusion. The underlined chromosomes represent biarmed chromosomes not present in metaphase spreads of the mouse erythroleukemia parent (B), and therefore are presumably of human origin. (B) Karyotype of the 6-thioguanine-resistant mouse erythroleukemia cell. (C) Karyotype of fetal hamster erythroblast X mouse erythroleukemia cell hybrid no. 1 six weeks after fusion. The underlined chromosomes represent ones not present in the metaphase spreads of the mouse erythroleukemia parent (B), and therefore are presumably of hamster origin. Several of these underlined chromosomes are identifiable hamster marker chromosomes.

on exposure to 2% Me2SO, reacted only slightly with the

human cDNA (Fig. 2A). Both human and mouse globin mRNA sequences were present in the hybrid cell RNA (Fig.

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Cell Biology: Deisseroth et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. Hemoglobin synthesis in erythroblast X mouse erythroleukemia cell hybrids

Cell lines

Erythroleukemia parent Human marrow cells Chinese hamster fetal erythroblasts Human marrow X mouse erythroleukemia hybrid No. 1 (1S EL x iS HM) No. 1 (iS EL X 1S HM) No. 2 (iS EL x iS HM) (2S EL x iS HM) N (2S EL x iS HM) EL x iS HM) No. 4 (1S (2S EL x 1S HM) Chinese hamster X mouse erythroleukemia hybrid No. 1 (2S EL x 1S CH) No. 2 (iS EL x iS CH) (2S EL x 1S CH)

Weeks after fusion

Total no. of chromosomes*

-

38.1 (34-42) 46 22

6 10 6 6 6 6 6

41.8 42.3 43.5 41.6 77.1 40.6 75.9

6 10 10

83.0 (57-103) 39.5 (35-44) 71.0 (59-77)

(35-47) (37-43) (40-56) (37-52)

(69-88) (35-43)

(61-107)

No. of biarmed chromosomes 4.0 (3-5) 36 16

6.0 3.7 5.4 5.4 11.3 4.5 9.5

(5-7) (1-5)

(4-8) (5-7)

(9-16) (4-7) (8-17)

14.3 (8-18) 3.9 (3-5) 6.7 (6-7)

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N.T., not tested; EL, erythroleukemia; CH, Chinese hamster; HM, human marrow. * All average chromosome numbers are based on analysis of at least 30 metaphase spreads for each cell line listed. t RNA-cDNA hybridization was used to analyze globin gene expression in HM x erythroleukemia hybrids nos. 1 and 4. Labeling of globin chains and CM-cellulose chromatography was used to analyze globin gene expression in HM X erythroleukemia hybrids nos. 2, 3, and 4, as well as CH X erythroleukemia hybrids nos. 1 and 2.

2B). Me2SO incubation of these cells resulted in marked increases in the levels of globin mRNA of both species (Fig. 2B). At 6 weeks after fusion, hybrid cell population no. 1 contained biarmed chromosomes (Fig. 1A) not present in the erythroleukemia parent (Fig. 1B) or in human marrow X erythroleukemia hybrid cell populations nos. 2, 3, or 4. Ten weeks after fusion, karyological analysis revealed loss of the extra biarmed chromosomes present at 6 weeks after fusion (Table 1), and RNA extracted at this stage contained mouse globin mRNA but little if any human globin mRNA (Fig. 2C). Hamster Fetal Erythroblast X Erythroleukemia Cell Hybrids. Karyological analysis of cells of CH fetal erythroblast X mouse erythroleukemia hybrid no. 1 (Table 1), when studied 4 weeks after fusion, revealed a large number of marker CH chromosomes (Fig. IC). A double complement of mouse chromosomes was present, as reflected by the total number of telocentric chromosomes (Table 1) and by the presence of two large telocentric marker chromosomes having secondary constrictions (Fig. iC). (Only one such marker chromosome is present, per diploid genome, in the parent mouse erythroleukemia cell, as shown in Fig. 1B) The hybrid nature of these cells was confirmed by the presence of heteropolymeric forms of 6-P-GA dehydrogenase. Twenty percent of the 1 X 109 cells of CH X erythroleukemia hybrid no. 1 available at 6 weeks after fusion (at least 29 population doublings) became benzidine-positive after 4 days of incubation in 2% Me2SO, indicating the presence of hemoglobin in these-cells. Analyses for globin chain synthesis revealed a peak of [3H]leucine-labeled protein (tubes 25-27) coinciding with the elution volume of the marker 14C-labeled CH beta globin (Fig. 3). In addition, peaks of activity

corresponding to the mouse beta major (tubes 31-34), mouse beta minor (tubes 37-41), and mouse alpha (tubes 48-54) globin chains were present in the [3H]leucine extracts of the hybrid cell (Fig. 3). The fractions containing the CH beta globin were recovered, and the 3H-labeled product was shown to comigrate with the authentic CH beta [14C]globin by alkaline urea/polyacrylamide gel electrophoresis (Fig. 4A), while the radioactivity of the mouse beta major chain migrated in the same position as mouse beta major globin (Fig. 4B). Comigration of the CH and mouse alpha globin chains on CM-cellulose prevented analysis of CH a globin gene expression. Cells of the CH fetal liver X mouse hybrid no. 2 contained CH marker chromosomes and a single complement of mouse chromosomes 6 weeks after fusion. By 10 weeks after fusion, most of the identifiable CH chromosomes had been lost. Analysis of the labeled proteins at 10 weeks by CM-cellulose chromatography demonstrated that only mouse globins were synthesized (Table 1). DISCUSSION These studies have established the potential for the globin genes from two different species to be coexpressed in a hybrid cell. Induction of both human and mouse globin mRNA by Me2SO was detected in a human marrow X mouse erythroleukemia cell hybrid by the highly specific molecular hybridization reaction with human and mouse globin cDNA (Fig. 2). Me2SO-dependent synthesis of mouse and CH beta globin in a CH fetal liver X mouse erythroleukemia cell hybrid was established by chromatographic purification of labeled hemoglobin synthesized in the hybrid cells followed

Cell Biology: Deisseroth et al. MOUSE GLOBIN cDNA

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Proc. Nat. Acad. Sci. USA 72 (1975)

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ythroleukemia cell hybrid no. 1 six weeks after fusion, which had been incubated in 296 Me2SO for 7 days and labeled for 20 hr with [3H]leucine. The hemoglobin from the hybrid cell had been purified by batch elution from a CM-cellulose column with 10 mM phosphate buffer after addition of the 14C-labeled hamster reticulocyte hemoglobin (see Methods).

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FIG. 2. Hybridization of human and mouse globin cDNA with RNA extracted from erythroleukemia cells and human marrow X mouse erythroleukemia cell hybrid no. 1. Reaction mixtures contained mouse or human globin cDNA (0.3 ng, specific activity 6000 cpm/ng) and one of the following: (A) RNA extracted from erythroleukemia cells incubated for 7 days in the presence (0) or absence (0) of 2% Me2SO; (B) RNA extracted from human marrow X erythroleukemia hybrid no. 1 six weeks after fusion (several human chromosomes still present) incubated for 7 days in the presence (-) or absence (0) of 2% Me2SO; (C) RNA extracted from human marrow X erythroleukemia hybrid no. 1 ten weeks after fusion (most of the human chromosomes lost by this time) after incubation for 10 days in the presence (0) or absence (0) of 2% Me2SO. (0.025 A260 = 1 ug of RNA). The fraction of cDNA resistant to nuclease in the absence of added RNA (10-20%) has been subtracted from each point. Where there was sufficient RNA to run two or more determinations at a given point, the range is shown.

conditions used. Since hybrid cells are isolated after distribution of the fusion products into 10 separate vials and grown through at least 29 population doublings before analysis, the contribution of remaining nonviable unfused parental cells to the hybrid cell population could be no more than 0. 1%.

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by demonstration that the putative CH globin comigrated with authentic CH reticulocyte beta globin, first on a CMcellulose column in 8 M urea/phosphate buffer at pH 6.8 (Fig. 3) and then on electrophoresis in a 7% polyacrylamide gel at pH 9.8 (Fig. 4). Whether the probable erythroid nature of the human and CH parent cells used in these fusions was important in obtaining these results is unknown. Nonetheless, we have shown that globin genes from differentiated hematopoietic cells can be introduced into a continuously proliferating cell of another species in a state that permits induced expression of these genes in the presence of Me2SO. Several lines of evidence have established that the 15-20% benzidine-positive cells present in the hybrid cell population are not unfused parental cells remaining in the population of cells analyzed for globin gene expression. All of the 30 to 50 metaphase spreads analyzed of each hybrid cell population contained chromosomes of both parents. The 6-thioguanineresistant erythroleukemia cell, the human marrow cells, and the chinese hamster fetal liver cells are incapable of cell division and are nonviable after 2 weeks under the culture

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Cell Biology: Deisseroth et al.

erythroleukemia hybrid cells only after they had been exposed to Me2SO. The relationship between the human globin gene expression and the general process that resembles erythroid differentiation on -exposure of either these hybrid cells or the parent erythroleukemia cell to Me2SO remains to be defined. Cell division appears to be necessary for Me2SOinduced expression of the mouse globin genes in an erythroleukemia cell (26). The ability to induce erythroid differentiation is not a unique property of Me2SO, for several other polar compounds are also effective (27). The probability that a given erythroleukemia cell will undergo erythroid differentiation on exposure to Me2SO may be characteristic for each erythroleukemia line (8). The mechanism of globin gene regulation, whether transcriptional, post-transcriptional, or translational, may vary between individual erythroleukemia lines (28, 29). Similar considerations as to the exact mechanism of globin gene induction apply to the hybrid cells we have generated. The simultaneous induction of globin genes on separate chromosomes from two species, observed in these hybrids, suggests that diffusible regulator substances may be involved. Human globin mRNA synthesis in the human marrow X erythroleukemia hybrid cell no. 1 was absent after loss of several biarmed human chromosomes. Whether the genetic locus lost was a structural locus or a regulatory locus is not known. Further study of these hybrid cells exhibiting both coexpression of globin genes of different species and progressive chromosomal loss may generate further information concerning the chromosomal locations of structural and regulatory loci involved in regulation of human globin genes. Also, hybrid cells of this type offer an opportunity to study control of expression of the three linked globin structural genes (gamma, delta, and beta) in man. Only the beta gene directs the synthesis of significant protein in the adult human erythroid cell. Study of the products of these genes in a hybrid cell might permit clarification of the mechanism for their differential activity.

Proc. Nat. Acad. Sci. USA 72 (1975) Davidson, R. L. & de la Cruz, F. (Raven Press, New York), pp. 137-150. 4. Friend, C., Scher, W., Holland, J. G. & Sato, T. (1971) Proc. Nat. Acad. Sci. USA 68,378-382. 5. Ostertag, W., Melderis, H., Steinheider, G., Kluge, N. & Dube, S. (1972) Nature New Biol. 239,231-234; 6. Ross, J., Ikawa, Y. & Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69,3620-3623. 7. Deisseroth, A., Burk, R., Picciano, D., Minna, J., Anderson, W. F. & Nienhuis, A. (1975) Proc. Nat. Acad. Sci. USA 72, 1102-1106. 8. Orchin, S., Hirosi, T. & Leder, P. (1975) Proc. Nat. Acad. Sci. USA 72,98-102. 9. Ruddle, F. H. & Kucherlapati, A. (1974) Sci. Am. 130,36-44. 10. Miller, R. A. & Phillips, R. A. (1969) J. Cell. Phys. 75, 191206. 11. Barker, J. E., Anderson, W. F. & Nienhuis, A. W. (1975) J. Cell Biol. 64,515-527. 12. Littlefield, J. W. (1964) Science 145, 709-711. 13. Fowler, J. H., McCulloch, E. A., Till, J. E. & Simmovitch, L. (1960) J. Lab. Clin. Med. 68, 523-530. 14. Khan, M. P. (1971) Arch. Biochem. Biophys. 145,470-483. 15. Nienhuis, A. W. & Anderson, W. F. (1972) Proc. Nat. Acad. Sct. USA 69,2184-2188. 16. Crystal, R. G., Nienhuis, A. W., Elson, N. E. & Anderson, W. F. (1972) J. Biol. Chem. 247,5357-568. 17. Clegg, J. B., Naughton, M. A. & Weatherall, D. F. (1966) J. Mol. Biol. 19,91-108. 18. Moss, B. & Ingram, V. M. (1968) J. Mol. Biol. 32,489-496. 19. Nienhuis, A. W., Falvey, A. & Anderson, W. F. (1974) Methods in Enzymology, eds. Moldave, K. & Grossman, L., (Academic Press, New York), Vol. 30, pp. 621-630. 20. Verma, I. M., Temple, G. F., Fan, H. & Baltimore, D. (1972) Nature New Biol. 235,163-167. 21. Kacian, D. L., Spiegelman, S., Bank, A., Terada, M., Metafora, F., Dow, L. & Marks, P. A. (1972) Nature New Biol. 235, 167-169. 22. Ross, J., Aviv, H., Scolnick, E. & Leder, P. (1972) Proc. Nat. Acad. Sci. USA 69,264-268. 23. Falvey, A. K., Kantor, J. A., Roberts, M. G., Picciano, D. J., Vavich, J., Weiss, G. & Anderson, W. F. (1974) J. Biol. Chem.

249,7049-7056. We thank Marcia Willing, Theresa Caryk, Bernard Kefauver, Eugene Young, and Patricia Turner for their excellent technical assistance, Drs. J. and D. Bear through the Office of Program Resources and Logistics, Viral Oncology, National Cancer Institute, for donation of the avian myeloblastosis virus, and Dr. Paul McCurdy of the Georgetown Medical School for referral of patients who donated bone marrow. 1. Harris, H. (1970) Cell Fusion (Harvard University Press, Cambridge, Mass.). 2. Ephrussi, B. (1972) Hybridization of Somatic Cells (Princeton University Press, Princeton, N.J.). 3. Davidson, R. L. (1974) in Somatic Cell Hybridization, eds.

24. Steggles, A. W., Wilson, G. W., Kantor, J. A., Picciano, D. J., Falvey, A. K. & Anderson, W. F. (1974) Proc. Nat. Acad. Sci. USA 71, 1219-1223. 25. Kacian, D. L. & Spiegelman, S. (1974) Anal. Biochem. 58, 531-540. 26. McClintock, P. R. & Papaconstantinou, J. (1974) Proc. Nat. Acad. Sci. USA 71, 4551-4555. 27. Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A. & Marks, P. A. (1975) Proc. Nat. Acad. Sci. USA 72, 10031006. 28. Gilmour, R. S., Harrison, P. R., Windass, J. D., Affara, N. A. & Paul, J. (1974) Cell Differ. 3,9-22. 29. Harrison, P. R., Gilmour, R. S., Affara, N. A., Conkie, D. & Paul, J. (1974) Cell Differ. 3,23-30.